Electrophysiological recordings from muscle and motor neurons can be obtained from a properly prepared oph embryo body wall. Muscles 6, 7, 12, or 13 can be easily recorded from the whole cell recording configuration and robust miniature amplitude currents can be measured after achieving a giga ohm seal to record from the neuromuscular junction. A suction electrode can be used to draw up a motor nerve near the CNS exit point and deliver pre-synaptic stimulation while whole cell currents are measured in the post-synaptic muscle.
Alternatively, a pipette may be used to deliver excitatory neurotransmitters near the recording electrode via iontophoresis or pressure ejection. Hi, I'm Ton from the Lab of Dave in the Department of Biological Sciences at University of Illinois at Chicago. Today I will show you electro physiologically recording the salate stage embryonic neuro musk junction and the central neurons.
These procedures we're using to study the function and development of joof neuromuscular system. These techniques were originally developed by Kendall Brody. Pass through down to David Fit Stone and then to me today I will pass it on to you.
This procedure requires that embryos be properly harvested and dissected in advance To see how this is accomplished, view the accompanying Jo of publication by Brody Featherstone and Chen Electrophysiological. Recordings from Jossa embryonic muscles and neurons utilizes standard whole cell patch clamping techniques. First, the dissected embryo is placed in a small plexiglass recording chamber.
The physiology recording area is electrically dampened by a Faraday cage and stationed on a vibrationally dampened table. Chamber is viewed under transmitted light using an upright compound microscope with KY optics and a 40 x long working distance water immersion. Objective recordings are typically made below room temperature as it becomes increasingly difficult to patch clamp oph muscles as temperatures increase.
The recording electrode should have a final resistance of five to 10 mega ohms and be heat polished to about one micrometer inner diameter. At the tip recording electrodes are backfilled with a physiological solution containing potassium chloride and sucrose. A chloride silver ground wire is placed in the bath containing standard saline, which is also used in embryo dissection.
Signals are amplified using a patch clamp amplifier filtered and stored digitally for later analysis. Any system suitable for patch clamp experiments can be used such as this equipment and software by axon. Instruments cells from three different tissues in the embryonic nervous system can now be stimulated and recorded from the muscles neurons at the NMJ and central motor neurons.
With this embryo preparation, any embryonic muscle may be recorded from most experiments. Focus on the group of four longitudinal ventral muscles 6, 7, 12, and 13, particularly muscle six in the anterior abdomen, A two to a three to record from the dorsal muscles. The embryo must be glued, dorsal side down, and dissected along the ventral midline in young embryos.
No enzymatic treatment is needed before muscle recording. However, in later embryos with developed cuticles, a muscle sheath also develops prior to patch recording. This sheath must be removed by brief immersion in a collagenase.
Most recordings are done in the whole cell recording configuration achieved easily from the cell attached state with slight suction or an electrical buzz. That's a zap. It's a electro stimulation to you know to rupture the cell membrane cuts.
A whole cell setting Seal resistances on the muscle are typically greater than one giga ohm whole cell currents are usually obtained less than three minutes after seal formation series. Resistance is markedly increased in most instances and cells can develop developed an increased leakage. Current for patch clamping, embryos must be at least 14 hours old at earlier developmental stages.
Extensive electrical coupling between myo tubes will make voltage clamping inaccurate to make patch clamp recordings of muscle fibers. Typical holding potentials are negative 60 to negative 80 millivolts and voltage clamp and current clamp methods can be used. Successful whole cell recordings that negative 60 millivolts are accompanied by robust spontaneous synaptic currents that range from a few to several hundred picoamps, such as those shown here.
Large NMJ currents can be evoked by stimulating the motor nerve with suction electrodes. These can be pulled from a variety of fiber filled glass electrodes and should be fire polished to achieve the desired configuration using gentle suction on the pipette draw in a small segment of the appropriate segmental motor nerve near the CNS exit point To form a tight seal brief stimulation works best with an experimentally determined intensity, usually between one to five volts. Reliable stimulation is achieved by setting the stimulus strength one to two volts above the experimentally determined threshold.
Post-synaptic NMJ glutamate receptors can be activated by applying the neurotransmitter glutamate charged molecules like glutamate can be delivered to the NMJ using iontophoresis. High resistance pipettes are used to map receptors and lower resistance pipettes are used to estimate the total glutamate receptor response. Ionophore pipettes are loaded with a 100 millimolar L glutamate solution.
To release the L glutamate, use a short pulse of negative current with a small positive backing current to prevent leakage, glutamate, or any uncharged molecule can also be delivered to the NMJ using pressure ejection. For pressure ejection use one to two millimolar glutamate and apply five to 10 PSI of outward force to the pipette. Using a pico spritzer.
NMJ currency evoked by nerve stimulation or glutamate application at negative 60 millivolts are typically 1500 to 2000 pico amps. Many embryonic neurons can be individually identified based on soma position and projection morphology. Most experimental recordings have concentrated on a group of five superficial dorsal neurons within the embryonic ventral nerve cord.
To record from these neurons, the ventral nerve cord neuroma sheath must be removed with brief focal treatment with protease, the cell bodies are des sheathed by delivering 0.5 to 1%protease in recording using a large diameter patch pipette suck the sheath material into the pipette containing the protease. Gentle mouth action can be used to repeatedly iterate the sheath in and out of the pipette. After one to two minutes, the sheath will rupture.
The cells are now exposed for whole cell patch clamping. This recording paradigm does not require that saline be circulated or warmed. The external recording saline in this preparation is different from what is used in recording from muscles.
The patch pipette can be loaded with Lucifer yellow to later visualize the position and morphology of the patch cell. Here is a central motor neuron that has taken in Lucifer yellow and visualized using fluorescence microscopy. Whole cell voltage clamp recordings are made at holding potentials of negative 60 millivolts at 18 degrees Celsius.
Current clamp recordings are also possible. Neurons can be stimulated with the neurotransmitters, acetylcholine, or gaba. These are applied at the cell soma using iontophoresis or pressure ejection as when recording from njs.
Once a whole cell connection has been established, spontaneous currents are first observed. This is one minute of raw data obtained from embryonic ventral longitudinal muscle six patch clamped at negative 60 millivolts in the absence of stimulation. A healthy preparation typically shows robust spontaneous synaptic activity represented here by the many downward current deflection.
Here's data from the same muscle with stimulation applied using a suction pipette on the motor nerve leading to that muscle. This resulted in multiple downward deflections of the current representing evoked junctional currents or e jcs. This is data from the same muscle stimulated by one millimolar glutamate applied using pressure ejection.
The glutamate opens receptors leading to large downward deflections. In the current two traces are shown representing two subsequent applications of glutamate. I just shown you how to electro theological record from late stage geo soft embryos electro theological record in generally use standard patch hunting techniques, which we could not describe here in any depths.
Ideally, anyone seeking to record from your softener embryos will have already learned patch campaign using an easier preparation. That's it. Thanks for watching and good luck with your experiments minutes.
