1. Introduction
The sense of direction, encoded by head direction (HD) cells in the brain, is critical for animals to recognize their surroundings and navigate effectively in space. An impaired sense of direction can cause a loss of the ability to find one’s way in locomotor environments [
1,
2]. When the head of an animal points in a specific direction, the corresponding HD cells discharge preferentially, with different cells having different preferred directions [
3]. Head direction cells are widely distributed in multiple brain regions and interconnect to form a head direction cell system which decodes the sense of orientation in animals [
4].
The retrosplenial cortex (RSC) is a crucial brain region for spatial navigation and is widely involved in memory and contextual encoding [
5]. It participates in the processing of visual information via numerous connections with multiple visual areas, including the primary visual cortex [
6]. In addition, the RSC has a wide distribution of angular head velocity (AHV) cells which encode angular head velocity and receive inputs from the vestibule [
7]. Furthermore, studies have shown that animals with lesions in the RSC exhibited defective pathway integration in the dark [
8,
9]. Therefore, RSC is encoded by both visual and vestibular signals and is essential to spatial navigation behaviors involving various visual cues or self-motion.
The attractor model, an important theoretical model of HD function, suggests that when rats face a specific orientation in a visual environment, the optical input cells activate corresponding HD cells, and the angular velocity cells provide angular velocity input to encode the self-motion signal to update the HD signal [
4]. In this way, HD cells can maintain a steady sense of direction even in darkness [
10]. The combination of visual and vestibular inputs contributes to increasing the head direction encoding accuracy of the HD system [
11,
12]. However, visual and vestibular inputs can become dissociated when we take transportation and use VR devices, which differ from traditional navigation in a stable environment. The way in which HD cells process these two types of dissociated sensory information has not yet been investigated. Recent studies have started to explore the difference between self-motion and visual input to investigate the relative contributions of two sensory inputs to the spatial activity of place cells in the RSC and hippocampus [
13,
14] and the temporal synchrony effects of optic flow and vestibular input on heading perception [
15]. Therefore, detection of the response of the HD system under visual and vestibular information dissociation remains a problem to be solved.
At present, extracellular recording of brain neurons includes neural electrodes and two-photon microscopic imaging. Two-photon imaging is a method of optical imaging to obtain neuronal discharge signals [
16], which features a wide field of view and high throughput [
17]. Neural electrodes are implanted into the brain of animals and directly contact with target neurons, which can obtain rich electrophysiological information such as spike discharge and local field potential [
18,
19], with high temporal and spatial resolution. At the same time, because of its small size and light weight, it is easier to detect electrophysiological information in the motion state. Compared to traditional wire electrodes, the MEA has good orientation and high integration, which are conducive to accurate implantation in the target brain area and the detection of high-density neurons with less implantation damage. Furthermore, the distribution of electrode sites can be customized according to the detection task, and a relatively fixed spatial distribution can be maintained during the implantation process to maximize the detection effect. In addition, MEAs prepared using MEMS technology have good uniformity and their planar site can be modified with nanomaterials to improve detection performance [
20]. Indeed, Pt nanoparticles (PtNPs) have the advantages of high surface area and high conductivity, which can significantly enhance the ability to detect weak signals [
21]. As a conductive polymer, poly(3,4-ethylene dioxythiophene) (PEDOT) has been verified as having a low Young’s modulus and good biocompatibility [
19], and has been widely used in the biomedical field. The PtNPs and PEDOT are electrodeposited on the microelectrode, which helps to reduce the impedance of the microelectrode and improve long-term detection stability.
In our study, an MEA suitable for microdriver stepping detection was designed to locate HD cells. PtNPs and PEDOT were electrodeposited on the MEA to protrude from the electrode sites and improve the detection performance and biocompatibility. Because of the sensitivity of HD cells to directional changes, we rotated a cylindrical arena to separate the visual and vestibular signals of the rats in the directional dimension. We measured the directionality of HD cells in the RSC at the three stages of before, during, and after the separation of the visual and vestibular input of the rats. We found that within a short time, visual information had a more substantial effect on HD cells under sensory dissociation than vestibular input. Furthermore, with prolonged sensory dissociation, the function of the HD cells was gradually impaired.
