| Brain science is an important field in modern life sciences in21st century. Exploring working patterns of neural network is one of the key issues of brain science research. In recent years, regulation of rhythmic activity by neural networks has been extensively examined. Compared to complicated neural network of brain, relatively small network of central pattern generator (CPG) has been widely addressed. CPG is the neural network that can produce rhythmicity even when no sensory input or timing input is present. CPG regulates simple physiological rhythmic activities such like walking, swimming, chewing, breathing and movements of pylorus and gastric mill.Stomatogastric ganglion (STG) of crayfish is the common model that has been used to study CPG because of many advantages. It has a clear anatomical structure and is composed of a small number of neurons, and the synaptic connections between neurons are relatively simple. Isolating STG is relatively easy and physiological condition of in vitro STG sample can sustain a relatively long time. Rhythmic activity of stomach is performed by contraction of muscles innervated by different neurons of STG. Pylorus has an obvious and stable rhythmicity which is a tri-phasic activity of discharge generated by three groups of neurons in succession. The generation of rhythmicity depends on the characteristics of neurons and their mutual interactions through synapse.STG is composed of thirty neurons among which sixteen neurons control gastric mill and fourteen neurons generate pyloric rhythm. Compared with rhythmicity of gastric mill, the rhythmicity of pylorus is more often to be studied because it is easy to be identified and recorded with a long stability. The fourteen neurons in pylorus consist of one interneuron anterior buster (AB) and thirteen motoneurons. According to their functions, these neurons can be divided into one AB neuron, two PD (pyloric dilator) neurons, one LP (lateral pyloric) neuron, eight PY (pyloric) neurons, one IC (inferior cardiac) neuron and one VD (Ventricular dilator) neuron. AB neuron plays a role of pacemaker in pyloric rhythm. However, AB is an interneuron without axon reaching to the muscles and the discharge activity of AB can not be recorded extracellularly. Since PD neuron and AB neuron are electrically coupled and can be regarded as an AB/PD assembly. All synaptic connections in pylorus network are inhibitory. AB/PD, LP and PY neurons inhibit each other and fire in succession, generating a tri-phasic pyloric rhythm.The maintenance and regulation of pyloric rhythm depends on excitability of each neuron. Activity of ion channels on neuronal membrane, therefore, is of great importance for maintenance and regulation of pyloric rhythm. IA and Ih, currents are activated near the resting potential and can regulate excitability of neurons.Though how IA and Ih, regulates excitability of individual neurons in STG network has been extensively studied, the role in maintenance and regulation of the whole pyloric rhythm played by IA and Ih, remains unknown. Using specific blocker4AP and ZD7288, this study examined how these blockers change the pylorus rhythm. The result contributes to the understanding of not only generation and regulation mechanism of pyloric rhythm, but also the maintenance and regulation mechanism of CPG activity.Results:1. Pyloric tri-phasic rhythm under normal conditionWhen isolated STG is under perfusion of normal extracellular fluid, STG pylorus network present tri-phasic rhythm, that is, LP, PY and PD neurons discharge one after another. LP neuron discharge with a high amplitude and a high frequency, PY neuron discharge with a high amplitude and a low frequency, PD neuron discharge with a low amplitude and a high frequency. Under15℃, the whole cycle of the tri-phasic rhythm is about6s-8s, in which LP cycle is about1s, PY cycle is about3.5s-5.5s and PD cycle is about1.5s.2. The influence on pyloric rhythm made by STG front-end ganglionIn a low-temperature condition, ice cold extracellular fluid is added on COG to maintain a stable pyloric rhythm. At room temperature, however, the pyloric rhythm becomes unstable. The result shows that STG front-end ganglion has a regulatory function on pyloric rhythm.3. The effect of different concentrations of4AP on pyloric tri-phasic rhythmWhen lmmol/L4AP is added on isolated STG, the of tri-phasic rhythm is unstable but its tri-phasic pattern remains. When2mmol/L4AP is applied, the cycle of pyloric rhythm decreases, the rhythm is stable and its tri-phasic pattern remains. When4mmol/L4AP is applied, the rhythm becomes periodic and the tri-phasic pattern vanishes. 4.2mmol/L4AP changes the cycle and phases of pyloric rhythmWhen2mmol/L4AP is applied, the cycle of pyloric rhythm decreases to about2s, among which LP cycle is about0.4s, PY cycle is about0.6s and PD cycle is about1s. Paired t-test revealed that the decrease is statistically significant (P<0.01, n=9). Compared to the cycle of whole rhythm, the PD ratio reduces (P<0.01) and the LP ratio remains unchanged, and the discharging phase of LP and PY neurons are delayed.5. The effect of different concentrations of ZD7288on pyloric rhythmWhen50μmol/L ZD7288is added on isolated STG, the pyloric rhythm is unstable and PD phase is lost. When100μmol/L ZD7288is applied, the cycle of pyloric rhythm is stable and the tri-phasic pattern remains but the cycle of pyloric rhythm decreases. When200μmol/L ZD7288is applied, tri-phasic pattern disappears.6.100μmol/L ZD7288changes the cycle and phases of pyloric rhythm10minutes after100μmol/L ZD7288application, the cycle of pyloric rhythm decreases to about1.5s, among which LP cycle is about0.45s, PY cycle is about0.5s and PD cycle is about0.55s. Paired t-test revealed that the decrease is statistically significant (P＜0.01, n=10). Compared to the cycle of whole pyloric rhythm, the PD ratio increases (P<0.01), the of LP ratio reduces (P＜0.01) and the phase of LP and PY activity is relative delayed. |
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