Peter Grafe

Introduction The neuropathy in patients with neurofibromatosis type 2 (NF2) is difficult to quantify and follow up. In this study we compared 3 methods that may help assess motor axon pathology in NF2 patients. Methods Nerve conduction studies in median nerves were supplemented by deriving motor unit number estimates (MUNEs) from compound muscle action potential (CMAP) scans and by high-resolution ultrasound (US) peripheral nerve imaging. Results CMAP amplitudes and nerve conduction velocity were normal in the vast majority of affected individuals, but CMAP scan MUNE revealed denervation and reinnervation in many peripheral nerves. In addition, nerve US imaging enabled monitoring of the size and number of schwannoma-like fascicular enlargements in median nerve trunks. Conclusion In contrast to conventional nerve conduction studies, CMAP scan MUNE in combination with US nerve imaging can quantify the NF2-associated neuropathy and may help to monitor disease progression and drug treatments. Muscle Nerve 55: 350–358, 2017

Background and purpose: Amitriptyline is often prescribed as a first‐line treatment for neuropathic pain but its precise mode of analgesic action remains uncertain. Amitriptyline is known to inhibit voltage‐dependent ion channels and also to act as an antagonist at ligand‐gated ion channels, such as nicotinic acetylcholine receptors (nAChRs). In the present study, we tested the effect of amitriptyline on nicotinic responses of unmyelinated axons in isolated segments of human peripheral nerve. In particular, a comparison was made between the concentrations of amitriptyline necessary for inhibition of nAChRs and those required for inhibition of the compound C‐fibre action potential.Experimental approach: Isolated axon fascicles were prepared from short segments of human sural nerve, and multiple measures of axonal excitability were recorded using computer‐controlled threshold tracking software.Key results: Amitriptyline (EC₅₀2.6 µM) reduced the nicotine‐induced increase in C‐fibre excitability but only slightly altered the amplitude and latency to onset of the compound action potential. In contrast, tetrodotoxin produced a clear reduction in the amplitude and a prolongation of action potential onset latency but was without effect on the nicotine‐induced increase in axonal excitability.Conclusions and implications: These data demonstrate that low concentrations of amitriptyline suppress the response of human peripheral C‐type axons to nicotine by directly inhibiting nAChRs. Blockade of tetrodotoxin‐sensitive, voltage‐dependent sodium channels does not contribute to this effect. An inhibitory action of amitriptyline on nAChRs in unmyelinated nociceptive axons may be an important component of amitriptyline's therapeutic effect in the treatment of neuropathic pain.

Compression and/or contusion of a peripheral nerve trunk can result in painful sensations. It is possible that release of ATP into the extracellular space may contribute to this symptom. In the present study, we used real-time measurements of ATP-induced bioluminescence together with electrophysiological recordings of compound action potentials to follow changes in the extracellular ATP concentration of isolated rat spinal roots exposed to mechanical stimuli. Nerve compression for about 8 s resulted in an immediate release of ATP into the extracellular space and in a decrease in the amplitude of compound action potentials. On average, a rise in ATP to 60 nM was observed when nerve compression blocked 50% of the myelinated axons. After the compression, the extracellular concentration of ATP returned to the resting level within a few minutes. The importance of ecto-nucleotidases for the recovery period was determined by exposure of isolated spinal roots to high concentrations of ATP and by use of inhibitors of ecto-nucleotidases. It was observed that spinal roots have a high capacity for ATP hydrolysis which is only partially blocked by βγ-methylene ATP and ARL 67156. In conclusion, acute nerve compression produces an increase in the extracellular concentration of ATP and of its metabolites which may be sufficient for activation of purinergic P2 and/or P1 receptors on axons of nociceptive afferent neurons.

Receptors for ATP in the peripheral nervous system may contribute to the transduction of sensory, including nociceptive, stimuli and are candidates in the pathogenesis of neuropathic pain. In a complex neural tissue, such as the human peripheral nerve trunk, ATP may activate P2X, P2Y, and adenosine receptors present on various cell types. Experiments were performed on segments of isolated human sural nerves. The experimental set-up enabled simultaneous recording of C fiber excitability, intracellular Ca²⁺ ([Ca²⁺]_i) and extracellular K⁺ activity (aK_e). The increase in excitability of unmyelinated fibers seen during bath application of both ATP and adenosine was reversed to a reduction in axonal excitability in the presence of 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolol[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385), an antagonist of adenosine A2 receptors. The pharmacological profile of the axonal subexcitability indicates the presence and activation of adenosine A1 receptors. Intracellular Ca²⁺ transients were observed during bath application of ATP but not of adenosine and were blocked by 2'-deoxy-N ⁶-methyladenosine 3',5'-bisphosphate (MRS 2179), an antagonist at P2Y(1) receptors. K⁺-sensitive microelectrodes were used to search for a possible activation of P2X receptors by ATP. In isolated rat vagus nerve, activation of P2X receptors by α,β-methylene-adenosine 5'-triphosphate (α,β-meATP) and by diadenosine pentaphosphate (Ap5A) resulted in a rapid, transient rise in the extracellular K⁺ activity. In contrast, in human nerve, application of P2X receptor agonists did not result in a detectable elevation of aK_e. The data suggest that ATP-induced changes in axonal excitability and of [Ca²⁺]_i result from activation of adenosine A2, A1 and P2Y nucleotide receptors in human nerve; a contribution of P2X receptors was not found with the methods used. It is suggested that antagonists of A2 receptors might suppress enhanced activity in human nociceptive afferent nerve fibers under conditions in which ATP and/or adenosine is released into the trunk of a human peripheral nerve.