2. Materials and Methods
2.1. Design and Fabrication of the MEA
In this work, we designed an MEA to detect HD cells in the RSC. It consisted of four 4 mm long needles with a 110 × 26 μm
2 (width × thickness) cross-section and a tip angle of 54°. Each needle contained four recording sites (diameter = 14 μm) and a reference site (14 μm × 146 μm). Driven by a microdriver, the MEA was lowered by 75 μm every day to monitor deeper cells. In order to monitor more cells, we designed the arrangement of electrode sites so that none of the forward tracks of the recording sites overlapped (
Figure 1B). In addition, the recording sites were close to the edge (8 μm) so that, after stepping, they exceeded the original outline range of the electrode (
Figure 1B). Studies have shown that approximately 12 h after MEA insertion, microglia begin moving toward the implanted MEA. After 24 h, they can surround the MEA [
22]. By designing the distance between the electrode sites and the edge and using stepping, our electrode sites could be reimplanted into an area with fewer microglia which contributes to improving the electrode recording effect. In summary, we designed the lateral distribution of electrode sites and their distance to the edge of the silicon needles and used microdriver stepping electrodes to improve the detection effect of HD cells.
The MEA was fabricated in a clean room using microelectromechanical systems (MEMS) technology, as previously reported [
23]. Three photo masks were designed, including conductive, insulating, and MEA probe shape layers. (1) An SOI wafer was thermally oxidized and spun using photoresist AZ5214. Then, we used an initial photolithography process to define the shape of the conductive layer. The microelectrode areas were patterned selectively by sputtered Ti/Pt (30 nm/250 nm), followed by the lift-off process (
Figure 1A(a–d)). (2) A SiO
2/Si
3N
4 (300 nm/500 nm) insulation layer was deposited on the conductive layer via plasma-enhanced chemical vapor deposition (PECVD, 300 °C). The insulating layer pattern was defined using the second photolithography process. Then, it was etched in a CHF
3 environment to expose the recording sites and bonding pads (
Figure 1A(e–f)). (3) The MEA probe shape layer pattern was transferred onto the wafer. We selectively deep-etched the silicon layer to form the electrode shape. Finally, the MEA was released using wet etching to remove the backer silicon (
Figure 1A(g–i)). The completed MEA is shown in
Figure 1B.
2.2. PtNPs/PEDOT:PSS Modification Method
To detect electrophysiological signals with a high signal-to-noise ratio and long time detection, the Pt nanoparticles (PtNPs) and PEDOT:PSS were electrochemically deposited on the MEAs successively. We changed the electroplating parameters to increase the protrusion of the recording sites and improve biocompatibility. Firstly, 48 mM H2PtCl6 and 4.2 mM Pb(CH3COO)2 were 1:1 mixed to obtain the PtNPs solution, and the EDOT solution was obtained by adding 20 mM EDOT to 0.1 M PSS solution and mixed ultrasonically for 30 min to ensure uniform dispersion. Next, the MEAs were electrodeposited (CA, −1.1 V, 60 s) in Pt solution to form PtNPs substrates with a protruding structure. Then, we electroplated a thick PEDOT layer in EDOT solution with parameters CV, 0~1.05 V, rate: 100 mV/s, 20 cycles to further improve the convexity and biocompatibility of the electrode sites. All the electrochemical deposition processes were carried out using electrochemical instruments (Gamry Instruments, Warminster, PA, USA).
2.3. Subjects and Surgery
Three male Sprague Dawley (SD) rats weighing 250 g were selected for the experiments. The rats were 3 to 6 months old and were individually housed under a 12 h light–dark schedule. Their food was slightly restricted to reduce their weight to 90% of normal free-feeding weight. All experiments were conducted with the permission of the Beijing Association on Laboratory Animal Care and approved by the Institutional Animal Care and Use Committee at the Aerospace Information Research Institute, Chinese Academy of Science (AIRCAS).
Before surgery, a self-made microdriver shuttle plate was mounted on the MEAs, enabling the MEAs to be lowered in order to detect deeper brain regions (
Figure S3B). Under 1–2% isoflurane anesthesia, the rats were fixed on a stereotaxic device. We cleaned the tissue and exposed the skull, and then implanted six stainless steel screws into the skull to serve as ground electrodes. Then, we opened the skull (AP: −4.44 mm, ML: −0.7 mm) and slowly implanted the MEAs in the RSC at 0.7 mm. After implantation, the shell of the microdriver was fastened to the skull using dental adhesive, and the shuttle plate was protected with Vaseline to ensure mobility. After surgery, the rats were allowed one week to recover before the experiment commenced.
