The overall goal of this procedure is to detect messenger RNAs in individual neurons. This is accomplished by patch clamping and recording individual neurons then completely aspirate the cytoplasm from each recorded neuron from the Mr.mRNA and the cytoplasm. CD NA is made and amplified by PCR.
Ultimately, specific mRNAs for receptors ion channels and enzymes can be detected through a conventional gel imaging system. My name is, I work at the University of Tennessee Health Science Center. Generally, individuals are new to this method will struggle because the amount of MRIs in a single neuro is extremely small and easily lost.
Begin this protocol by using a vibrator to cut 300 micrometer thick coronal brain slices containing the mid rostral part of the substantial nigra. Brains can be isolated from 15 to 40 day old spra dolly rats. The slice cutting procedure is performed ice cold in a high sucrose cutting solution, oxygenated with 95%oxygen and 5%carbon dioxide, and at a pH of 7.4, incubate the slices in artificial cerebral spinal fluid and bubbled with a 5%carbon dioxide, 95%oxygen gas.
Maintain the temperature at 34 degrees Celsius and leave the slices there for 45 minutes. Then keep the brain slices at room temperature until recording. Begin by transferring one brain slice to a patch clamp recording chamber.
Continuously perfused with oxygenated artificial cerebral spinal fluid at 32 degrees Celsius. Fill the patch pipettes with autoclave intracellular solution prepared with DNAs and RNAs free water. Lower a loaded pipette into the bath.
Check that the resistance is between two and three mega ohms. First, locate the substantial nigra by its distinct and anatomical location. Then using namar optics and a 60 x objective, select large cells in the SNC and SNR for recording.
These are possible dopaminergic or GABAergic neurons without hesitation. Use a conventional giga tight seal whole cell configuration to make current clamp and voltage clamp. These recordings generally should be performed in less than five minutes to minimize mRNA degradation.
In about a minute, it is possible to provide electrophysiological fingerprints for the neurons of interest by recording just their membrane and spiking properties. After obtaining data from a neuron of interest, use gentle mouth suction to aspirate out the cytoplasm. The tight seal should not be broken as otherwise.
Extracellular debris, including mRNA, may be sucked into the patch pipette and contaminate the cell content. Expel the cell content into a 0.2 milliliter PCR tube by breaking the pipette tip and applying light positive pressure. When finished, immediately place the tube in a negative 20 freezer to rule out contamination of extracellular debris that may contain mRNAs.
Make a negative control by collecting the contents of a patch pipette lowered into the tissue without using any aspiration. After collecting cell content from between 10 to 20 neurons immediately begin the reverse transcription reaction. To minimize the degradation of the mRNAs, begin by digesting any contaminant genomic DNA with DNAs, one for five minutes, A 25 degrees Celsius.
This step is critical in reducing potential false positives and should not be circumvented. Then inactivate the DNAs one with 1.2 microliters of 25 millimolar EDTA and incubate the samples at 70 degrees Celsius for five minutes. Now, use a commercially available kit to synthesize CD NA to verify that the genomic DNA was completely removed.
Run a control reaction of each sample without reverse transcriptase. While all other reaction components remain the same. Store the products at minus 20 degrees Celsius until they are needed for PCR design.
Primers according to the sequences in Gen bank, whenever possible, make T intro spanning prime repairs to help detect genomic DNA contamination. To check the effectiveness of the primer pairs, test them on whole brain CD NA for the first stage. PCR amplify five microliters of the 30 microliter CDNAs for 35 cycles using a standard thermal cycling protocol.
For the second stage, PCR amplify one microliter of first stage PCR product with the same prime repair and use a slightly modified cycling profile. Now separate the products on a 2.5%AGROS gel and visualize them using AUM bromide or gel green. Under UV light, excise the positive DNA bands and sequence them using a commercially available kit to collect the DNA.
The electrophysiological characteristics of neurons in the substantial nigra, pars, compacta, and pars reticulata were compared. SNR GABAergic neurons exhibited higher frequency, spontaneous spiking at around 10 hertz. The spikes had a base duration around one millisecond upon hyperpolarizing current injection.
SNR GABAergic neurons displayed a subtle SAG response indicating a weekly activated current. In contrast, the nigro dopaminergic neurons exhibited low frequency spontaneous spikes with a base duration of around three milliseconds. Dopaminergic neurons also showed a pronounced SAG in response to hyperpolarization indicating a strong expression of IH current in SNR GABAergic neurons, but not in dopaminergic neurons.
Single cell R-T-P-C-R detected mRNA for glutamic acid decarboxylase one, a key enzyme in GABA synthesis. In contrast, an identified SNC and SNR dopaminergic neurons, but not in GABAergic neurons. Single cell R-T-P-C-R detected mRNA for tyrosine hydroxylase a key enzyme for dopamine synthesis.
Next single cell R-T-P-C-R was used to profile the expression of voltage activated KV three channel subunits, which vary the properties of voltage gated potassium channels. In the example SNR gaba, neuron mRNAs for KV 3.1, 3.2, 3.3, and 3.4 were detected in an example S-N-R-D-A neuron only. KV 3.2, 3.3, and 3.4 were detected in pooled data.
KV 3.1 was more frequently detected in SNR GABAergic neurons than in nigro dopaminergic neurons indicating a higher expression level of KV 3.1 in the fast Viking SNR GABA neurons. While attempting this procedure, it's important to remember to wear gloves and keep the tools science free after its development. This technique paved the way for researchers in the field of neuroscience to explore the expression and the function of receptors and the ion channels in the nervous system.
