| Most insects have gained amazing flight skills during the long-term evolution, and their high maneuverability of flight has drawn great attention from human for a long time, to explore their flight mechanisms and imitate them by man-made micro air vehicles (MAVs). Although this traditional bionic study has developed for decades, it still confronts significant challenges in many aspects, such as payload mass, flight range, speed and so on. In recent years, a novel approach called the insect-machine interface (IMI) has been developed and promises to solve the bottle-neck in the study of MAVs by directly controlling the flight behavior of insects. IMI implements remote control to electrical stimulation on either neural or neuromuscular systems of insects through the built hybrid system between insects and micro-electronic mechanical systems.The most important technique in IMI, which controls insect flight by neural stimulation, is named brain-machine inteface (BMI). So far, studies on BMI have achieved successful control of flight initiation and cessation in both beetles (Cotinis texana and Mecynorhina torquata) and moth (Manduca sexta), as well as the "throttling" (frequency and stroke amplitude of wing oscillation) modulation. But all previous studies have laid emphasis on establishing methods to achieve control results from an engineering point of view. In addition, brain areas that were stimulated before only included two primary processing brain subregions for sensory input — optic lobe and antennal lobe, also the position of electrodes was lack of accuracy.The honeybee (Apis mellifera L.), as a eusocial insect, owns a rich repertoire of behaviors but a simple brain, so that it has been taken as an emerging model organism for studies especially on brain function and neural regulation mechanisms for the social behaviour, learning and cognition, flight et al..Taking honeybee as the controlled object, by establishing a technology to precisely locate several brain subregions and an optimized brain stimulation scheme, the neuromechanism underlying flight control by BMI was studied. The contents and major results are listed as follow.(1) The honeybee brain subregion locating method. After design, assembly and test, we had set up an equipment system used for honeybee fixation, electrode implantation and electrical stimulation. Meanwhile, a stereotyped procedure for fixing honeybee head in consistent position had been established. Furthermore, positional data for six brain subregions in two — medio-lateral and antero-posterior directions were obtained from measurement of brain sections of nine honeybees. And, we identified some new brain landmarks by stereomicroscopy on brain surface and light microscopy on brain sections. In the end, by combining these brain landmarks with positional data, we had established a new brain locating method for embedding microelectrodes reproducibly into targeted brain subregions. By prussian blue verification using the optimized electrode dissociation method we had proved this method was adequate to locate seven brain subregions in honeybees reproducibly.(2) Optimizing parameters for electrical stimulation. We chosed the unilateral lobula as a constant stimulation site, and took the success rate for flight initiation and flight duration induced by stimulation as evaluation basis. Four parameters of stimulus pulses were optimized including amplitude, pulse width, frenquency and duration. As a result, the optimal parameters for stimulation were amplitude at 30 μA, pulse width at 1 ms, frequency at 200 Hz, and duration at 0.3 s.(3) Effects of stimulating different brain subregions on flight initiation. We used the optimized parameters for electrical stimulation with frequency at 200 Hz, pulse width at 1 ms, and duration at 0.3 s to stimulate different brain subregions of honeybees and compare the success rate for flight initiation. Meanwhile, we had set up two intensity groups which applied current amplitude at 10 μA and 30 μA, respectively. Results showed that current intensity had significantly affected the success rate for flight initiation, which was higher in high intensity group for most brain subregions. Also, stimulation of different brain subregions had shown different success rate for inducing honeybee flight which decreased in the sequence of a-lobe (or β-lobe), ellipsoid body, lobula, medulla and antennal lobe. Among which, stimulation of the mushroom body peduncles, including α-lobe and β-lobe, showed the highest success rate for flight initiation (100%). Followed by the ellipsoid body of the central complex, which obviously required higher stimulus intensity than the mushroom body peduncles. To the medulla or lobula — two optic lobe neuropils, only when high intensity current was used, flight could be induced in 43% and 81% of honeybees, respectively (N= 100). Other regions such as the antennal lobe and the mushroom body calyx failed to induce honeybee flight even with a higher current intensity. However, stimulation of the subesophageal ganglion rear to the antennal lobe induced flight initaition in 17.5% and 62% of honeybees in the low and high intensity group, respectively.(4) Neurotransmitters or neuromodulators related to flight inducing. By dripping a biogenic amine, biogenic amine receptor antagonist or acetylcholine solution onto the brain surface of honeybee, we had achieved to manipulate the content or action course of this neurotransmitter/neuromodulator. Then the success rate for flight initiation and flight duration were compared respectively between the stimulation of α-lobe given before and after drug delivery. Results showed except octopamine, neither the manipulation of dopamine nor of seretonin had obvious influence on flight inducing. While the addition of octopamine elongated flight duration and increased success rate, application of an octopamine-receptor antagonist shortened flight duration and reduced success rate. Besides, application of acetylcholine had not shown obvious influence on flight inducing too.In conclusion, this thesis includes the study of accurate method for locating multiple brain subregions in honeybees, optimizing parameters for electrical stimulation for inducing honeybee flight, effects of stimulating different brain subregions on flight initiation, and neurotransmitters/neuromodulators related to flight inducing. The results of this thesis provide an enlightening significance to continue developing the locating method for insect brain subregions. Also we have proved there are some other brain subregions except the optic lobes that can be used to control insect flight by BMI. There may be a group of neurons in each of these brain subregions mediating flight initiation through a cluster of descending neurons which arrive to the thoracic ganglia to contact with motor neurons innervating flight muscles. Meanwhile, through drug delivery experiments, we have proved octopamine as an important neurotransmitter in the neural circuit for flight control by BMI. These results will support us to further study the neurons involved in specific brain areas via a neurophysiological approach, and fully reveal the flight control neuromechanism underlying BMI. |
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