Methodology Study Of Detecting Ca²⁺ Signal Of Neurons In The Hypothalamic Ventromedial Nucleus In Vivo Using Two-photon Fluorescence Microscopy

Background Two-photon fluorescence imaging has been considered as a powerful tool for monitoring intracellular calcium signal and cell membrane potential changes.With the technical improvements,this technique can be used to monitor anesthesia mice in vivo in recent years.However,the conventional imaging in vivo with two-photon fluorescence microscopy can only be used to observe the upper cortical layer neurons of brain,but not to detect deep layer and the surface layer in the bottom of brain.Because the fluorescence signals of the ventromedial hypothalamic nucleus(VMH)at the bottom of brain can not be detected in vivo with two-photon fluorescence imaging according to the conventional method.Objective To invent a novel method for recording the calcium activity of VMH neurons in vivo with a kind of surgery exposing skull base combines two-photon fluorescence imaging in order to study activity routines of hypothalamus.Methods1.Adhering a titanium headpost All hair at the parietal centre of the anesthetized mouse was removed,and a titanium headpost was adhered at the center on the skull with cyanoacrylate glue.The rest of the exposed skull was covered with dental cement in order to fix the mouse during monitoring.2.Monitoring mainly physiological index of mice The physiological status of mouse was monitored by Mouse Ox pulse oximeter.The body temperature was measured by a rectal probe and an infrared sensor was placed on the thigh of the mouse by using the half arc shape of the sensor to grasp tissue during surgery.Mouse was placed on a heating mat which was maintained at approximately36 °C.3.Intratracheal intubation The mouse was placed in the supine position and immobilized on a warm pad.A longitudinal incision(about 1 cm)was made on the ventral midline of the neck to expose the submandibular glands.The sternothyroid muscles was bluntly exposed by haemostatic forceps.A silicone rubber hose(about 3 cm)was inserted into the trachea and ligated with sterile sutures.4.The surgery of skull base exposing The tips of forceps were put close to the mylohyoid muscle,and then a high-frequency electrosurgical generator was put on the forceps vertically to burn surface vessels with bipolar electric coagulation from top to bottom in a slow and constant speed.The incisor teeth of mouse were cut a small hole with surgical scissors and then were ioslated by high-frequency electrosurgical generator.The mylohyoid muscles were cut from top to bottom and the tongue was excised in circum using a high-frequency electrosurgical generator.The soft surface of palate was removed using a high-frequency electrosurgical generator and a hole about 2.5 mm x5.0 mm was trepanninged by a high-speed drill.The skull around the hole was ground near translucence.The dura mater was then carefully lifted from the edges of bone with tweezers and all of crushbone was removed by forceps.5.Injecting calcium indicator Calcium indicators,Oregon Green 488 BAPTA-1,were slowly injected into the ventromedial hypothalamic to detect the changes of calcium.Then a piece of coverglass was carefully covered on the tissue and then filled with 1.5% low-melting-point agarose to minimize muscle tremors and breathing jitter of mouse.6.In vivo two-photon fluorescence imaging Mouse loaded with calcium indicator OGB-1 were measured with a two-photon laser scanning microscope.The imaged depth of VMH is approximately-350 ~-550 ?m with a shoot speed at 20.4 frames/s per region(768 × 768 pixels).Results1.We have successfully established a set of operative method to expose the skull base.The mice surviving after the surgery has a common feature in physiological index,including core temperature,arterial O2 saturation and heart rate.All physiological indexes were a slow increase as time goes by.2.The spontaneous calcium activity of the ventromedial hypothalamic nucleus were different.Some of these at high-frequencies(<0.0204Hz),others at low-frequencies(>0.004Hz)in the imaging time.Even if the same neuron,time intervals for each activity was not equal.The spontaneous calcium activity frequency range from0.004 Hz to 0.0204 Hz.3.Glial cells gathered together and formed a dense network with blood vessels and neurons,and other neuronal activities associated with it can also induce the glial cells in the increase of calcium ion concentration,indicating that glial cells also have similar properties to neurons and may play a role in the process of information transmission.4.With this method,cell activities of hypothalamic ventromedial nucleus are still in good response in 2-3 hours monitored by two-photon fluorescence microscopy,the cell fluorescence can not be quenched during the time,which could achieve stable functional imaging.Conclusion We creat a method allows three-dimensional and real-time monitoring of the functional changes of neurons in large area of the ventromedial hypothalamic nucleus in vivo by combining the surgery that exposure of skull base and a fluorescent labeling technique as well as a two-photon in vivo imaging technique.It also can be applied to many other important nuclei of the hypothalamus.