| BackgroundMany physical factors,such as electromagnetic,sound and light,exist everywhere in people's life and working environment.Thus,it is worthy to investigate the biological effect of these factors,especially on the nervous system.A large number of studies have shown that the hippocampus plays an extremely significant role in learning memory and spatial navigation.Simultaneously,the hippocampus is connected to multiple sensory cortices through the entorhinal cortex,which enables the hippocampus to function as a center of multi-information integration,so the hippocampus can deal with various sensory information.As an important stimulating target of various external physical factors,how neurons respond is worth attention but is rarely concerned in vivo.Patch clamp is an important technique in electrophysiology used in recording individual neuronal activity and illustrating the state of membrane ion channels and the change of membrane potential.These physiological activities are closely related to features of neurons;as such patch clamp can help us to identify the specific brain function at cellular level.The present study attempts to investigate the effects of laser stimulation on the brain function,where the laboratory classic laser stimulation with different wavelengths were used to observe the reaction of hippocampal neurons,with the hope that it can provide experimental evidence for the research into physical stimuli affecting brain function.To observe the real-time effect on neural function in live animals caused by the flash stimulus,we use in vivo patch clamp technique to record the electrical activity changing process of single neuron at the same time when the rat receives external stimuli.ObjectiveTo explore the effect of laser flash stimulus on the electrical activity of hippocampal neurons in rats,and provide direct experimental evidence for the study of the effects of physical stimulus on brain function.Part I Exploring the experimental technology of in vivo patch clamp to record the electrical activity of hippocampal neuronsContent and Methods1.Contents: The experimental methods were established for the in vivo whole cell patch clamp to record the electrical activity of hippocampal neurons.2.Methods: The experimental methods were divided into two stages: animal operation and electrophysiological recording.2.1 The animal operation was practiced industriously to refine experimental steps and reinforce the researchers' experimental skills.An exposed clean surface was obtained through careful operation,with endotracheal intubation performed and the body temperature maintained to ensure the good condition of animals.2.2 The key to electrophysiological record lies in the sealing and rupture of the membrane,and the operation steps of each critical link are repeatedly performed and optimized in these experimental processes.Results1.Successful recordings of spontaneous and evoked discharge of hippocampal neurons were achieved after several improved operations,including cleaning the brain surface,improving the overall state and adjusting the parameters during sealing and rupture of membrane.In spontaneous discharge,the mean membrane potential is-62.25 ± 2.13 m V,the amplitude of action potential is 63 ± 4.23 m V,and the mean action potential half-width is 1.26 ± 0.38 ms.2.Spontaneous discharge occurred in about 80% of the hippocampus neurons with an average frequency of 4.82 ± 1.79 Hz.The spontaneous discharge is divided into two modes: single action potential firing pattern and explosive discharge firing pattern.Part II Effects of different monochromatic light flash stimulus on hippocampal neuronal activity in rats.Content and Methods1.Content: The effects of different monochromatic light flashes investigated on the activity of the hippocampal CA1 pyramidal neurons in rats.2.Methods: In vivo patch clamp was applied to record the discharge activity of rat hippocampal neurons,examined retinal histological structure that received the flash stimulus.Details are as follows:2.1 After the anesthesia of healthy adult Sprague Dawley rats,endotracheal incubation and craniotomy at the skull directly above the hippocampus were carried out,the in vivo whole-cell patch clamp recording was than conducted.During the stable cell recording,the monochromatic laser with four different wavelengths were used to stimulate the rat eye and the response of hippocampal neurons were recorded.The rat eye was stimulated by using purple laser with different power to examine the changing rule of neurons' activity in hippocampus.2.2 The retinal frozen section and HE staining on the eyeball were carried out and observed after the flash stimulation,to identify how far retinal structure was damaged by the flash stimulus.Results1.Exposure to transient laser stimuli showed that the red and the green laser light could not lead to the hyperpolarization of membrane potential in hippocampal neurons but purple and blue laser light could.2.The amplitude and duration of hyperpolarization change induced by blue laser light were 9.05 ± 0.71 m V and 116.4 ± 11.90 ms respectively,with no difference from purple light group(9.78 ± 1.67 m V and 99.23 ± 13.31 ms).3.The hyperpolarization caused by purple laser light is positively correlated with the laser power,and under the power of 125 m W,there is no structural damage on different layers of retinal tissue in rats.Part ?The Biocytin labeling and neuron type identification of hippocampal neurons recorded by in vivo whole-cell patch clamp.Content and Methods1.Content: The biocytin labeling of hippocampal neurons recorded by in vivo whole-cell patch clamp was performed,and the neuron type was preliminarily identified.in vivo2.Methods: The morphology of the effect neurons was observed by using the biocytin labeling method,and the type of neuron was identified by VGlu T1 immunofluorescence staining.Details are as follows:2.1 Electrophysiological recording was performed with biotin simultaneously injected into the recorded neurons,after which,the rats' brain tissue was removed for frozen section and immunofluorescence staining was applied to present the labeled neurons in red color.2.2 The VGlu T1 immunofluorescence staining was performed on neurons labeled by biocytin.Results1.In the hippocampus neurons with biocytin labeled in vivo,a comparison between morphological staining with different thickness of frozen section r by the pasting and floating suggests that the best staining could be obtained on the slice of 60 ?m thick floating section.And the labeled neurons resemble classical hippocampus pyramidal neurons by judging on the morphology of their dendrites and cell somas.2.After detection on the hyperpolarization reactive neurons which had been carried out co-staining of biocytin and other cell markers,the cells were ascertained to be VGlu T1 immunoreactive neurons.Conclusions1.The experimental approach of in vivo whole-cell patch clamp was established,and the spontaneous discharge and evoked firing of hippocampus neurons were successfully recorded.2.After transient exposure on particular wavelength of laser light,an obvious hyperpolarization response of hippocampus pyramidal neurons appeared and was found to be positively correlated with the purple laser power,indicating an inhibitory effect on hippocampus neurons of such monochromatic light.In comparison,no significant effect was found evoked by red and green laser stimuli.3.Biocytin labeling and the immunofluorescence staining were adopted with results suggesting that the hyperpolarization reactive neurons labeled by biocytin was preliminarily proved to be VGlu T1-immunoreactive neurons.4.Because of the low incidence of in vivo whole-cell patch clamp,the present investigation only demonstrated the concentration-response relationship between the hyperpolarization response and laser power strength.Presumably,more data are needed in future studies to confirm the effect and we should conduct in-depth discussions about the neurochemical properties,fiber connections and functional significance of the target neurons. |
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