The overall goal of this procedure is to link cell type specific action potential spiking in the sensory cortex during information processing to the morphological identity of the recorded neurons. This is accomplished by first surgically preparing the rat for juxta somal recordings. Once prepared, an electrode records spontaneous and sensory evoked spiking activity, and the recorded neuron is biocidin loaded using current and injections.
Afterwards, the brain is processed for biotin staining. Finally, the biotin filled neuron is digitally reconstructed from subsequent sections for cell type classification. Ultimately, juxta somal recordings of individual neurons allow for the study of sensory processing and structural information within the cortical network.
This method can help to answer key questions in neuroscience such as what is the role of individual cell types and layers during sensory processing? Generally, individuals need this method will struggle because it's very easy to kill neurons during the filling procedure. The chances of successful biocidin filling is increased by preparing optimal quality pipettes, and additionally, a trained eye is necessary to control the narrow band current injections.
Therefore, it takes experience to increase the success rate of recovering well filled neurons. To begin place an anesthetized wistar rat onto a stereotaxic frame equipped with a heating pad. Confirm the depth of sedation by testing the toe pinch reflex.
Then insert a rectal probe to maintain the correct body temperature. Secure the animal's head and remove hair from the surgical site. Apply ointment to the eyes to prevent dryness.
Wait three to five minutes after injecting lidocaine at the operation site and remove the skin covering the surface of the skull. Next, clear away any remaining tissue and clean the skull extensively with 0.9%sodium chloride ride. Determine the coordinates for the primary somatosensory cortex on the left hemisphere and mark the location on the skull with a surgical skin marker pen.
Use a dental drill to scrape the bone from the region of interest. Continue scraping the bone until it becomes transparent and blood vessels are clearly visible. Next, cut a small window into the thin skull with a scalpel.
Taking care not to damage the dura mater and blood vessels. Mark the edges of the craniotomy using a surgical skin marker pen. To improve visibility, apply a thin layer of super glue on the dry skull, and then dental cement to construct a bath.
In closing the craniotomy, trim the whiskers on the contralateral side to exactly five millimeters to aid in their visibility. Highlight the trimmed whiskers with black mascara before beginning the recordings make patch pipettes from Boro Silicic glass. The ideal pipette morphology is a gradual slender taper, a low cone angle, and an inside tip diameter of approximately one micron.
Load the patch pipette with normal rad ringer, supplemented with 2%biotin, and mount the patch pipette onto a pipette holder, fixed to a head stage, and connected to a microm manipulator. To specifically target the D two column of rat S one, set the angle of the electrode holder to 34 degrees with respect to the sagittal plane. Next, position the patch pipette in close proximity to the craniotomy.
Fill the bath with 0.9%sodium chloride. With the amplifier in bridge mode, apply square pulses with positive current injection to determine the electrode resistance. The optimal electrode resistance lies between three and five mega.
Establish over pressure of 100 to 150 millibar and advance the patch pipette in one micron steps while applying positive current in the form of square pulses. Monitor the robust resistance change upon establishing contact with the dura mater. At this point, set the coordinates of the micro manipulator to zero to allow accurate depth measurements.
Advance in one micron step mode until the patch pipette penetrates the dura mater indicated by a sudden drop in electrode resistance. Remove the holding pressure of the pipette. Next, search for single units while advancing in one micron steps.
Monitor the electrode resistance continuously by applying 200 millisecond on off pulses and increase in electrode resistance typically indicates the proximity of a single neuron advance the electrode until positive going action. Potential waveforms of approximately two millivolts are recorded. Optionally record spontaneous spiking activity and determine the whisker revoked spiking activity by coddly deflecting individual whiskers using a pizo electric device to obtain optimal conditions for juxta somal filling.
Advance the electrode until the resistance is 25 to 35. Mega ohms and spikes have amplitudes of three to eight millivolts to start the juxta somal filling. Apply positive square current pulses starting at one nano amp.
Slowly and gradually increase the current by steps of 0.1 nano amps while monitoring the AP wave form and frequency. Monitor the membrane opening as a clear increase in AP frequency during the on phase of the block pulse the spike wave form during filling shows an increased width and reduced after hyperpolarization increase or decrease the current while filling to maintain stable biotin infusion, reduce or stop the current pulses upon sudden increase of the AP frequency. To avoid toxicity by excess influx of extracellular ions such as sodium ions.
It's important to note that every neuron will respond differently to the filling procedure. Therefore, the juxta somal labeling parameters have to be adjusted depending on the recording condition. Closely monitor the signal after stopping the current injection.
The spike wave form after a filling session is usually broadened and shows a strongly reduced after hyperpolarization wait for recovery of the neuron, which is apparent when the spike waveform returns to its original properties to reduce any mechanical stress to the cell. Retract the patch pipette in steps of one micron until the spike amplitude decreases wait at least one hour for the biotin to diffuse intracellularly while closely monitoring the animal's body temperature and breathing. Finally, brain samples are obtained and processed to reveal the quality of juxta somal labeling.
These images show images of juxta mally filled neurons in primary somatosensory cortex. This tangential view of a parametal neuron demonstrates the difference in size between dendrites and axons. In this digital reconstruction of a juxta Somali labeled neuron, the neuron projection image is shown at high resolution, but low magnification, and with automated digital reconstruction overlaid axon in blue, dendrite in red respectively.
Here the box region is expanded to show axon bhuton throughout the length of the axon. In this final animation, we exemplify the reconstruction pipeline for one layer five thick tufted parametal neuron from rat primary somatosensory cortex. The pipeline entails the reconstruction of dendritic and axonal morphology at 100 times magnification and barrel contours at four times magnification.
The result is a full 3D reconstruction, which is subsequently registered to a standardized reference frame to perform quantitative analysis of morphological characteristics. Thus juxta somal labeling in vivo allows exceptional labeling quality for reliable reconstruction of full 3D morphology. Here we showed the method in urethane anesthetized animals.
However, Jux Somal recordings can also be used in awake head restrained animals to study the role of individual cell types and layers during active exploration of the environment. After watching this video, you should have a better understanding of how to perform Biocidin labeling on individual neurons for post-war identification and morphological reconstruction.
