The overall goal of this procedure is to record the physiological response of single neurons during movement and further characterize the morphology and neurochemistry of these neurons. This is accomplished by first anesthetizing and surgically preparing the animal. The next step is to electrophysiological record the intracellular response of single neurons during movement.
After perfusing the animal and processing the brain, the final step is to visualize and analyze the physiological response and neuronal morphology. Ultimately, the techniques of intracellular neuronal recording, immunochemistry and analysis combine to show the modulations in the membrane potential of single neurons during movement. The main advantage of this technique over existing methods like cell culture, is that neuronal connections and synaptic inputs are preserved, and the neurons are not ex autotomized nor placed in artificial media On the day before, the experiment inject a neuronal tracer, such as horse radish, peroxidase into the peripheral target regions to retrograde label motor neurons.
The next day anesthetize the rat and place it on a heating pad, shave the skin, overlying the posterior skull with animal clippers using aseptic technique. Make an incision along the inner thigh, insert a cannula into the femoral vein for additional anesthesia. Insert another cannula into the femoral artery to monitor blood pressure.
Finally, insert a cannula into the trachea. Next, place the rat into a stereotaxic frame. Perform a craniotomy to expose the cerebellum being careful to avoid the large venous sinus located directly under the junction between the parietal and inter parietal bones.
Next, cover the surface of the brain with warmed mineral oil. Now glue the mandible to a rod coupled to an electromagnetic vibrator. Movement of the mandible is controlled by command signals to the vibrator from a signal generator.
Next place a grounding electrode under the skin adjacent to the craniotomy. To prepare for the intracellular electrodes, pullen fill micro electrodes with a IDE or tetraethyl rod Domine dye solution tests. That electrode impedance is 60 to 80 mega ohms for large diameter axons and 80 to 150 mega ohms for small afferent axons and inter neurons.
Next, place the micro electrode into the head stage of the electrometer using a small telescope that is fixed in position behind the animal's head. Visualize the micro electrode through the telescope's 20 x enclosed redle. The target area is about 0.5 millimeters coddle to the inferior border of the inferior colliculus.
Now make a small opening in the pia mater to allow accurate location of the surface of the brain. Overcompensate the capacitance feedback so that when the electrode touches the brain, a feedback signal is produced. Advance the electrode until it enters the brain.
The next step is to activate repeated displacement of the mandible. Then using a stepping motor, advance the electrode into the brain. When a neuronal impalment is indicated by a sudden drop in dc potential and ongoing synaptic activity, stop advancing the electrode.
When the penetration is deemed stable, initiate ramp and hold and sinusoidal jaw movements. Record the neuronal response and characterize the neuron based on its response. Next, to map the receptive field of the neuron, use a blunt probe to explore the skin around the head and intraoral cavity.
Explore the response of the neuron to other stimuli such as muscle contraction or noxious stimuli. When the recordings are complete, inject one to four nano amp, DC current for a total injection of 15 to 70 nano amp minutes. Monitor the electrode penetration and discontinue current injection.
If the membrane potential becomes more positive than minus 30 millivolts, the next step is to section the brain tissue. After euthanizing and perfusing the animal, remove the brain. Then cut 50 to 100 micron sections in either the frontal, sagittal, or horizontal plane.
To visualize the relationship between the labeled sensory neuron and a variety of motor neurons, process the tissue for H-R-P-D-A-B or Texas red, depending on the tissue, additional analyses can be done with a fluorescent or confocal microscope or using quantitative colocalization software. As desired immunochemical processing can be done for synaptophysin to accurately locate synapses within labeled neurons. This figure shows a brainstem neuron that was injected with IDE after electrophysiological characterization.
Based upon the neuronal response during movement, this neuron can be identified as a secondary muscle spindle afferent neuron. The brown outline indicates the location of the trigeminal motor nucleus. This figure shows the physiological response of a sensory neuron during jaw displacement.
The response of the neuron is represented as instantaneous firing frequency. Note that the response closely mimics mandibular displacement indicating that this particular neuron provides sensory feedback related to mandibular position. This figure shows a sensory neuron that responded during muscle probing.
The neuron was stained with ide following immunochemical processing. The synaptic batons appearing as red swellings are seen co localized with the yellow synaptophysin. Green is a fluorescent missile stain.
This figure is an animation of an axon from a muscle spindle primary afferent neuron. Multiple optical sections of the labeled neuron were used to generate the animation. Following this procedure, other methods like electromicroscopy, confocal microscopy, and anterior grade or retrograde neuronal labeling can be performed to answer additional questions about the ultra structural characteristics of the neuron colocalization of neurotransmitters with synaptic bns and neuronal connectivity.
