Laiche Djouhri

The TTX-Resistant Sodium Channel Nav1.8 (SNS/PN3): Expression and Correlation with Membrane Properties in Rat Nociceptive Primary Afferent Neurons

We have examined the distribution of the sensory neuron‐specific Na+ channel Nav1.8 (SNS/PN3) in nociceptive and non‐nociceptive dorsal root ganglion (DRG) neurons and whether its distribution is related to neuronal membrane properties. Nav1.8‐like immunoreactivity (Nav1.8‐LI) was examined with an affinity purified polyclonal antiserum (SNS11) in rat DRG neurons that were classified according to sensory receptive properties and by conduction velocity (CV) as C‐, Aδ‐ or Aα/β. A significantly higher proportion of nociceptive than low threshold mechanoreceptive (LTM) neurons showed Nav1.8‐LI, and nociceptive neurons had significantly more intense immunoreactivity in their somata than LTM neurons. Results showed that 89, 93 and 60 % of C‐, Aδ‐ and Aα/β‐fibre nociceptive units respectively and 88 % of C‐unresponsive units were positive. C‐unresponsive units had electrical membrane properties similar to C‐nociceptors and were considered to be nociceptive‐type neurons. Weak positive Nav1.8‐LI was also present in some LTM units including a C LTM, all Aδ LTM units (D hair), about 10 % of cutaneous LTM Aα/β‐units, but no muscle spindle afferent units. Nav1.8‐LI intensity was negatively correlated with soma size (all neurons) and with dorsal root CVs in A‐ but not C‐fibre neurons. Nav1.8‐LI intensity was positively correlated with action potential (AP) duration (both rise and fall time) in A‐fibre neurons and with AP rise time only in positive C‐fibre neurons. It was also positively correlated with AP overshoot in positive neurons. Thus high levels of Nav1.8 protein may contribute to the longer AP durations (especially in A‐fibre neurons) and larger AP overshoots that are typical of nociceptors.

Spontaneous pain, both neuropathic and inflammatory, is related to frequency of spontaneous firing in intact C-fiber nociceptors

Spontaneous pain, a poorly understood aspect of human neuropathic pain, is indicated in animals by spontaneous foot lifting (SFL). To determine whether SFL is caused by spontaneous firing in nociceptive neurons, we studied the following groups of rats: (1) untreated; (2) spinal nerve axotomy (SNA), L5 SNA 1 week earlier; (3) mSNA (modified SNA), SNA plus loose ligation of the adjacent L4 spinal nerve with inflammation-inducing chromic gut; and (4) CFA (complete Freund’s adjuvant), intradermal complete Freund’s adjuvant-induced hindlimb inflammation 1 and 4 d earlier. In all groups, recordings of SFL and of spontaneous activity (SA) in ipsilateral dorsal root ganglion (DRG) neurons (intracellularly) were made. Evoked pain behaviors were measured in nerve injury (SNA/mSNA) groups. Percentages of nociceptive-type C-fiber neurons (C-nociceptors) with SA increased in intact L4 but not axotomized L5 DRGs in SNA and mSNA (to 35%), and in L4/L5 DRGs 1–4 d after CFA (to 38–25%). SFL occurred in mSNA but not SNA rats. It was not correlated with mechanical allodynia, extent of L4 fiber damage [ATF3 (activation transcription factor 3) immunostaining], or percentage of L4 C-nociceptors with SA. However, L4 C-nociceptors with SA fired faster after mSNA (1.8 Hz) than SNA (0.02 Hz); estimated L4 total firing rates were ∼5.0 and ∼0.6 kHz, respectively. Similarly, after CFA, faster L4 C-nociceptor SA after 1 d was associated with SFL, whereas slower SA after 4 d was not. Thus, inflammation causes L4 C-nociceptor SA and SFL. Overall, SFL was related to SA rate in intact C-nociceptors. Both L5 degeneration and chromic gut cause inflammation. Therefore, both SA and SFL/spontaneous pain after nerve injury (mSNA) may result from cumulative neuroinflammation.

Aβ-fiber nociceptive primary afferent neurons: a review of incidence and properties in relation to other afferent A-fiber neurons in mammals

The existence of nociceptors with Abeta-fibers has often been overlooked, and many textbooks endorse the view that all nociceptors have either C- or Adelta-fibers. Here we review evidence starting from the earliest descriptions of A-fiber nociceptors, which clearly indicates that a substantial proportion of cutaneous/somatic afferent A-fiber nociceptors conduct in the Abeta conduction velocity (CV) range in all species in which CV was carefully examined, including mouse, rat, guinea pig, cat and monkey. Reported proportions of A-fiber nociceptors with Abeta-fibers vary from 18% to 65% in different species, usually >50% in rodents. In rat, about 20% of all somatic afferent neurons with Aalpha/beta-fibers were nociceptive. Distributions of CVs of A-fiber nociceptors usually appear unimodal, with a median/peak in the upper Adelta or lower Abeta CV range. We find no evidence to suggest discontinuous differences in electrophysiological or cytochemical properties of Adelta and Abeta nociceptors, rather there are gradual changes in relation to CV. However, some functional differences have been reported. In cat, A-fiber nociceptors with lower mechanical thresholds (moderate pressure receptors) tend to have faster CVs [P.R. Burgess, D. Petit, R.M. Warren. Receptor types in cat hairy skin supplied by myelinated fibers. J. Neurophysiol. 31 (1968) 833-848]. In primate (monkey) A-fiber nociceptors that responded to heat were divided into type I A mechano-heat (AMH) units (Adelta and Abeta CVs) with lower mechanical and higher heat thresholds and may include moderate pressure receptors, and type II AMH units (Adelta CVs) with higher mechanical/lower heat thresholds. It is important that the existence of Abeta nociceptors is recognised, because assumptions that fast conducting, large diameter afferents are always low threshold mechanoreceptors might lead/have led to misinterpretations of data.

