The Improved Method Of Acute Spinal Cord Slices In Electrophysiological Studies

The method of recording the functional spinal cord slices by Whole cell patch-clamp technique has become an important tool to study the neural network structure. Compared with the method of recording electrical activity in cultured cells, this method has distinct advantages. Recently, scientists have identified the community neurons by molecular genetics, and then realized that these communities can be self-generating activities, which for the neural network structure and the mechanism is very important. If this technology can be developed, it will bring great benefits to physiology and anatomy.Due to the complex steps and approach this technique is more difficult. In recent years, the reported about vitro spinal cord slices of the patch clamp technique is not much. There are many difficulties in the preparation of the spinal cord slices, such as spinal cord is elongated, this will take a long time to dissect, it hardly fixed on a slicing machine; spinal cord nerve fibers aligned make them more difficulty to cut smoothly; spinal cord neurons,especially motor neurons, are easily damaged. Thus improved the method of preparing vitro spinal cord slices and increased survival rate of neurons in the spinal cord slices is the basis for further study of the electrical activity of the spinal cord neural network. The purpose of our research is to establish a reliable method of spinal cord slices, thereby reducing the spinal cord injury, increasing the motorneuros survival rate.Objective: Prepared the spinal cord slices usually take a long time, easily being damaged during slicing, motor neurons have poor tolerance to environmental changes, the low survival rate of the cells. In order to Study electrophysiology and neural network structure in vitro slice, we improved the method of spinal cord slices. This can shorten the time of spinal cord slices, reduce the injury of spinal cord, increase survival rate of spinal neuronal cells.Methods: In order to prepare vitro spinal cord slices, firstly, we should prepared artificial cerebrospinal fluid and 2% agar block. The ACSF is divided into two parts.Take one part of artificial cerebrospinal fluid in a glass bottles, glass bottles into the ice and then prepared in ACSF 0?, and another placed in a beaker and the beaker was placed in a water bath case. With two parts fully filled with the prepared 95% O₂ and 5% CO₂ gas mixture for 30 minutes. Cut with an agar block about 1×0.5×0.7cm?length× width × height?,in the middle of the long side open semi-circular grooves, for fixing the spinal cord.Then 8 to 12 days of C57 BL/6J inbred mice were anesthetized and placed on ice 5min, decreasing the metabolic rate of mice, to better maintain the vitality of its cells. After decapitated, the skin incision was cut along the ridge using micro scissors. Under dissecting microscope cut up the thoracic vertebrae one by one along the dorsal midline from the sacral, the spinal cord is exposed by microscopic tweezers. The both sides of the ribs and soft tissue were cut away using micro scissors and the arachnoids membrane attached on spinal cord was removed in oxygenated ice-cold ACSF. Next, the prepared spinal cord together with the agar block was glued to the platform. At last prepare 450?m spinal cord slices through vibration. The slices were placed in 95% O₂ and 5% CO₂ saturated ACSF in 34 water bath for 60 minutes to ?spare.In this study, firstly, C57 BL/6 mouse?8¹2 days old? were anesthetized and placed on ice 5min, decreasing the metabolic rate of mice, to better maintain the vitality of its cells. After decapitated, the skin incision was cut along the ridge using micro scissors, rapidly remove the whole spine into ACSF prepared in advance, the thoracic spinal cord was removed, reserve to thoracic segments to above L5 organization, with a 5ml syringe with a plastic pipette 200 ul suction head filled with artificial cerebrospinal fluid, blown out of the spinal cord from the caudal vertebrae hole, cut with microsurgical scissors lumbar spinal cord. The prepared spinal cord in the groove, together with the agar block was glued to the platform. At last prepare of 450?m spinal cord slices through vibration. The slices were placed in 95% O₂ and 5% CO₂ saturated ACSF in 34 ?water bath for 60 minutes to spare. we use the whole-cell patch clamp recording;the voltage clamped-65 70 mv,interval time of 1s, given 0p A, 50 p A, 100 p A and 150 p A current stimulus program persists 500 ms, each stimulus interval 1s to record action potentials in spinal motor neurons.Results: After repeated experiments we found that about a month to be able to master the lamina incision after removing the spinal cord and prepared the spinal cord slices. But about 10 days you can master the method of injection method artificial cerebrospinal fluid to vertebral hole and prepared slices. The improved method can be easier to master.Injection of artificial cerebrospinal fluid to remove the spinal cord is more complete than laminotomy, a little injury, a shorter time. The average time of preparation spinal cord slices with traditional method is 25.60 ± 2.51 minutes, The average time of preparation spinal cord slices with improved methods is 8.60 ± 1.14 minutes,the two method used in the preparation of spinal cord sections time by t test P <0.01.The number of motor neurons in the slices of anterior horn prepared by injection increased significantly than the number of motor neurons prepared by laminotomy in the anterior horn in high magnification. The average numbers of preparation spinal cord slices with traditional method is 3.00±1.58. The average numbers of preparation spinal cord slices with improved method is 8.20 ± 0.84, the two methods used in the preparation of spinal cord sections time by t test P <0.01.C57 was determined in mice spinal motor neurons threshold potential, The threshold potential of C57 mice spinal motor neurons is average of-54.2 ± 6.1mv, amplitude averaged 37.7 ± 8.7mv, single action potential average time 2 ± 0.3 ms, the action potential rise in the rate of an average of 148.6 ± 4.8mv/ms, the action potential average rate of decline- 65.6 ± 7.6mv/ms.Conclusions: The time of preparing spinal cord slices by injection artificial cerebrospinal fluid to the vertebral hole is shorter than the time of preparing spinal cord slices by laminotomy. The method of injection artificial cerebrospinal fluid to the vertebrae hole to prepare spinal cord slices has obvious advantages than the method of Laminotomy.