Presynaptic inhibition is one of the most powerful inhibitory mechanism in the spinal cord. Pathological changes in presynaptic inhibition are imported in several disease pattern mechanisms. For example, neuropathic and inflammatory pain, as well as abnormal central pain processing.
It is important in spinal cord injury and in central nervous system disorders with motor hyperexcitability. So when animal models for these disorders are investigated, knowing the strengths of the presynaptic inhibition would provide significant information. We present here a method how to assess presynaptic inhibition in a mouse model.
In vivo presynaptic inhibition is mediated by depolarization of primary ens, which is induced by GABAergic Axo axonal synapsis onto sensory presynaptic fibers. Gaby, A receptor activity leads to an increase in the chloride conductance, which then elicits primary affluent depolarization due to the local ion con distribution. This depolarization blocks the propagation of action potentials into the exome terminals and reduces their strengths leading to a decreased calcium influx and the reduction of transmitter release.
So the excitatory postsynaptic potentials in monos, synaptically exci, modern neurons can be inhibited without changing the postsynaptic membrane potential and the excitability of the modern neurons volume conducted potential of this primary ENT depolarization give a direct measure of the presynaptic inhibition in the spinal cord, and these potentials can be measured from spinal cord, dorsal roots after stimulation, and these are called dorsal root potentials. Measurement of dorsal root potentials are well established and have been widely used in cats, rats, and frogs, but in mice, recordings have been almost exclusively performed in ex vivo, isolated spinal cord preparations. Here we describe a method how to record those root potentials in anesthetized mice in vivo, allowing a direct measure of presynaptic inhibition in the intact organism.
For DRP recording in mice in vivo, the following equipment is needed. Standard sugar instruments, a stereo microscope three manual for positioning stimulation electrode, recording electrode and reference electrode respectively. Further, a mouse frame and a heating pattern for controlling the body temperature of the mouse for stimulation and recording alone or stimulator.
A standard amplifier and a data collections is Tim needed to improve signal to noise. During recording of the DRP custom made section electrodes are needed for the fabrication of them. Pull conventional glass tubing with an standard pipet puller.
Break the tip of the electrode using a diamond file to widen the tip opening fire. Polish the tip with a standard lab torch to smoothen the tip. Before starting the preparation, anize the animal with an IP injection of keils in and fix the head of the mouse within the stereotactic frame.
Then test the depth of the anesthesia by pinching the rear PO of the mouse. Start the preparation only when no withdrawal of the PO is visible, the depth of the anesthesia should be checked repetitively during the procedure. Repetitive injections of ketamine sine in the rear quadriceps muscle.
Prolong the anesthesia, monitor the body temperature of the animal with the rectal probe. For the preparation of the dorsal column. Open the skin along the dorsal midline.
Loose the skin from the underlying tissue to separate the vertebra from the surrounding connective tissue. Make two cuts on both sides of the spine and widen the cuts by pushing the tissue apart with the scalpel. Then remove the S spinous processes using a small bone nipple.
Starting at lumbar levels. Proceed to mid thoracic levels to prevent drying out of this operational wound. Play small pieces of gauze around the wound and moisten them with 0.9%sodium chloride.
Then start preparing the spinal cord starting at the lumbar region. Carefully push the tip of the bone nipple in the space between two adjacent vertebra. Take special care to reduce the pressure applied to the spinal cord as far as possible.
Proceed to thoracic levels moist in the spinal cord with artificial cerebral spinal fluid. Do this repetitively during all subsequent steps. Open the durometer carefully using a 30 gorge needle.
Then loosen and separate two adjacent ipsilateral roots from the spine. Then cut the roots as distal as possible. During the procedure.
Prevent excessive pulling of the roots as this will ultimately hamper a successful recording of DRP. After positioning the reference electrode close to the spinal cord, move the tip of one section electrode in close proximity to the proximal end of one dorsal root. Then position the dorsal root over the tip opening of the suction electrode.
Afterwards sack the dorsal root into the electrode by applying a short negative pressure through a syringe connected to the electrode. Then fill the electrode with A CSF while applying low continuous negative pressure until the solution surrounds both the doer root and the chloride silver wire within the electrode. Subsequently, elevate the tip of the electrode from the spinal cord so that no liquid bridge short circuits the electrode.
Repeat the steps for second ipsi adjacent root. Once both electrodes are positioned, start the recording of DRP by stimulating one route while recording from the other one, increase the voltage of the stimulus to supra maximal levels. Typical recordings of dorsal root potentials look like the one shown in the left panel.
A short stimulation artifact is followed by a short positive deflection and a prolonged negative deflection. The last deflection is the actual dorsal root potential. Its amplitude can be calculated with respect to the baseline recorded directly in advanced of the stimulus record.
Several traces from one pair of roots as artifacts from breathing movements and or EKG artifacts might interfere with the recorded DRP. After a successful recording, it is possible to switch stimulation and recording site and conduct a second recording. The presented method of recording.
Do root potentials in my in vivo allows the assessment of primary ent, anti polarization, and the intact animal, especially in combination with genetically engineered mice. It can be a powerful tool to investigate mechanisms underlying spinal inhibition. A spinal integration is critical for motor function and multimodal sensory perception.
This method is useful in research addressing pain, spinal cord injury, peripheral nerve injury, and inflammation. The recording of DRP in vivo enables the analyzers of interactions of supraspinal activity and pad. This is not possible in isolated spinal cord Preparations usually used to measure DRP in mice.
In principle, drug application to the spinal cord is possible during the recording. Local application through additional pipettes could enhance the and fasten control of a local drug concentration.