Mputerised threshold tracking technique with photometric and confocal measurements of the free intracellular Ca2+ concentration revealed differences in the effect of ATP and related compounds. Pyridoxal-phosphate-6-azophenyl-2′,5′-disulphonic acid (iso-PPADS, an antagonist of ionotropic P2X receptors) completely blocked the excitatory effect of α,β-meATP on unmyelinated axons, whereas the effects of ATP and 2-Cl-ATP were only slightly changed. Moreover, the threshold lowering effects of ATP and 2-Cl-ATP, but not of α,β-meATP, were accompanied by intracellular Ca2+ transients. In confocal imaging experiments, the lectin IB4 was used to identify unmyelinated nerve fibres and their ensheathing Schwann cells. The Schwann cells were identified as the cellular elements underlying ATP-induced Ca2+ transients. In addition, an increase in axonal excitability of C fibres was seen during a rise in [Ca2+]i induced by inhibition of the endoplasmic Ca2+ ATPase with cyclopiazonic acid. These data show that an increase of the extracellular ATP concentration in an intact peripheral nerve trunk activates both axons and Schwann cells. It appears that P2 nucleotide receptors on Schwann cells may contribute to the excitatory effect of ATP observed on unmyelinated, including nociceptive, axons....

There is considerable interest in the possibility that ATP may function as a peripheral pain mediator. Most of the studies underlying this idea are electrophysiological recordings from sensory cell bodies, as nociceptive axons with diameters of 1 to 3 μm are not accessible by intracellular electrophysiological methods. However, the most important region for conduction of nociceptive information is thin axons in the peripheral nerve trunk. These nociceptive axons are in close contact with ensheathing Schwann cells, which also cannot easily be studied in situ by conventional recording techniques. In this review, we describe new methods that have been used to study the effects of ATP on unmyelinated axons and Schwann cells in intact peripheral nerve preparations. Threshold tracking of compound C fibre potentials and confocal calcium imaging revealed that extracellular ATP has rapid excitatory effects on both axons and Schwann cells in peripheral nerve trunk. Thereby, at least four different ionotropic and metabotropic P2 receptors were separated by pharmacological profiles. These receptors (P2X_2/3 on unmyelinated axons; P2Y₁, P2Y₂, and P2X₇ on Schwann cells) would allow for rapid interactions between axons and Schwann cells. It is suggested that purinergic signalling in peripheral nerve trunk contributes not only to pain transduction but might be involved in mechanosensitivity, inflammation, proliferation, and demyelination. Drug Dev. Res. 52:83–88, 2001.

1. The paranodal Schwann cell region is of major importance for the function of a myelinated axon. In the present study we searched for a possible ionotropic effect of extracellular ATP in this Schwann cell compartment. 2. Whole-cell patch-clamp recordings from cultured rat Schwann cells revealed that ATP and 2'-3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP) induced a non-specific cation current. The effect of ATP was much enhanced in a Ca2+- and Mg2+-free solution. ADP, UTP and alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-meATP) had no effect. 3. Confocal Ca2+ imaging of myelinating Schwann cells in isolated rat spinal roots showed a BzATP-induced rise in the free intracellular Ca2+ concentration in the paranodal Schwann cell cytoplasm whereas alpha,beta-meATP and 2-(methylthio)-adenosine 5'-triphosphate were without effect. In contrast to the known metabotropic effect of UTP on these Schwann cell regions, the BzATP-induced Ca2+ signal was not transient, was unaffected by depletion of intracellular Ca2+ stores and dependent on the presence of extracellular Ca2+. 4. These results suggest that an ionotropic ATP receptor with electrophysiological and pharmacological characteristics of the P2X7 subtype of nucleotide receptors is functionally active in myelinating Schwann cells of peripheral nerves. Such a receptor might contribute to Schwann cell reactions in nerve injury or neuropathy.