After the test, the electrode was removed, and another MEA with red fluorescent dye DiI (Beyotime, Shanghai, China) was replanted at the same position. After half an hour, the rats were anaesthetized and sequentially infused with saline and 4% paraformaldehyde (PFA) throughout the body to fix the brain tissue. Afterwards, the brain was taken and dehydrated in the sucrose solution (20% sucrose in PB 0.1 M, 30% sucrose in PB 0.1 M). Finally, the brain was sliced into 50-micron slices using a Rotary Microtome Cryostat to obtain the implantation traces of the MEA (
Figure S15).
2.4. Apparatus
The recording environment was a cylindrical arena made of semilucent milky white acrylic (diameter: 80 cm, height: 80 cm), separated from the rest of the room by a cylindrical curtain with a diameter of 120 cm. The curtain had the same center as the arena. A black cue card (42 cm wide × 41 cm high) was attached to the inside of the wall. Opposite the cue card, a triangular LED strip (color temperature: 6000 K, powered by USB) was fixed on the outside of the wall, and its light could illuminate the inside of the arena through the wall as another visual cue. A motor was placed under the cylindrical arena and this could rotate the arena at a speed of 1.4 r/min.
2.5. Recording Setup
The electrical activity and behavioral data of the rats were captured simultaneously by the recording. Sixteen-channel headstage and recording cables were attached to the MEA connector. The electrical signals were amplified and stored by a neuron data recording system (AIRCAS-128, China) at 30 kHz. Highpass (>250 Hz) and lowpass (0–250 Hz) filters were used to separate neural spikes and LFPs, respectively. Two LEDs (one red and one green, spaced 6 cm apart) fixed to the headstage assembly provided the behavioral information, including the location and direction of the rat’s head. An overhead camera recorded the LEDs at 30 frames per second. Further behavioral information processing was performed using behavioral processing software (EthoVision XT16, Noldus, Beijing, China).
2.6. Behavioral Training and Testing Protocol
Rats were pretrained for two weeks before surgery to familiarize them with the recording environment. They were trained to forage for millet that was randomly scattered around the arena once every three minutes. For the first ten days, the rats were trained for 20 min per day in the stable arena. For the next four days, the arena was stable for 15 min and rotated for 15 min to help the rats adapt to the rotating environment. When the arena was spun, the visual and vestibular inputs of the rats dissociated so that the rats observed that they remained stationary in relation to their surroundings, but in the vestibular perception, they felt they were rotating in relation to the ground. Then, the MEA implantation surgery was performed, and the rats were allowed a week to recover.
After the rats had recovered from surgery, the electrophysiological recording sessions were initiated. Before and after recording, the cue cards and the recording arena were wiped with 75% ethanol to scramble olfactory signals. The roughness of the cylinder base was uniform, and, before recording every day, the whiskers of the rats were trimmed to within 1–2 mm above the skin surface using blunt surgical scissors. The recording sessions consisted of three groups of Control–Rotating–Recovery trials (
Figure S1d–f), with each trial lasting 10 min. In Control, the arena was stable, and rats ran freely in the open field. We recorded neural activity in the RSC under these normal conditions. During Rotating, we rotated the arena to separate the visual and vestibular information of the rats, and recorded changes in the neural activity of the brain under the navigation of visual and vestibular separation. Then, in Recovery, the area was again stable, and we recorded the neural activity of the rats in their recovered state. Two hours before the daily tests, the microdriver was used to drive the MEA down in steps of 75 μm.
2.7. Identification and Analysis of HD Cells
The relative orientation of the green and red LEDs was used to estimate the direction of the rats’ heads. The HD tuning curve was used to describe the distribution of neuronal firing rate with direction in each bin of 10 degrees, which was measured using the following formula:
where
,
was the total number of spike trains in one bin, and
was the total amount of time that the head was pointed in that bin. Then, the binned tuning curve was smoothed with a Gaussian kernel (standard deviation of 30°). The peak firing rate was defined as the rate in the bin with the highest rate, and the peak angle was defined as the angle of this bin.
Directional specificity was evaluated using Rayleigh vectors. It was determined by calculating the mean vector length from the circular distributed firing rates. Cells were considered to be head direction cells if the mean vector of the data recorded in Control was longer than the 95th percentile of the mean vector lengths in the distribution generated from the shuffled data. The value of the 95th percentile was 0.26.
To better describe the orientation specificity of HD cell tuning curves. We proposed the following formula to quantify it:
where
was defined as the highest rate in tuning curve, and
was defined as the average firing rate in all directions.
