Ron Amir

Tactile allodynia in the absence of C-fiber activation: altered firing properties of DRG neurons following spinal nerve injury

&NA; We examined the relation between ectopic afferent firing and tactile allodynia in the Chung model of neuropathic pain. Transection of the L5 spinal nerve in rats triggered a sharp, four‐ to six‐fold increase in the spontaneous ectopic discharge recorded in vivo in sensory axons in the ipsilateral L5 dorsal root (DR). The increase, which was not yet apparent 16 h postoperatively, was complete by 24 h. This indicates rapid modification of the electrical properties of the neurons. Only A‐neurons, primarily rapidly conducting A‐neurons, contributed to the discharge. No spontaneously active C‐neurons were encountered. Tactile allodynia in hindlimb skin emerged during precisely the same time window after spinal nerve section as the ectopia, suggesting that ectopic activity in injured myelinated afferents can trigger central sensitization, the mechanism believed to be responsible for tactile allodynia in the Chung model. Most of the spike activity originated in the somata of axotomized DRG neurons; the spinal nerve end neuroma accounted for only a quarter of the overall ectopic barrage. Intracellular recordings from afferent neuron somata in excised DRGs in vitro revealed changes in excitability that closely paralleled those seen in the DR axon recordings in vivo. Corresponding changes in biophysical characteristics of the axotomized neurons were catalogued. Axotomy carried out at a distance from the DRG, in the mid‐portion of the sciatic nerve, also triggered increased afferent excitability. However, this increase occurred at a later time following axotomy, and the relative contribution of DRG neuronal somata, as opposed to neuroma endings, was smaller. Axotomy triggers a wide variety of changes in the neurochemistry and physiology of primary afferent neurons. Investigators studying DRG neurons in culture need to be alert to the rapidity with which axotomy, an inevitable consequence of DRG excision and dissociation, alters key properties of these neurons. Our identification of a specific population of neurons whose firing properties change suddenly and synchronously following axotomy, and whose activity is associated with tactile allodynia, provides a powerful vehicle for defining the specific cascade of cellular and molecular events that underlie neuropathic pain.

Multiple interacting sites of ectopic spike electrogenesis in primary sensory neurons.

Ectopic discharge generated in injured afferent axons and cell somata in vivo contributes significantly to chronic neuropathic dysesthesia and pain after nerve trauma. Progress has been made toward understanding the processes responsible for this discharge using a preparation consisting of whole excised dorsal root ganglia (DRGs) with the cut nerve attached. In the in vitro preparation, however, spike activity originates in the DRG cell soma but rarely in the axon. We have now overcome this impediment to understanding the overall electrogenic processes in soma and axon, including the resulting discharge patterns, by modifying the bath medium in which recordings are made. At both sites, bursts can be triggered by subthreshold oscillations, a phasic stimulus, or spikes arising elsewhere in the neuron. In the soma, once triggered, bursts are maintained by depolarizing afterpotentials, whereas in the axon, an additional process also plays a role, delayed depolarizing potentials. This alternative process appears to be involved in “clock-like” bursting, a discharge pattern much more common in axons than somata. Ectopic spikes arise alternatively in the soma, the injured axon end (neuroma), and the region of the axonal T-junction. Discharge sequences, and even individual multiplet bursts, may be a mosaic of action potentials that originate at these alternative electrogenic sites within the neuron. Correspondingly, discharge generated at these alternative sites may interact, explaining the sometimes-complex firing patterns observed in vivo.

Spike‐evoked suppression and burst patterning in dorsal root ganglion neurons of the rat

1 A low level of spontaneous impulse discharge is generated within dorsal root ganglia (DRGs) in intact animals, and this activity is enhanced following nerve injury. Many physiological stimuli present in vivo are capable of augmenting this ectopic discharge. Whatever their cause, episodes of sharply accelerated DRG firing tend to be followed by ‘after‐suppression’ during which discharge falls below baseline rate. In this study we examined the process of postexcitation suppression of firing rate, and how it shapes spike patterning in primary sensory neurons. 2 We recorded intracellularly from sensory neurons in excised rat DRGs in vitro. Trains of spikes triggered by intracellular current pulses evoked a prolonged hyperpolarizing shift. This shift appeared to be due to activation of a Ca2+‐dependent K+ conductance (gK(Ca)). Spikes evoked by just‐suprathreshold pulses triggered a hyperpolarizing shift and spike cessation. As the shift decayed, spiking was restored. The net result was bursty (on–off) discharge, a previously unexplained peculiarity of ectopic discharge in some DRG neurons in vivo. 3 Conditioning nerve tetani delivered to axons of neurons which share the DRG with the impaled neuron evoked transient depolarization (‘cross‐depolarization’). However, when stimulus strength was increased so as to include the axon of the impaled neuron, the net result was a hyperpolarizing shift. Nerve stimulation that straddled the threshold of the axon of the impaled neuron drove it intermittently, but it always drove axons of at least some neighbouring neurons. The result was dynamic modulation of the membrane potential of the impaled neuron as cross‐depolarization and spike‐evoked hyperpolarizing shifts played off against one another. Membrane potential shifted in the hyperpolarizing direction whenever the axon was activated, and shifted in the depolarizing direction whenever it was silent. Dynamic modulation of this sort probably also occurs in vivo when stimuli are drawn over the surface of the skin.

