We thank Manfred Heckmann for helpful discussions during establishing of the method. Further, we thank Claudia Sommer for technical assistance and Frank Schubert for support producing the video. The work was supported by the Federal Ministry of Education and Research (BMBF), Germany, FKZ: 01EO1002 and the Interdisciplinary Center for Clinical Research (IZKF) of Jena University Hospital.

All experimental procedures mentioned in the following protocol were approved by the Thuringian state authorities (Th&#252;ringer Landesamt f&#252;r Verbraucherschutz, Reg.-Nr. 02-044/12).
1. Preparations for Experiment
<ol>
	<li>Fabrication of suction electrodes
	<ol>
		<li>Pull a micropipette using a standard borosilicate glass capillary with a micropipette puller, e.g. a standard patch electrode.</li>
		<li>Brake the electrode to tip diameter of 0.5-1 mm (slightly larger than the diameter of the dorsal roots) using a diamond file.</li>
		<li>Heat polish the tip so it will not harm the dorsal root when it is sucked in. A standard lab torch will be suitable.</li>
		<li>Mount the glass filament on an electrode holder connected to a syringe through which negative pressure can be applied.</li>
	</ol>
	</li>
	<li>Preparation of solutions
	<ol>
		<li>Injection anesthesia for mice: Mix 0.75 ml ketamine 10%, 0.24 ml xylazine 2% and 5 ml 0.9% saline. 10 &#181;l/g bodyweight (BW) of the solution will be injected intraperitoneally (i.p.).</li>
		<li>Artificial cerebrospinal fluid (aCSF): Dilute in double distilled water the following salts (in mM): NaCl 134; KCl 3; KH2PO4 1.25; MgSO4&#8226;H2O 2; NaHCO3 25; CaCl2 2; D-glucose 10. Add H2O2 to a final concentration of 0.003%. Use always freshly prepared solution.</li>
	</ol>
	</li>
	<li>Preparation of the recording setup (Figure 2)
	<ol>
		<li>Connect the amplifier to a PC interface for digitizing data.</li>
		<li>Use a PC-based program or an analogue timer to trigger a square pulse stimulator and data acquisition on PC hard drive.</li>
		<li>Use chlorided silverwires connected to standard glass electrode holders as for stimulation and recording.</li>
		<li>Assemble three manipulators for stimulation, recording and reference electrode respectively around a stereotactic frame for mice, so that all electrodes have access to the prepared spinal cord later on.</li>
		<li>Connect tubings and syringes to the electrode holders to be apple to apply negative pressure.</li>
	</ol>
	</li>
</ol>
2. General Comments for Animal Experiments and Animal Preparation for Recording Procedure
<ol>
	<li>Perform all experiments using mice according to the guidelines of the respective institutional animal care and use committee. Perform all surgery and recordings under deep anesthesia ensuring that animal suffering is minimized.</li>
	<li>Anesthetize the animal by i.p. injection of ketamine/xylazine (125mg and 8mg per g BW of ketamine and xylazine, respectively; 10 &#181;l/g BW of the prepared solution as described above). If necessary during long-term recordings, additional injections can be made i.p. or i.m. 
	Note: Repetitive i.m. injections of 0.05-0.1 ml of ketamine/xylazine solution in the upper thighs have proven to be suitable to keep the animal in deep anesthesia for up to 3 hr.</li>
	<li>Use vet ointment on eyes to prevent dryness while under anesthesia.</li>
	<li>Fix the head of the animal in a stereotactic frame and use a heating pad with rectal probe and reflex loop to control body temperature of the animal during the experiment. Fixation of the spinal cord in a stereotaxic frame is not necessary.</li>
	<li>Before starting the preparation, check depth of narcosis by eyeblink reflex and twitching between toes of the mice. Reflexes should be abolished.</li>
	<li>Open the skin along the midline above the spinal cord from the upper thoracic level to lower lumbar areas to get a clear operational field using a scalpel. Do not use scissors to make skin incisions. Carefully loose the skin from the underlying tissue. During subsequent steps keep the wound moistened by 0.9% saline.</li>
	<li>Cut tendons and connective tissue on both side of the vertebrae from lumbar to thoracic levels using scalpel and scissor.</li>
	<li>Remove the spinous processes and rest of connective tissue around the vertebrae using a small nipper.</li>
	<li>Carefully crack vertebrae with the nipper starting from lumbar levels (L4/L5) under a dissecting microscope. Shove the tip of the nipper in the space between the vertebrae and the spinal cord and lift bone pieces apart. Do not harm the dura and avoid pressure to the spinal cord. Both are critical for success. Keep the spinal cord moistened during the whole procedure. Proceed to mid-thoracic levels.</li>
</ol>
3. Separating of Dorsal Roots and DRP Recording (Figure 2)
<ol>
	<li>Open the dura mater carefully using a thin needle (30 G) with a bent tip. Use aCSF for moistening from now on.</li>
	<li>Separate the dorsal roots as far as possible using the gauge and cut two adjacent roots as distal as possible. Vigorous pulling on the roots during separation affects the success of the experiment.</li>
	<li>Lower the suction electrode down to the dorsal roots. Add as much aCSF to the spinal cord as possible because liquid helps to suck in the dorsal roots.</li>
	<li>Suck the cut end of one dorsal root into one glass pipettes by applying negative pressure through a syringe. If needed, move the dorsal root in front of the electrode opening using a fine needle.</li>
	<li>If the dorsal root lies dry within the suction electrode, add some aCSF to its tip while carefully sucking until the pipette is sufficiently filled with aCSF.</li>
	<li>After the dorsal root is sucked in, raise the electrode tip from the spinal cord. Take care that no &#8220;water bridge&#8221; between the pipette tip and the spinal cord short-circuit the recording/stimulation electrode.</li>
	<li>Repeat steps 3.3-3.6 for an ipsilateral adjacent root.</li>
	<li>Position the reference electrode as close as possible to the dorsal roots and keep it moistened by applying aCSF.</li>
	<li>By establishing the recording setup, one root is already chosen for stimulation, the other for recording. Increase voltage stepwise while recording from the second dorsal root is done in current clamp mode. One may notice a short downward deflection which is followed by a slow, long-lasting upward deflection representing the DRP.</li>
	<li>Adjust the stimulus voltage to supramaximal levels and record several sweeps (at least 20 - 30 sweeps/dorsal root at 0.1 Hz, filter between 0.3 Hz-3 kHz).</li>
	<li>Record short trains of three stimuli (100 Hz) to get information about the time-dependent summation of the DRP.</li>
	<li>Switch recording and stimulation site for additional contralateral recordings.</li>
	<li>Animals are not intended to survive the procedure. Sacrifice animals after last recording by decapitation while still under deep anesthesia (ketamine/xylazine, see above, step 2.2).</li>
</ol>
4. Data Analysis
<ol>
	<li>Transfer data to analysis program (e.g. Sigma Plot, Igor Pro, or MATLAB).</li>
	<li>Calculate average traces from 20-30 sweeps.</li>
	<li>Take the maximal amplitude of the voltage deflection (from baseline; DRP peak amplitude) for further analysis (Figure 3).</li>
	<li>Calculate the ratio of the DRP peak amplitude after three pulses and single pulse to gain a measure of the time dependent summation of subsequent potentials.</li>
</ol>