4. Discussion
In this study, an MEA was designed and fabricated to reliably find HD cells. The PtNPs/PEDOT:PSS-modified sites, characterized by a three-dimensional convex structure, increase the surface area and contact with tissues and effectively improve the signal-to-noise ratio and electrical performance. Three approaches were used to improve the ability to find HD cells, including the design of the lateral distribution of the electrode sites, a reduction in the distance between the recording sites and the edge of the silicon needle, and the daily use of a microdriver stepping electrode.
To explore the influence on the HD system, the visual and vestibular signals of the rats were dissociated by rotating the arena. We found that when subjected to a short period of sustained sensory dissociation, the HD cells anchored to the visual reference frame and their directional preference drifted. With prolonged and continuous dissociation of the two signals, the directional tuning of the HD cells gradually degraded. After the dissociation ended, they slowly reverted to the direction they established based on visual information rather than to the original direction.
During short periods of visual and vestibular input conflict, changes in the directionality of the HD cells were used to explore the contributions of the two signals. The inputs of both visual and vestibular sensory information play an essential role in the directional tuning of HD cells in the RSC [
33,
34]. In our experiment, when the two sensory inputs were dissociated for a short time, the apparent directional tuning and consistent drift of the peak directions of the HD cells in the visual frame of reference contrasted with the implicit directionality and scrambled shift of peak angles in the vestibular frame of reference (
Figure 5). We suggest that the HD cells retain their directional tuning and remain anchored to the optical frame of reference during short periods of sensory dissociation. The HD cells bound more strongly to visual signals than vestibular inputs. A recent study used calcium imaging to record the place cells firing when mice watched VR virtual images to mediate their vision and vestibular senses, finding that visual signals overrode locomotion signals [
13]. Using an MEA to detect the electrophysiological signals of neurons, we also observed that head direction cells were dominated by visual information input compared with vestibular perception. This result is consistent with our navigation in the internal environment of buses in daily life.
After entering the rotating environment, HD cells anchored to a new direction based on visual information (
Figure 5A), which may be due to the sudden rotation of the arena forming a new environment, and the change in environmental conditions causing a drift in the anchored orientation of the HD cells.
In response to long-term and continued visual–vestibular dissociation, the directional tuning of HD cells degraded significantly. The RSC brain region receives both visual and vestibular information [
33,
35] and is the critical brain region integrating the two types of sensory information. At the same time, HD cells also integrate information in brain regions such as the ADN and project between HD cells in multiple brain regions [
36]. In addition, visual and vestibular information play different roles in HD cell circuits in different brain regions [
4]. It is possible that long-term information asynchronism increases the difficulty of information integration in the brain, which gradually causes dysfunction of the information integration function and thus leads to the degeneration of the directional tuning ability of the HD system (
Figure 6A,B). This result is consistent with the poor orientation caused by car sickness or the prolonged use of VR devices.
Similar to the report from a previous experiment [
37], we noticed a shift in the firing direction of HD cells in the RecoveryIII compared to Control stage. In this study, the firing direction shift occurred at the moment of transition from a stationary to a rotating environment. Throughout the experiment, the HD cells showed obvious directionality in three periods: Control, RotatingI, and RecoveryIII. In the Recovery stage, the preferred direction of the HD cells reverted to the direction newly established in the RotatingI stage (
Figure 6A,C), indicating that the new direction established during the RotatingI stage was stable and the rats remembered the area environment in its rotating state. Finally, the HD cells reverted to the direction newly established in the RotatingI stage rather than the original direction in the Control stage. This phenomenon may be consistent with the example of people experiencing disorientation when suddenly getting out of a car after having been asleep during the journey. If we do not correct the directional deviation between ourselves and the external environment in time due to falling asleep or other reasons, our internal direction will be different from the actual direction of the outside world. When we get out of the car, we will feel disoriented and need to correct our sense of direction. So, in the navigation of both internal and external environments, it is necessary to constantly update the direction with visual information to maintain correct awareness of direction.
In this study, we revealed the phenomenon of orientation anchoring and degradation of HD cells under sensory dissociation. However, we need to acknowledge that this experiment also has some limitation, rotation speed is an influential factor in studies of visual and vestibular dissociation. A slowdown or increase in rotation speed might have a different effect on the behavior of HD cells. In this experiment, we revealed the behavior of HD cells under sensory inconsistency at an appropriate rate. It would be interesting and useful to further explore the effects of different rotation speeds on HD cells and to find a speed threshold that allows the HD system to deal with sensory inconsistencies below it. In addition, it would be meaningful to explore ways to enhance the HD system’s ability to deal with sensory inconsistencies in combination with electrical stimulation therapy.