The overall goal of the following experiment is to isolate neurons that retain attached pinched off synaptic batons that allow for pharmacological control and improved space clamp while eliminating the influence of neighboring cells. This is achieved by preparing acute brain slices from rat or mice from postnatal day P one to P 21 containing the brain area of interest to isolate individual neurons as a second step. The acute slices are placed in a dish and a flame sealed glass micro pipette is vibrated within the area of interest while moving the tip through the slice, which liberates individual neurons.
The neurons are allowed time to attach to the bottom of the dish. Next, the dish containing fibro dissociated neurons is moved to a microscope in order to examine synaptic physiology, pharmacology, modulation, plasticity, as well as real-time imaging and characterization of pre and post-synaptic elements in the living cell results are obtained that show the physiologic and pharmacologic characteristics of synaptic responses and associated changes in presynaptic terminal function through tight seal, whole cell recording and fluorescence microscopy. The main advantage of this technique over existing methods like recording from brain slices, cultured neurons and enzymatically dissociated neurons, is that the attached pinch tough synaptic bns allows for the control and visualization of presynaptic elements, while the relatively compact postsynaptic structure allows for improved voltage clamp and outstanding control of the extracellular environment.
Generally, individuals new to this method will struggle because the yield to get healthy neuros can vary dramatically depending on several conditions, such as the age of animal, the vibration parameters, and the area of interest in the slice. Also, individual who cell recordings persist for less time than in conventional slides.Recordings. Visual demonstration of this method is critical as the VI dissociation steps are difficult to learn because it is difficult to accurately describe the movement distance and dissent through the slice I To prepare for making the brain slices, prepare artificial cerebral spinal fluid bubble A CSF with carbogen for 15 minutes.
Next, add two millimolar calcium chloride, and one millimolar magnesium chloride to it. After anesthetizing and decapitating an animal of P one to 21 postnatal age, remove the brain, then cut it in the desired orientation so as to include the region of interest. A fix the dissected brain to a stage and submerge it into a chamber filled with A CSF.
Then mount the chamber on a brain slicer and section the brain at 250 to 400 micrometer thickness per slice with a vibrating blade. Place slices an A CSF and bubble it with carbogen in a pre incubation chamber for at least one hour. To prepare a flame sealed glass micro pipette, first pull a standard patch clamp micro pipette with two micrometer tip diameter on a flaming brown or equivalent micro pipette puller.
Place the micro pipette tip into the flame from a bunsen burner for about two seconds to form a 200 to 300 micrometer diameter fused ball. Next place the flame sealed patch pipette onto a holder of the micro manipulator that can be rapidly vibrated side to side with travel distance of 100 to 200 micrometers using a piso electric bimorph relay or equivalently effective device. Prepare heaps buffered saline solution.
Adjust the solution with sodium hydroxide to pH 7.4 and the osmolarity with sucrose to about 300 milli osmoles. Then fill a 35 millimeter diameter culture dish with heaps buffered saline solution. Place a slice in the culture dish, visualize it with a dissecting stereoscope at 250 x magnification with a bent wire.
Hold the slice to the bottom of the dish to dissociate the cells. Position the tip of the flame sealed micro pipette on the slice surface. Activate the micro manipulator so that the tip vibrates laterally at 10 to 30 hertz with an excursion distance of about 100 micrometers.
Lower the micro pipette tip into the slice with the microm manipulator such that it cuts through the entire depth within 30 seconds. Repeat this step as needed to maximize the number of isolated neurons. Next, remove the tip from the slice.
Pick up the slice with forceps and gently shake it in the solution. Then remove the slice from the solution completely. Allow the cells to settle to the dish bottom for at least 10 minutes.
Place the dish containing the neurons on the stage of an inverted microscope and visualize them with a one x to 63 x objective. Look for the neurons which have smooth membranes with no blabbing and detectable, but not oversized nuclei. If phase contrast optics is used, look for the phase bright neurons with a yellowish tinge, but not too blue.
Pull a standard patch micro pipette using a flaming brown or equivalent puller with the pipette tip resistance of two to four mega ohms. Fill the patch pipette with a cesium chloride based intracellular solution for measurement of synaptic responses or a potassium chloride based intracellular solution for measurement of intrinsic membrane properties and action potentials. Perform whole cell patch clamp on the neuron using standard electrophysiological techniques.
First record spontaneous post-synaptic currents using a gap free data acquisition protocol. Next, apply 200 nan animal or tetro toxin and or low calcium containing extracellular solution to record miniature post-synaptic currents to change extracellular molecular content. Drugs of interest are individually loaded into one of the barrels of a fused square tipped glass tube, which is positioned close to the neuron of interest.
An individual barrel is exposed to the neuron by a stepper motor driven micro manipulator that moves laterally to allow solution exchange 10 to 100 milliseconds. Look at the downloaded bns at high magnification with high numerical aperture objective in a camera based or multi photon microscope. Shown here is the image of a neuron and bns captured with an electron multiplying charge coupled device camera.
The power of the light source for excitation was attenuated to 1.2%with a neutral density 1.0 filter and an iris filter adjusted to 12%output during voltage clamp recordings. With a cesium chloride based intracellular solution, spontaneous synaptic currents are observed to visualize vesical fusion. In the presynaptic batons, we have used mice expressing the synaptic florin construct under the control of the TH one promoter.
Synaptic florin is a molecular construct in which ecliptic Florin, A GFP mutant with enhanced pH sensitivity is linked to the vesical associated membrane protein upon vesical fusion. This motif is exposed to the more neutral extracellular environment with a resultant increase in fluorescence at puncta that correspond to vesicles and presynaptic terminals. Fibro dissociation of hippocampal neurons from these mice allows the visualization of fluorescent puncta of the size and location expected for GABAergic terminals While attempting this procedure.
It is important to remember that in order to have healthy neurons, you must make sure that you have healthy slices. Therefore, the preparation of acute brain slices is an important step in this procedure Following this procedure. Other methods like immunohistochemistry or single cell RT PCR R can be performed in order to answer additional questions like what proteins and mRNA species are expressed in the synaptic batons or postsynaptic neurons.
After watching this video, you should have a good understanding of how to get healthy isolated neurons and use them for electrophysiology as well as real time imaging.