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The authors declare no competing financial interests.

Extra- and intracellular electrophysiological recordings of neuronal activity and synaptic potentials in vivo are state of the art techniques in investigating CNS neuronal functions and pathophysiology. Spinal integration is critical for motor function, e.g. limb movement and for multimodal sensory perception. Presynaptic inhibition is one critical mechanism in this computational process ensuring appropriate responses to sensory inputs. GABAergic synapses on Ia afferent fibers inhibit the excitation of ...

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. It inhibits excitatory postsynaptic potentials (EPSPs) in monosynaptically excited motoneurons without changing the postsynaptic membrane potential and the excitability of the motoneurons1-3. Primary afferent depolarization (PAD) induced by GABAergic axo-axonal synapses onto sensory presynaptic fibers is the underlying mechanism4-7 (see also Figure1a). These synapses contain GABAA- and GABAB-receptors (GABAAR and GABABR). GABAAR activity leads to an increase in chloride conductance which elicits PAD due to the local ion distribution. This depolarization blocks the propagation of action potentials into the axon terminals and reduces their strength leading to a decreased Ca2+-influx and a reduction of transmitter release. Activation of GABAB receptors does not contribute to PAD but leads to a reduction of Ca2+-influx thereby enhancing presynaptic inhibition. While the activation of GABAAR seems to be involved in short term inhibition, GABABR are involved in long-term modulation8-10. In addition to GABA, which accounts for the major part of PAD and presynaptic inhibition, other transmitters systems might also modulate and contribute to this mechanism11,12.
Pathological changes in presynaptic inhibition seem to be crucial in several disease states e.g. peripheral inflammation and neuropathic pain13,14, as well as abnormal central pain processing15, spinal cord injury16, and CNS disease with motor hyperexcitability mediated by defective GABAergic transmission17,18. Thus, estimating presynaptic inhibition is worthwhile to investigate experimental pathological conditions on the spinal cord level in vivo. PAD gives rise to volume conducted potentials providing a direct measure of the presynaptic inhibition in the spinal cord. Those potentials are called dorsal root potentials (DRP) and can be measured from spinal cord dorsal roots after stimulation of adjacent dorsal roots7.
First measurements of DRP have been reported in cats and frogs19 and were intensively studied in cats by Eccles, Schmidt, and others in the early 1970s3,4,20,21. While in vivo recordings of DRP in cats22 and rats23 have been widely used, measurements in mice have been almost exclusively performed in ex vivo isolated spinal cord preparations15,24. Here, we describe a method to record DRP in anesthetized mice in vivo allowing a direct measure of presynaptic inhibition in the intact organism.

