The aim of the following experiment is to use voltage sensitive dyes to optically record membrane potential changes and synaptic interactions between neurons in the central pattern generators of the crustacean St Somatic asterisk ganglion. This is achieved by loading identified neurons with the DD eight and at PQ by ion retic injection using sharp micro electrodes and simultaneous extracellular recording of motor nerve activity as a second step, the fluorescence of the voltage sensitive dye is excited by green light, which allows the imaging of fluorescence changes that correspond to the changes in the cell membrane potential. Next, the image data is analyzed by selecting a region of interest in the neuron's membrane and comparing the optical signal to the recorded extracellular bike activity in order to identify action potentials and synaptic potentials.
In the image neurons results are obtained that show the optical recording of synaptically interacting pattern generating neurons can supplement traditional recording techniques and open new opportunities for understanding how central pattern generator neural networks function. The main advantage of this technique over existing methods, like extra and intercellular recordings, is that after dive filling, optical recordings of member potential changes and synaptic interactions can be achieved without the steric restrictions imposed by electrode placement and without further damage to the neurons during long-term recordings. Though this method can provide additional insight into pattern generation in the crustacean stomach gastric ganglion.
It can also be applied to other systems such as locomotion and respiration in vertebrates. Generally, individuals new to this method will struggle because D selection injection and preparation are difficult, and the parameters for the analysis of the image data have to be set adequately to detect membrane potential changes. Begin the preparations by making the fluorescent dye solution in a plastic vial mix five milligrams of the fluorescent voltage sensitive dye D eight and FPQ with one milliliter of F1 27 onic acid in DMSO solution.
Next, centrifuge the dissolved dye for 20 minutes at 12, 000 rotations per minute. In order to separate larger D particles that may clog the micro electrode following centrifugation user prepared to aspirate the top 200 microliters of the supinate, expel the supinate into a separate plastic vial. Wrap the vial in aluminum foil to prevent exposure to light.
Store the D solution in a minus 20 degrees Celsius freezer on the day of the experiment. Defrost the dye solution by placing the wrapped vial in 25 degrees Celsius water for 10 to 15 minutes. To prepare for the recordings, start by isolating the STS somatic gastric nervous system of a crab and de sheathing the somatic gastric ganglion.
Apply petroleum jelly around a section of the lateral ventricular nerve or LVN to create an electrically isolated compartment. Use modeling clay to attach the Petri dish to the operating platform of the imaging microscope. Place a stainless steel electrode wire in the petroleum jelly compartment to record from the LVN place, another stainless steel electrode in the bath as a reference electrode.
When the D solution is defrosted, draw up enough of the D solution to fill approximately two thirds of a microfill needle. Inject the D solution from the microfill needle into the tip of a glass micro electrode. Cover the dye filled electrode with a box and wait 15 minutes.
Then backfill the micro electrode with a three molar potassium chloride solution. Place the micro electrode back into a dark electrode storage box for 10 to 20 minutes to allow capillary action to pull the dice solution into the fine tip of the electrode. After 10 to 20 minutes, place the micro electrode into an electrode holder and attach it to the head stage of the intracellular amplifier.
The intracellular amplifier is connected to the data acquisition board that provides a pulse signal to drive current pulses into the micro electrode. Next, adjust the micro electrode to an oblique angle and position its tip above the somatic gastric ganglion. Lower the microscope objective until the electrode tip is in focus.
Then lower the objective, being careful to stop before touching the micro electrode. Then lower the micro electrode until it reappears. In the focus of the objective monitoring progress on the oscilloscope, gently pierce the membrane of the selected somatic gastric ganglia neuron.
At this point, compare intracellular and extracellular recorded action potentials. In order to identify the impaled neuron, begin dye injection by switching the intracellular amplifier into current injection mode and injecting 10 nano positive current steps into the micro electrode. The current steps should last for one second, separated by one second gaps of no current.
Continue this current injection for 30 minutes. After 10 minutes of dye injection, use the MYCA zero two imaging system to check the dye spread within the neuron. Repeat this, check after 30 minutes.
If the dye filling is successful, the dyed patch around the micro electrode should grow between the 10 minute and 30 minute images. Pull the electrode out of the neuron and allow the neuron to relax for at least 20 to 30 minutes to allow the dye to spread further in the cell. During this time, another neuron can be injected with dye.
Switch to the highlight transmission efficiency 20 times objective on the MYAM zero two imaging system. Reposition the microscope's operating stage as needed, and focus on the D field neuron. Feed the extracellular recording data into the imaging system through its analog input channel.
Turn off the room lighting and all remaining light sources. In addition, close a black curtain around the recording rig to prevent light exposure from external sources. Set the imaging system to the appropriate pixel resolution and imaging time shown in the chart.
If the dye in the neurons is not very strong, use the H bin setting. Set the microscopes light level as low as possible to avoid photo modulation and photo damage of the recorded neurons. Set the imaging sessions to between four to 16 seconds long.
Then record the spontaneous activity of the DY neurons over several imaging sessions. To begin analysis of the optical responses using the BV A NA imaging software, select an appropriate circular region on each recorded neuron if temporal smoothing of the data is necessary. For example, if the D generates a relatively low amount of fluorescence, use the software to apply temporal smoothing with three to five time units.
The two dye injected neurons in the upper left of this figure are identified as a PD neuron and an LP neuron on the basis of their intracellular recordings. The voltage traces to their right show the spontaneous optical responses from each neuron. The extracellular recording of the LVN is shown at the bottom of the figure.
The LP and PD neurons are part of the pyloric central pattern generator of a crab somatic gastric ganglion and a mutually inhibitory. Their optical recordings can be matched well to the extracellular recordings of the LVN. The inset at the bottom shows an overlay of several sweeps of LP action potentials and their average demonstrating the match between optically and electrically recorded activities.
Once mastered, this technique can be done in one and a half hours if it is performed properly. Following this procedure, other methods like immunostaining can be performed in order to answer additional questions such as which type of synapsis was involved in eliciting the observed membrane potential changes. After watching this video, you should have a good understanding of how to record optically, multiple identified and ethal interacting neurons in the stomach gastric ganglia.
