The overall goal of this procedure is to study gene expression and electrophysiological changes in single identified neurons. This is accomplished by first carefully identifying the neurons of interest from the ganglion of plegia, following the protease treatment and des sheathing of the ganglion to expose neurons. The second step is to record electrophysiological properties of the identified neurons.
Next, isolate single neurons following electrophysiological measurements and then isolate RNAs from these neurons for linear amplification. The final step is to characterize gene expression by quantitative PCR. Ultimately analyze the electrophysiological and gene expression data from the treatments and controls to identify specific changes.
The main advantage of this technique over existing methods like organs, tissue, or ganglion analysis, is that it provides analysis on a single neuron level. This method can help answer key questions in the cognitive neuroscience field, such as how exertion of genes change in single neurons during aging, or in response to different pharmacological reagents. We first had the idea for this method when we wanted to investigate how aging affects functioning of individual neurons and circuits To isolate the abdominal ganglia.
First, anesthetize the animal by injecting 380 millimolar magnesium chloride solution and wait for five to 10 minutes. Next, identify the abdominal ganglia based on its position in the central nervous system. Then remove the ganglia by surgical operation and immediately store in the artificial sea water for protease treatment.
Incubate the abdominal ganglia in a Petri dish with 0.1%protease diluted in a SW at 34 degrees Celsius for 30 minutes. Note that the amount of protease used needs to be standardized with the age and weight of the animals to remove the ganglion sheath. After the enzyme treatment, pin the ganglion down to the cigar silicone base placed on the top of a polycarbonate glass cylinder, perfuse it with a SW at 150 microliters per minute at 18 degrees Celsius.
In order to increase the visibility of the identified neurons, the polycarbonate glass cylinder can be easily illuminated from the bottom using a binocular stereo microscope with 20 x and 40 x magnification. Next, carefully remove the sheath from the ganglion using forceps and micro scissors in this procedure. First, identify the neuron of interest.
Next, impale the neuron with a single intracellular sharp micro electrode filled with three molar potassium chloride solution. Apply the drug of choice into the cell chamber by gently pipetting or injecting directly into the neurons. Then record the neuronal activity using an intracellular recording system.
The recording data is processed with electrophysiology software such as Axo graph to isolate single neurons. After the electrophysiological experiments, stop the A SW perfusion. Subsequently wash the ganglion with 100%ethanol in order to petrify the neurons.
Then remove the single neurons with a pair of fine forceps and transfer them to a small einor tube containing 500 microliters of ice cold. RNA extraction reagent. The isolated single neurons can be stored in RNA extraction reagent at minus 80 degrees Celsius for future analysis to isolate RNAs from single neurons using the standard TRIOL protocol for RNA isolation.
Briefly add 20%volume per volume of chloroform to the triol and mix well by briefly. Vortexing Next, place the tubes on a rotator for 10 minutes at four degrees Celsius. Spin the Triol chloroform mix at 12, 000 times gravity for 15 minutes at four degrees Celsius.
Then collect the aqueous phase and transfer it to a fresh tube To precipitate RNA. Add sodium acetate solution to a final concentration of 0.3 molar 100 nanograms per milliliter of the co precipitant glyco blue and 2.5 volumes of 100%ethanol to the aqueous phase. After that, mix the sample well and incubate at minus 80 degrees Celsius overnight.
The next day, spin the sample at 12, 000 times gravity for 20 minutes. At four degrees Celsius, a small blue pellet will be visible at the bottom of the tube. After that, remove the supernatant and wash the pellet with 850 microliters of 75%ice cold ethanol at 12, 000 times gravity for 10 minutes at four degrees Celsius.
Then remove the supernatant carefully and air dry the RNA pellet for seven to 10 minutes. Make sure that RNA is not left for long to dry as over drying. The RNA pellet makes solubilization very difficult.
Before preparing for quantitative real-time PCR analysis, synthesize A RNA from single neurons and amplify it using a commercially linear RNA amplification system. Then generate CDNA using one microgram of A RNA in 20 microliters of a reverse transcription reaction. Next, dilute CDNA in nuclease free water.
After that, add eight microliters of the QPCR master mix containing two microliters of H2O five, microliters of two x cyber green master mix, and one microliter of 10 micromolar forward and reverse primer to two microliters of diluted CDNA. Subsequently pipette the CDNA and master mix. Then close the tubes and seal the plate.
Mix the contents by gently tapping and spinning down at 1, 500 RPM for 30 seconds before setting up the QPCR machine. This cartoon shows the simultaneous recording from L seven and R 15 neuron of abdominal ganglia. And here are the representative intracellular recordings from L seven and R 15.
With the arrow indicating the time of a CH application, one could continue these measurements for eight to 10 hours. This figure shows that a CH induced changes in the action potential of R 15 AP waveforms were analyzed before, during, and after a CH exposure. Here is the QPCR analysis of expression of CREB one in abdominal ganglia and single identified neurons.
This graph shows the absolute quantification of CREB one transcript in the young and adult abdominal ganglia of plegia. And this graph shows the absolute quantification of CEB one transcript in the identified single neurons of the adult plegia. Once master, this technique can be done in four, six hours and the RNA isolation amplification and QPCR can be completed in three days if it is performed properly.
While attending this procedure, it's important to remember to standardize the protease treatment and the removal of the ganglion sheath such that the neural activity is preserved. Also, take precautions while working with RNA, such as wearing gloves at all times. Use RNAs free drip tubes and water and treat the working space with RNAs removing reagents.
This technique will also help researchers in the field of neuroscience to explore the physiological and molecular basis of behavior in other invertebrate model organisms such as Triton, heli and limba.