<table><tbody><tr><td>Glass tubing (inner diameter 1.16 mm)</td><td>Science Products (Hofheim, Germany)</td><td>GB200F-10</td><td>Other glass tubing might also be suitable</td></tr><tr><td>Superfusion solution (sterile, 0,9% NaCl)</td><td>Braun Melsungen AG</td><td>3570350</td><td></td></tr><tr><td>Rompun 2% (Xylazine)</td><td>Bayer Animal Health GmbH (Leverkusen, Germany)</td><td></td><td></td></tr><tr><td>Ketamine 10%</td><td>Medistar GmbH (Ascheberg, Germany)</td><td>KETAMIN 10%</td><td></td></tr><tr><td>30 G Microneedle/ Sterican</td><td>Braun Melsungen AG</td><td>4656300</td><td></td></tr><tr><td>Salts for aCSF</td><td>Sigma-Aldrich</td><td>Diverse</td><td></td></tr><tr><td>S88 Dual Output Square Pulse</td><td>Grass Technologies (Warwick, USA)</td><td>S88X</td><td></td></tr><tr><td>SIU5 RF Transformer Isolation Unit</td><td>Grass Technologies (Warwick, USA)</td><td>SIU-V</td><td></td></tr><tr><td>InstruTECH LIH 8+8</td><td>HEKA (Lambrecht, Deutschland)</td><td>LIH 8+8 + Patchmaster software</td><td></td></tr><tr><td>Universal amplifier</td><td>NPI (Tamm, Deutschland)</td><td>ELC-03X</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments (Novato, USA)</td><td>P-1000</td><td></td></tr><tr><td>Dissecting microscope</td><td>Olympus (Tokyo, Japan)</td><td></td><td></td></tr><tr><td>Micromanipulator</td><td>Sutter Instruments (Novato, USA)</td><td>MPC-200/MPC-325</td><td>Mechanical micromanipulators also possible</td></tr><tr><td>Homeothermic Blanket System</td><td>Stoelting (Wood Dale, USA)</td><td>50300V</td><td></td></tr><tr><td>Intra-/extracellular recording electrode holder</td><td>Harvard Apparatus (Holliston, USA)</td><td>641227</td><td></td></tr></tbody></table>

measuring spinal presynaptic inhibition in mice by dorsal root potential recording in vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Typical DRP traces are shown in Figure 3. The prominent stimulation artifact is usually followed by a short downward deflection. Thereafter a slow, long-lasting upward deflection, representing the DRP is clearly distinguishable. In a subset of recordings, dorsal root reflexes are visible as small spikes on top of the DRP. In normal wild-type mice, dorsal root reflexes appear most often when stimulation voltage is excessive. As the dorsal root reflexes cannot be elicited with a high reproducibility in thi...

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Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

measuring-spinal-presynaptic-inhibition-mice-dorsal-root-potential

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13.9K Views.  Jena University Hospital, Jena, Germany. GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Video: Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e. the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice.
For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4 , including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice5,6. However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9, or reproducting human disease10,11, we expect this technique to be an essential tool for the study of spinal motor system.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system.

The  spinal motoneuron has long been a good model system for studying neural  function because it is a neuron of the central nervous system with the ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the different organ systems from the periphery towards the central nervous system. As such, the increased activity or &#39;sensitization&#39; of mesenteric afferent nerves has been allocated an important role in the pathophysiology of visceral hypersensitivity and abdominal pain syndromes.
Mesenteric afferent nerve activity can be measured in vitro in an isolated intestinal segment that is mounted in a purpose-built organ bath and from which the splanchnic nerve is isolated, allowing researchers to directly assess nerve activity adjacent to the gastrointestinal segment. Activity can be recorded at baseline in standardized conditions, during distension of the segment or following the addition of pharmacological compounds delivered intraluminally or serosally. This technique allows the researcher to easily study the effect of drugs targeting the peripheral nervous system in control specimens; besides, it provides crucial information on how neuronal activity is altered during disease. It should be noted however that measuring afferent neuronal firing activity only constitutes one relay station in the complex neuronal signaling cascade, and researchers should bear in mind not to overlook neuronal activity at other levels (e.g., dorsal root ganglia, spinal cord or central nervous system) in order to fully elucidate the complex neuronal physiology in health and disease.
Commonly used applications include the study of neuronal activity in response to the administration of lipopolysaccharide, and the study of afferent nerve activity in animal models of irritable bowel syndrome. In a more translational approach, the isolated mouse intestinal segment can be exposed to colonic supernatants from IBS patients. Furthermore, a modification of this technique has been recently shown to be applicable in human colonic specimens.