Electrical Excitability of the Soma of Sensory Neurons Is Required for Spike Invasion of the Soma, but Not for Through-Conduction

The cell soma of primary sensory neurons is electrically excitable, and is invaded by action potentials as they pass from the peripheral nerve, past the dorsal root ganglion (DRG) and toward the spinal cord. However, there are virtually no synapses in the DRG, and no signal processing is known to occur there. Why, then, are DRG cell somata excitable? We have constructed and validated an explicit model of the primary sensory neuron and used it to explore the role of electrical excitability of the cell soma in afferent signaling. Reduction and even elimination of soma excitability proved to have no detectable effect on the reliability of spike conduction past the DRG and into the spinal cord. Through-conduction is affected, however, by major changes in neuronal geometry in the region of the t-junction. In contrast to through-conduction, excitability of the soma and initial segment is essential for the invasion of afferent spikes into the cell soma. This implies that soma invasion has a previously unrecognized role in the physiology of afferent neurons, perhaps in the realm of metabolic coupling of the biosynthesis of signaling molecules required at the axon ends to functional demand, or in cell-cell interaction within sensory ganglia. Spike invasion of the soma in central nervous system neurons may play similar roles.

Ongoing activity in neuroma afferents bearing retrograde sprouts

Electrophysiological recordings were made from axons teased from the sciatic nerve 17-34 mm (mean 26.8 mm) central to a chronic nerve-end neuroma in adult rats. 23 fibers (2% of those sampled) appeared to have had a sprout(s) that grew in the retrograde (central) direction for at least this distance. Nine of the 23 (39%) carried spontaneous ongoing discharge. The parent fiber was myelinated (an A-fiber) in most instances, but unmyelinated (a C-fiber) in some. Thus, following nerve injury, a subset of afferent axons undergo retrograde sprouting, and many of them fire spontaneously. These contribute, along with the afferents whose trapped ends terminate at the injury site, to the ectopic afferent barrage generated in neuromas.

Simulation in Sensory Neurons Reveals a Key Role for Delayed Na+ Current in Subthreshold Oscillations and Ectopic Discharge: Implications for Neuropathic Pain

Somata of primary sensory neurons are thought to contribute to the ectopic neural discharge that is implicated as a cause of some forms of neuropathic pain. Spiking is triggered by subthreshold membrane potential oscillations that reach threshold. Oscillations, in turn, appear to result from reciprocation of a fast active tetrodotoxin-sensitive Na+ current (INa+) and a passive outward IK+ current. We previously simulated oscillatory behavior using a transient Hodgkin-Huxley-type voltage-dependent INa+ and ohmic leak. This model, however, diverged from oscillatory parameters seen in live cells and failed to produce characteristic ectopic discharge patterns. Here we show that use of a more complete set of Na+ conductances--which includes several delayed components--enables simulation of the entire repertoire of oscillation-triggered electrogenic phenomena seen in live dorsal root ganglion (DRG) neurons. This includes a physiological window of induction and natural patterns of spike discharge. An INa+ component at 2-20 ms was particularly important, even though it represented only a tiny fraction of overall INa+ amplitude. With the addition of a delayed rectifier IK+ the singlet firing seen in some DRG neurons can also be simulated. The model reveals the key conductances that underlie afferent ectopia, conductances that are potentially attractive targets in the search for more effective treatments of neuropathic pain.

Cross-excitation in dorsal root ganglia does not depend on close cell-to-cell apposition

ABOUT 90% of neurons in dorsal root ganglia (DRGs) of rats 2–5 weeks of age are depolarized and excited by impulse activity in neighboring neurons that share the same DRG. Synaptic contacts are extremely rare in DRGs, but instances of close membrane apposition between pairs of neuronal somata are not uncommon, especially in prenatal rats. Close membrane apposition could permit electrotonic interactions among neighboring DRG neurons. We carried out an ultrastructural examination of DRGs taken from rats 2–5 weeks of age and found that by this age <2% of cells remain in close apposition with neighbors. The remainder are separated by one or two layers of satellite glial cytoplasm. It is, therefore, unlikely that close apposition between adjacent neurons contributes significantly to functional cross-excitation in the DRG.

Subthreshold oscillations facilitate neuropathic spike discharge by overcoming membrane accommodation.

We have used computer simulation to better understand how the prolonged époques of repetitive discharge that underlie chronic neuropathic pain are generated. When subjected to step depolarization the cell soma of most primary afferents produces a single spike, or a brief spike burst, and then falls silent. Slow ramp depolarization typical of physiological stimuli does not evoke any spikes, due to the pronounced membrane accommodation of these neurons. Prior work in live DRG neurons suggests that prolonged neuropathic discharge occurs in neurons that generate subthreshold membrane potential oscillations; the rising (depolarizing) phase of the oscillation sinusoid triggers discharge that can last indefinitely. The specific contribution of oscillations to prolonged electrogenesis is not fully understood, however, as they typically add no more than approximately 5 mV to overall cell depolarization. We constructed a computer simulation of a dorsal root ganglion neuron that generates subthreshold oscillations and prolonged electrogenesis. We found that the slope of the rising phase of the oscillation sinusoid, in addition to its amplitude, is an important factor in the ability of oscillations to generate spike trains. The relatively steep slope of oscillation sinusoids facilitates electrogenesis by transiently overcoming the membrane accommodation associated with physiological slow ramp depolarization.