| Background and Objective:There are two cell types in the central nervous systems (CNS) of mammals----neurons and glia. The structure and function of neurons have been thoroughly studied. While, compared with neuron, the role of glia in information processing has not been systematically studied because they cannot produce action potentials like neuron. During the past decades, they have been considered to play a supportive role in CNS instead of information processing.Recent studies suggest that brain should no longer be regarded as a circuitry of neuronal contacts, but as an integrated network of interactive neurons and glial cells. Glial cells, and astrocytes in particular, are much more actively involved in brain information processing than previously thought. However, these results were mainly got from in vitro experiments, lacking of persuasive. While in intact animals, the connection of glial cells and neurons is intact and the environment is more stable, so the results are much closer to the reality. As a result, we want to investigate the roles of astrocytes in sensory processing in vivo. Barrel cortex, as a principle part of primary sensory cortex, whose layer IV has an exquisite somatotopic map where each individual whisker in rodent face is represented in a discrete anatomical unit, the "barrel". Each neuron in the barrel mainly receives information input from its corresponding whisker and generates evoked responses. So we can record the electrical activities of neurons when the whisker is stimulated. In the present study we want to investigate the roles of astrocytes in sensory processing at layer IV of barrel cortex.Methods:Sprague-Dawley (SD) rats weighing 220-280g and C57BL (C57) weighing 25-30g were used in this study, ignoring sex distinction. The rats were anaesthetized by intraperitoneal injection of urethane (20%,0.8ml/100g). The rat was fixed in a stereotaxic instrument. A small craniotomy was made according to the atlas and a tungsten electrode (FHC) was inserted into the layer IV of the barrel cortex (rat: centered 1.9mm to bregma and 5.5mm lateral to the midline, and a depth of about 0.8-0.9mm from surface of cortex; mice:centered 1.5mm to bregma and 3mm lateral to the midline, and a depth of about 0.4mm from surface of cortex). A whisker was stimulated by an air puff from glass microelectrode which was generated from a picospritzer controlled by an electrical stimulator. The direction of air puff is vertical to the stimulated whisker. The amplitude of vibration of the whisker caused by airflow is about lmm. We recorded the evoked responses of barrel cortex to identify the PW. The intracerebral microinjection volume of drug to barrel cortex's layer IV is also 1μl. The injection is finished in 3-5 min at average rate. In whisker deprivation experiment, we trimmed all the whiskers during the first postnatal week and the fifth postnatal week in SD rats. When they became adult, we recorded their responses evoked by whisker stimulation and compared the differences among three groups. After the experiment, the animals were perfused and the brains were taken out for further identification of the recorded brain area. For some rats, we did cytochrome oxidase histochemistry reaction to show the architecture of the barrel cortex.Results:1. Stimulation width would change the evoked responses in barrel cortex. The responses could not be evoked when the stimulation width was Oms, 1ms or 2ms; while we could record the evoked responses when the stimulation width was 3ms,4ms,5ms,6ms,7ms,8ms,9ms,10ms and the amplitudes of evoked responses were nearly same from 3ms to 10ms.2. The effects of various interstimulus intervals (ISI) between two whiskers ipsilateral or contralateral on the evoked responses were examined. When the ISI was 0.2s,2s or 5s, the ratio of the two evoked responses was about 1, which means stimulating one whisker would not affect the evoked response of stimulating another whisker ipsilateral or contralateral at the same time.3. Rhythmic vibrissal stimulation at 100 Hz applied to the whisker (PW) for 1 s caused a long-lasting increase in the vibrissa-to-cortex evoked responses. This long-lasting enhancement also occurred in the unstimulated barrel. Stimulation at 100 Hz was followed by a significant long-lasting increase in the peak amplitude of responses to whisker deflection on the stimulated side (recorded in the hemisphere contralateral to 100 Hz stimulation) In contrast, responses to whisker deflection on the unstimulated side (recorded in the hemisphere ipsilateral to 100 Hz stimulation) were not potentiated. 4. Similar to the control group, LTP can be evoked by 100Hz1s stimulation of the PW on the rats whose whiskers were trimmed during the fifth postnatal week and allowed to re-grow after that. But the LTP of the evoked responses in the barrel cortex can not be evoked by 100Hzls stimulation of the PW on those whose whiskers were trimmed during the first postnatal week and allowed to re-grow after that. The size of barrels and septa between barrels of rats whose whiskers were trimmed during the first postnatal week were bigger than the other two groups.5. After injecting CBX (200mM, 1μl) into layer IV of barrel cortex, the evoked responses of barrel cortex decreased from 1.0±0.02mV to 0.52±0.03mV(P<0.01, n=6).6. After injecting DHK (1mM, 1μl) into layerⅣof barrel cortex, the frequency of spontaneous responses of barrel cortex increased from 7.8±3.57Hz to 21.4±9.53Hz(P<0.01, n=7); the frequency of evoked responses of barrel cortex increased from 2.07±0.93Hz to 2.95±1.76Hz(P<0.05, n=7).7. After injecting AA (6.4mM, 1μl) into layerⅣof barrel cortex, the frequency of spontaneous responses of barrel cortex increased from 9.6±2.28Hz to 24±8.65Hz(P<0.01, n=9); the frequency of evoked responses of barrel cortex increased from 9.7±2.51Hz to 19.7±6.32Hz(P<0.01, n=9); The immunohistochemical results showed the number of astrocytes in the barrel cortex injected by AA decreased of 58±17% compared with control (P<0.01, n=8).8. After injecting FC (1mM, 1μl) into layer IV of barrel cortex, the frequency of spontaneous responses of barrel cortex increased from 6.5±1.69Hz to 42±14.3Hz (P<0.01, n=6); the frequency of evoked responses of barrel cortex increased from 6.5±1.83Hz to 20.7±7.78Hz (P<0.01, n=6); The immunohistochemical results showed the number of astrocytes in the barrel cortex injected by AA decreased of 43±25% compared with control The immunohistochemical results (P<0.01, n=9)Conclusion:Rhythmic whisker stimulation of 100Hz1s can evoked long term potentiation of the evoked response in the corresponding barrel. This plastic change can diffuse to another barrel at the same side, but not to another barrel at the other side. The first postnatal week plays an important role on the development and plasticity of the barrel cortex of rat. Astrocytes may regulate the activity of neuron during the sensory processing in the barrel cortex. |
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