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity.

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological characteristics in the intact spinal network and holds significant advantages relative to classical in vitro or extracellular recording techniques. An advantage of in vivo intracellular recordings is that this method can be performed on adult animals with a fully mature nervous system, and therefore many observed physiological mechanisms can be translated to practical applications. In this methodological paper, we describe this procedure combined with externally applied constant current stimulation, which mimics polarization processes occurring within spinal neuronal networks. Trans-spinal direct current stimulation (tsDCS) is an innovative method increasingly used as a neuromodulatory intervention in rehabilitation after various neurological injuries as well as in sports. The influence of tsDCS on the nervous system remains poorly understood and the physiological mechanisms behind its actions are largely unknown. The application of the tsDCS simultaneously with intracellular recordings enables us to directly observe changes of motoneuron membrane properties and characteristics of rhythmic firing in response to the polarization of the spinal neuronal network, which is crucial for the understanding of tsDCS actions. Moreover, when the presented protocol includes the identification of the motoneuron with respect to an innervated muscle and its function (flexor versus extensor) as well as the physiological type (fast versus slow) it provides an opportunity to selectively investigate the influence of tsDCS on identified components of spinal circuitry, which seem to be differently affected by polarization. The presented procedure focuses on surgical preparation for intracellular recordings and stimulation with an emphasis on the steps which are necessary to achieve preparation stability and reproducibility of results. The details of the methodology of the anodal or cathodal tsDCS application are discussed while paying attention to practical and safety issues.

This protocol describes in vivo intracellular recording of rat lumbar motoneurons with simultaneous trans-spinal direct current stimulation. The method enables us to measure membrane properties and to record rhythmic firing of motoneurons before, during and after anodal or cathodal polarization of the spinal cord.

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological ...

Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an increasingly detailed view of the circuits underpinning spinal pain processing. However, the effects of these connections for broader network activity in the DH remain less well understood because most studies focus on the activity of single neurons and small microcircuits. Alternatively, the use of microelectrode arrays (MEAs), which can monitor electrical activity across many cells, provides high spatial and temporal resolution of neural activity. Here, the use of MEAs with mouse spinal cord slices to study DH activity induced by chemically stimulating DH circuits with 4-aminopyridine (4-AP) is described. The resulting rhythmic activity is restricted to the superficial DH, stable over time, blocked by tetrodotoxin, and can be investigated in different slice orientations. Together, this preparation provides a platform to investigate DH circuit activity in tissue from na&#239;ve animals, animal models of chronic pain, and mice with genetically altered nociceptive function. Furthermore, MEA recordings in 4-AP-stimulated spinal cord slices can be used as a rapid screening tool to assess the capacity of novel antinociceptive compounds to disrupt activity in the spinal cord DH.

The combined use of microelectrode array technology and 4-aminopyridine-induced chemical stimulation for investigating network-level nociceptive activity in the spinal cord dorsal horn is outlined.

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an ...

Advanced Imaging of Dorsal Root Ganglion Neurons for Sensory Modality Research

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the protocol for in vivo calcium imaging of the exposed DRG in response to various visceral and somatic stimuli. Pirt-GCaMP6s mice or C57BL6 mice intrathecally injected with AAV viruses packaged with GCaMP6s were utilized to capture Ca2+ transients. The amplitude of these transients indicates sensitivity to specific sensory modalities. Afferent fibers originate from internal organs, with primary neuronal cell bodies in spinal or vagal ganglia. Studies on visceral nociception and acupuncture analgesia can potentially be conducted on primary sensory neurons using advanced imaging technologies like in vivo calcium imaging, allowing for the recording of neuronal activity ensembles in the intact animal during stimulation or intervention. The responses of DRG neuron ensembles to somatic and visceral stimuli applied to their corresponding receptive fields were recorded. This technique illustrates how neuronal populations react to various types of somatic and visceral stimuli. It is possible to comprehensively compare neuronal ensemble responses to different stimuli, which is a particularly valuable approach in research on visceral pain and segmental mechanisms of somatic stimulation, such as acupuncture.

The present protocol outlines in vivo calcium imaging for measuring the responses of ensembles of lumbar-6 DRG neurons to somatic and visceral stimuli. Thorough comparisons can be made among neurons responding to different stimuli. This protocol is valuable for investigating mechanisms of visceral pain and somatic stimulation, such as acupuncture.

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

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Live Imaging of Dorsal Root Axons after Rhizotomy

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli