A group of homing pigeons (Columba livia) were bred and housed in a loft under a normal day/night light cycle with food and water ad libitum. They were trained by flying around the loft twice a day with a load of 33 g that is heavier than the control module for robo-pigeons (16.8 g, see later detail). The load was normally attached to the pigeon’s back by gluing Velcro straps to the body of the pigeon and then attaching the load to the Velcro. Twenty pigeons, all between 2 and 3 years old, were selected for long-distance homing training twice per week. The robo-pigeon release sites were approximately 30 km from the loft to ensure enough time for testing the hierarchy stimulation algorithm. Pigeons were transported to the release sites in a van in carrier baskets allowing adequate ventilation. Two male pigeons with considerable homing experience, weighing 458 g and 476 g, were chosen from the twenty for the field investigation.

The preparatory surgical operation and electrode implantation was the same as previously reported [17]. Every effort was made to minimize animal suffering and minimize the number of pigeons used. This study was conducted in accordance with the Guide of Laboratory Animal Management Ordinance of China, and was approved by the Jiangsu Association for Laboratory Animal Science (Jiangsu, China).

Two nuclei, the nucleus intercollicularis (ICo) and the formation reticularis medialis mesencephali (FRM), were chosen for the deep brain stimulation (DBS), as they have been proved effective in the laboratory for electrical microstimulation of motion modulation of robo-pigeons under light anesthesia and ground moving [17]. Stimulation of both ICo and FRM reliably evoked motor behavior such as wing flapping and/or lateral body movments that might affect flight trajectory outdoors.

To precisely implant microelectrodes into the ICo or FRM, a standard stereotaxic instrument (type 68001, RWD Life Science, Shenzhen China) integrated with a Revzin adaptor [25] was employed. When fully inserted, the distance between the tips of the ear bars of the adaptor was 10.6 mm and 10.8 mm for the two chosen pigeons. After properly stabilizing the pigeon’s head in the adaptor, reliable stereotaxic coordinates were established following convention [25]. The location of the two stimulation targets are given in Table 1. Fig. 1 illustrates the distribution of ICo and FRM at a 3.50 mm coronal section of by comparing the stereotaxic atlas with a Magnetic Resonance Imageing. A total of eight electrodes were implanted into the ICo or FRM, while only two with reasonable stimulation efficacy (one for each hemisphere) were chosen for the final outdoor investigation.

Fig. 1.

Distribution of ICo and FRM at the coronal section of 3.50 mm. Left panel is cited from the stereotaxic atlas of the pigeon brain [25], while the right panel is a corresponding MRI image. The enclosed area in the MRI image indicates ICo (above) and FRM (below).

A posteriori histochemical analysis is required to check the accurate position of implanted microelectrodes to determine whether the tips have been correctly placed. However, subjects do not survive this procedure. Given the precision the stereotaxic instrument employed for electrode insertion (0.01 mm) and the success rate of electrode implantation in previous studies [17], the posteriori procedure was not conducted in this study. Correct electrode placement was determined by observing the motor behavior elicited by the electrical microstimuli during the implantation procedure. For each half brain of a robo-pigeon, four electrodes were implanted within the range given in Table 1. The best performing electrode was chosen for the outdoor investigation.

A robo-pigeon is comprised of three components: a well-trained pigeon provides the robot-moving carrier, a miniature light-weight control module, and a sustainable brain machine interface (BMI) that connects the control module to the pigeon’s brain [26]. Typically, users interact with the control module by wireless communication [20-23], that restricts the working radius of robo-animals and interferes with outdoor investigation. To eliminate this restriction, the control module employed for this study was designed to use onboard preprogramming.

The system architecture of the control module was designed on an embedded computer system based on ARM [27]. It contained four main components Fig. 2.3. The GPS sub-module (ATK-NEO-6M) with a ceramic antenna provided the real-time position and timing updated per second, using signals from satellites. The stimulation sub-module generated brain stimulation signals by means of pulse width modulation (PWM). The micro-SD card storage sub-module logged the GPS signals and the stimulation commands for data post processing. The CPU sub-module (STM32f103rbt6) was used to activate the stimulation sub-module by the preprogrammed control logic (such as given in Fig. 3.1a), making use of the signal from the GPS sub-module.

Fig. 2.

