Neural Mechanisms Underlying The Generation Of Multiple Forms Of Locomotion And The Coordinated Expression Of Locomotion And Feeding In Aplysia

In face of a complex and uncertain environment and to meet diverse internal needs,motivational behaviors,such as locomotion and feeding,display multiple forms and are coordinated with each other to produce an optimal behavior strategy.Although there are separate studies on the mechanisms about the generation and modulation of locomotion and feeding,the generation of diverse forms of locomotion and the coordination between these behaviors remain poorly understood.In this thesis,we use an experimentally advantageous system,mollusk Aplysia,to study these problems.Since there is little prior knowledge about the Aplysia locomotor pattern-generating circuit,we will start the research by identifying the neuronal elements in the locomotor circuit,and then study the mechanisms of the higher-order neural center regulating the diversity of locomotor forms and the coordination between locomotion and feeding.We describe these studies in details in the following three sections:First,we seek to identify the elements in the pedal locomotor pattern-generating circuit.Aplysia locomotion is a kind of typical rhythmic behavior.The forward movement of the animal is achieved by the front-to-back rolling waves generated on its foot,and there is a progressive phase shift among the contractions of different regions of foot muscles.Previous studies showed that there are a number of rhythmic neurons on the dorsal surface of the pedal ganglion exhibiting the phase shift in the P9-stimulation locomotor program,but they have identified neither the motoneurons nor the pattern-generating elements in the pedal ganglion.Given the rhythmicity of the pedal wave,we first provided direct evidence that the PPCN(parapedal commissural nerve)bursts correspond to the tail contraction in intact animal using implanted electrodes in behaving animals,establishing PPCN as a monitor of locomotion.Then we identified a cluster of motoneurons(about 20 cells),named P1 root motoneurons,located at the root of the P1 nerve on the ventral surface of pedal ganglion,and showed that they are motoneurons by electrophysiological experiments in neuromuscular preparations.Their axons project to the periphery through pedal nerves P1,P7,P8,and P9,and their activation can elicit contraction of different regions of foot muscles when stimulated.In addition,compared with the firing phase of PPCN,the firing phases of these motoneurons covered most of the phases in one locomotor cycle.In addition,we also found that there is electrical coupling between motoneurons that fire in a similar phase,but not between motoneurons that fire in different phases,which may contribute to the phase shift of the pedal wave.Therefore,a series of bursting activities of P1 root motoneurons and PPCN can represent the pedal waves during locomotion in vitro.This provided the basis for the subsequent exploration of the neural mechanisms related to locomotion in the isolated nervous system.Moreover,we found two types of potential interneurons in the locomotor central pattern generator on the ventral surface of the pedal ganglion.These types of interneurons can elicit or regulate locomotor programs and have synaptic connections to P1 root motoneurons.However,they differ in their axonal projections.The axons of the first type of interneurons(2 of them)only projected within the ipsilateral pedal ganglion,which may be related to the generation of the unilateral locomotor rhythm;the axons of the second type of interneurons projected to the contralateral pedal ganglion through the pedal commissure,which may be related to the coordination of the locomotor rhythm between two sides.Second,we studied the roles and circuit mechanisms of the cerebral high-order neurons CC9/10 and CPR(cerebral-pedal regulator)in inducing different forms of locomotion.Previous work showed that the serotonergic CC9/10 can elicit defensive locomotor programs(escape).Here we for the first time found that CPR which mediates food-induced arousal can also elicit locomotor programs.Previous work indicates that CPR is activated by food stimuli from the head.We now used a semi-intact feeding preparation to show that CPR is strongly activated before feeding,but becomes quiescent during the consummatory feeding behavior.The CPR-elicited locomotor programs were significantly different from the CC9/10-elicited programs.Comparison between the programs elicited by CPR and CC9/10 showed that the CC9/10-elicited programs were stronger.More importantly,the CPR-elicited programs would terminate soon after terminating stimulation,whereas the CC9/10-elicited programs were persistent for more than 2 minutes.This phenomenon is one of the typical differences between appetitive and defensive locomotion.Then,we studied the synaptic mechanisms underlying it.Immunohistochemical experiments showed that CPR is glutamatergic.We found that CPR elicits fast EPSPs to P1 root motoneurons,which could be blocked by the AMPA receptor antagonist CNQX;while CC9/10 elicits slow EPSPs to P1 root motoneurons,which could be blocked by the mollusk 5-HT receptor blocker Methysergide.This suggested that the ionotropic AMPA receptor and the metabotropic 5-HT receptor may play important roles in mediating the CPR and CC9/10-elicited locomotor programs.In order to verify the hypothesis above,we perfused 5-HT agonist and AMPA agonist in the isolated pedal preparations and found that the agonist-induced programs match the programs elicited by CPR and CC9/10,respectively,in terms of program intensity and persistence.Moreover,the CPR and CC9/10-elicited programs could be curtailed significantly by AMPA or 5-HT receptor blockers respectively.Finally,we construct a simple computer model showing that synaptic dynamics from CPR or CC9/10 and their ionotropic or metabotropic receptors could account for the different persistence in motor programs.In short,utilizing Aplysia nervous system,we,for the first time,clarified that the higher-order neurons CC9/10 and CPR can use 5-HT and glutamate as neurotransmitters respectively to elicit defensive and appetitive locomotor program by activating the ionotropic AMPA receptor or metabotropic 5-HT receptors in Aplysia.Third,we performed a preliminary study about the role of neuropeptides in the coordinated expression of locomotion and feeding in Aplysia.A series of previous promote different feeding programs.The first type,such as APGWamide and FCAPb(feeding-circuit activating peptide,type b),can promote ingestive programs,while the other type,such as ap NPY(Aplysia Neuropeptide Y),can promote egestive programs.Here,we used behavioral and physiological experiments to show that APGWamide and FCAPb can inhibit locomotion,whereas ap NPY can promote locomotion.Moreover,we found that CC9/10 may be one of the potential targets of these neuropeptides.In short,our findings reveal the important roles of neuropeptides in the orderly and coordinated expression of locomotion and feeding in Aplysia.In summary,we used various approaches,including behavioral,physiological,pharmacological,immunohistochemical,and computational experiments,from macroscopic behavior to mesoscopic neural circuits,systematically demonstrate that higher-order neurons can induce different forms of locomotion through distinct receptors activated by different neurotransmitters in Aplysia,and neuropeptides are involved in coordinating the orderly expression of locomotion and feeding.Since the diversity of behavioral expression and the coordination between motivated behaviors are present broadly in the animal kingdom,our findings are expected to contribute to the discovery of the novel neural mechanisms underlying these phenomena and provide clues to the relevant research in other animals,including vertebrates.