The overall goal of this procedure is to record electrical activities from neurons in an intact fish brain preparation. This is accomplished by first anesthetizing, a four to six month old transgenic meca fish in MS 2 22, and to then dissect out its brain. Next, one of the neurons of interest labeled by GFP is contacted with a recording electrode and its electrical activities are recorded by loose patch or whole cell patch clamp electrophysiology.
Ultimately, results can show changes in the firing frequency of action potentials or changes in membrane potential in response to different treatments in the intact brain. The main advantage of this technique over existing methods, such as electrophysiology of cells and culture or brain slice preparations, is that we can characterize the electrical properties of neurons in a whole brain preparation with intact neural circuits. Though this Measured provide insight into reproductive neuroendocrinology, it also can be applied into the other neuro systems by using resene fish such as a neurons control motivation and affective behaviors.
This demonstration uses transgenic meca fish in which GnRH three neurons are genetically labeled with GFP To begin anesthetize an adult maka by immersing it in five milliliters of MS 2 22. A couple of minutes after the gill movements have ceased, transfer it to a 60 millimeter Petri dish with fish saline. Now use scissors to decapitate the fish at the coddle end of the alum.
Transfer the head to a 35 millimeter diameter Petri dish, half filled with fish saline, and lined with yl guard. The dish requires a central depression in which to position and secure the head dorsal side up with insect pins with fine scissors. Carefully cut the dorsal part of the skull open around its perimeter.
Then remove the skull gently with forceps exposing the dorsal part of the brain, including the anterior part of the spinal cord. Now, gently lift the spinal cord with fine tip forceps, then sever the bilateral connective nerves using fine tipped scissors. This includes severing the cranial nerves, spinal nerves, ventral side optic nerves, and the anterior olfactory nerves.
Place the fully dissected brain in a new Petri dish filled with fresh fish saline under a microscope. Carefully check that the whole brain and pituitary gland are intact and are not cut or punctured. Discard any damaged brains and start again using a needle.
Place a small drop of fast-acting glue like cyanoacrylate at the center of the recording chamber. Spread the drop of glue over a square centimeter. Then quickly transfer the brain to the glue.
Secure it ventral side up. Now fill the recording chamber with about a milliliter of fish saline to cover the brain. Using the fine tip forceps, carefully remove the meninges over the surface of the recording area of the brain going forward.
Continuously perfuse the brain with aerated fish saline at a rate of at least 200 microliters per minute. Make some six to 10 mega ohm glass micro electrodes and backfill them about halfway with loose patch recording solution. Using an upright fluorescent microscope with a cooled CCD camera, find the GFP expressing terminal nerve gn RH three neurons starting with the 10 x objective.
Then switch to the 40 x objective and locate the neurons at or near the ventral surface of the olfactory bulbs just anterior to the cephalon. Now switch to a video monitor that is providing real-time images from the microscope. The neuron cluster contains eight to 10 GnRH neurons.
Position the micro electrode into the recording chamber bath with the tip of the electrode above the target neuron. Apply constant slight positive pressure so the micro electrode doesn't clog. Gently approach the target neuron with the tip of the electrode check and monitor the electrode resistance with Axo graft software.
Using the seal test in voltage clamp mode, when the tip of the electrode is on the surface of the neuron, the electrode resistance changes slightly at this point. Release The positive pressure to make a low resistance seal if necessary applies light negative pressure. The most critical step for good recording is to select a clean neuro arm, so can help you to make a ideal seal between the electrodes and the same membrane.
Now switch to current clamp mode. Open chart, a power lab program, and begin recording the membrane voltage continuously without any current input, adjust the scale of voltage and rate of recording if necessary, record the baseline electrical activity in normal fish saline for about five minutes before applying any treatments. Bath perfusion of drugs, hormones, peptides, et cetera, should be added to the brain preparation, flowing at 200 microliters per minute.
Then use normal fish saline for a washout period after locating a cell check and monitor the electrode resistance in voltage clamp mode. When the tip of the electrode is on the surface of the neuron and the electrode resistance changes slightly. Release the positive pressure and apply a little negative pressure to make a high resistance seal on the neuron.
Apply gentle mouth suction. Rupture the patched cell membrane. The resistance will instantly drop to about 120 to 250 mega ohms when the membrane breaks.
Now switch to current clamp mode and record the membrane voltage continuously using chart without any current input. Again, adjust the scale of voltage and rate of recording as needed After making a baseline recording of electrical activity in normal fish saline for about five minutes, apply treatments followed by a washout period. The spontaneous electrical activities of moca terminal nerve GNRH three neurons were recorded in current clamp mode.
The typical firing rate is 0.5 to six hertz action. Potentials typically fire in a tonic or beating pattern with a fairly regular interspike interval, interspike membrane potential averages about minus 48 millivolts. This experimental approach is also successful in recording from GFP labeled GNRH neurons located in the forebrain of adult transgenic.
Zebra fish neuron electrical activity in the excised intact brain of transgenic. Zebra fish was recorded in a similar way as described for a DACA by loose patch. The same techniques provided whole cell recording as well.
Unlike with meca terminal nerve GnRH neurons, the pattern of spontaneous action, potential firing of zebrafish GnRH neurons in the pre-op area, ventral cephalon and hypothalamus is often irregular Once mastered. This technique can be done in one hour for a 15 minutes recording if you perform properly. Good luck.