A. The system block diagram of the control module. CPU is the kernel of the system with the preprogrammed code. According to the signal from GPS, CPU judges whether to send stimulation commands to BMI. All these information processed by CPU is logged in SD-card; B. The side photo of the control module. It is in size of 34 $\mathit{mm}\times$ 24 $\mathit{mm}\times$ 20 $\mathit{mm}(L\times W\times H)$ and in mass of 16.8 g including a rechargeable battery. During experiments, it is attached by the Velcro to the back of the tested bird.

Fig. 3.

A. The robo-pigeon is landing on the loft. The pigeons are trained to carry a similar sized and weighted gauge block in daily flight. During experiments, the gauge block is replaced by the control module. B. The close-up view of the control module mounted on the robo-pigeon. The module is connected by loose winding wires to the BMI on the robo-pigeon’s head, by which the stimulation commands are transmitted.

Rectangular biphasic pulses were employed for brain stimulation of the robo-pigeons due to their lower threshold [17] and lack of charge-accumulation [28], that both reduced the risk of injury during electrical stimulation. The hierarchy of three levels of stimulation (single-, periodic- and multi periodic-stimuli) was designed to ensure the effectiveness of the stimulus. Single-stimuli were defined as a one second 80 Hz 3.0 V biphasic square wave pulse train. Periodic-stimuli were as constituted of two successive single-stimuli followed by a two second resting potential of zero volts giving a duration of 4 s. Multi periodic-stimuli comprised 5 groups of periodic-stimuli with total duration of 20 s. This hierarchical approach to stimulation has proved practicable for outdoor investigation (see below).

The two robo-pigeons flying with simple control logic (Fig. 3.1a) were piloted around their loft to estimate the efficiency of stimulation. Whether stimulation had the desired effect was determined by the variation in flight direction at two successive sampling points before and after stimulation. For each robo-pigeon, two half brains and a total of 10 sequences of multi periodic-stimuli (thus, 50 periodic-stimuli and 100 single-stimuli) were tested. Fig. 3.1b gives one recording example, which shows the robo-pigeon’s orbiting flight trajectory was coincident with the brain stimulation control at the scale of multi periodic-stimuli. The left stimulation was applied to the target in the left half brain, which was expected to elicit a left turn; while the right stimulation was applied to the target in the right half brain, which was expected to elicit a right turn. The success ratios of the deep brain stimulation are given in Table 3.

Fig. 4.

A. Simple control logic for robo-pigeons flying around loft. After takeoff of a robo-pigeon, the control module waits 5 minutes to make sure the pigeon flying under stable conditions, and then generates the directional-stimuli alternatively by a specified number of times to test the stimulation efficiency. B. One example of flight trajectory under this control logic. GPS sampling frequency was 1 Hz, and the flight direction was indicated by black arrows on the trajectory. Right-pointing and left-pointing triangles indicate the timing when neural stimulation applied, respectively.

Fig. 5.

A. One example of robo-pigeon flight long distance outdoor. The color coded curve is the flight trajectory from south to north, and the color represents the robo-pigeon’s flight speed. The background is the local map referring to Google Map. Two white squares indicate two ROIs, where the multi periodic-stimuli were applied and the orbiting flight was elicited. B. & C. The detail of the flight trajectory in the squares indicated in A.

A long distance outdoor flight control investigation was conducted using two robo-pigeons. The birds were transported to a release site 30 km away from their loft. The release site was familiar to the birds as they had been regularly trained on homing flight twice per week. Two regions of interest (ROI) were selected according to the weekly training flight trajectories of the birds. The control logic was designed such that if the robo-pigeon’s GPS location was detected in the ROI, the multi periodic-stimuli would be applied to the brain stimulation target.

Fig. 3.1 gives an example of the robo-pigeon’s homing flight from the release site 30 km away. It covered from the south of north latitude 31.94 ${\mathrm{}}^{\circ }$ to the north of north latitude 31.97 ${\mathrm{}}^{\circ }$ . Within the latitude range, two ROIs were chosen, predefined for the DBS tests, and preprogrammed into the control module. When the robo-pigeon flew into the specified region, orbiting flight was successfully elicited by DBS so as to increase the time spent over the ROIs. Both robo-pigeons showed robust controllability for elicitation of orbiting flight.