Intense Isolectin-B4 Binding in Rat Dorsal Root Ganglion Neurons Distinguishes C-Fiber Nociceptors with Broad Action Potentials and High Nav1.9 Expression

Binding to isolectin-B4 (IB4) and expression of tyrosine kinase A (trkA) (the high-affinity NGF receptor) have been used to define two different subgroups of nociceptive small dorsal root ganglion (DRG) neurons. We previously showed that only nociceptors have high trkA levels. However, information about sensory and electrophysiological properties in vivo of single identified IB4-binding neurons, and about their trkA expression levels, is lacking. IB4-positive (IB4+) and small dark neurons had similar size distributions. We examined IB4-binding levels in >120 dye-injected DRG neurons with sensory and electrophysiological properties recorded in vivo. Relative immunointensities for trkA and two TTX-resistant sodium channels (Nav1.8 and Nav1.9) were also measured in these neurons. IB4+ neurons were classified as strongly or weakly IB4+. All strongly IB4+ neurons were C-nociceptor type (C-fiber nociceptive or unresponsive). Of 32 C-nociceptor-type neurons examined, ∼50% were strongly IB4+, ∼20% were weakly IB4+ and ∼30% were IB4−. Aδ low-threshold mechanoreceptive (LTM) neurons were weakly IB4+ or IB4−. All 33 A-fiber nociceptors and all 44 Aα/β-LTM neurons examined were IB4−. IB4+ compared with IB4− C-nociceptor-type neurons had longer somatic action potential durations and rise times, slower conduction velocities, more negative membrane potentials, and greater immunointensities for Nav1.9 but not Nav1.8. Immunointensities of IB4 binding in C-neurons were positively correlated with those of Nav1.9 but not Nav1.8. Of 23 C-neurons tested for both trkA and IB4, ∼35% were trkA+/IB4+ but with negatively correlated immunointensities; 26% were IB4+/trkA−, and 35% were IB4−/trkA+. We conclude that strongly IB4+ DRG neurons are exclusively C-nociceptor type and that high Nav1.9 expression may contribute to their distinct membrane properties.

Sensory and electrophysiological properties of guinea‐pig sensory neurones expressing Nav 1.7 (PN1) Na+ channel α subunit protein

The TTX‐sensitive Nav1.7 (PN1) Na+ channel α subunit protein is expressed mainly in small dorsal root ganglion (DRG) neurones. This study examines immunocytochemically whether it is expressed exclusively or preferentially in nociceptive primary afferent DRG neurones, and determines the electrophysiological properties of neurones that express it. Intracellular somatic action potentials (APs) evoked by dorsal root stimulation were recorded in L6/S1 DRG neurones at 30 ± 2 °C in vivo in deeply anaesthetised young guinea‐pigs. Each neurone was classified, from its dorsal root conduction velocity (CV) as a C‐, Aδ‐ or Aα/β‐fibre unit and from its response to mechanical and thermal stimuli, as a nociceptive, low threshold mechanoreceptive (LTM) or unresponsive unit. Fluorescent dye was injected into the soma and Nav1.7‐like immunoreactivity (Nav1.7‐LI) was examined on sections of dye‐injected neurones. All C‐, 90 % of Aδ‐ and 40 % of Aα/β‐fibre units, including both nociceptive and LTM units, showed Nav1.7‐LI. Positive units included 1/1 C‐LTM, 6/6 C‐nociceptive, 4/4 C‐unresponsive (possible silent nociceptive) units, 5/6 Aδ‐LTM (D hair), 13/14 Aδ‐nociceptive, 2/9 Aα/β‐nociceptive, 10/18 Aα/β‐LTM cutaneous and 0/9 Aα/β‐muscle spindle afferent units. Overall, a higher proportion of nociceptive than of LTM neurones was positive, and the median relative staining intensity was greater in nociceptive than LTM units. Nav1.7‐LI intensity was clearly positively correlated with AP duration and (less strongly) negatively correlated with CV and soma size. Since nociceptive units tend overall to have longer duration APs, slower CVs and smaller somata, these correlations may be related to the generally greater expression of Nav1.7 in nociceptive units.

Association of somatic action potential shape with sensory receptive properties in guinea‐pig dorsal root ganglion neurones

1 Intracellular voltage recordings were made from the somata of L6 and S1 dorsal root ganglion (DRG) neurones at 28.5–31 °C in young guinea‐pigs (150–300 g) anaesthetized with sodium pentobarbitone. Action potentials (APs) evoked by dorsal root stimulation were used to classify conduction velocities (CVs) as C, Aδ or Aα/β. Units with overshooting APs and membrane potentials (Vm) more negative than −40 mV were analysed: 40 C‐, 45 Aδ‐ and 94 Aα/β‐fibre units. 2 Sensory receptive properties were characterized as: (a) low‐threshold mechanoreceptive (LTM) units (5 C‐, 10 Aδ‐ and 57 Aα/β‐fibre units); (b) nociceptive units, responding to noxious mechanical stimuli, some also to noxious heat (40 C‐, 27 Aδ‐ and 27 Aα/β‐fibre units); (c) unresponsive units that failed to respond to a variety of tests; and (d) C‐fibre cooling‐sensitive units (n= 4). LTM units made up about 8 % of identified C‐fibre units, 36 % of identified Aδ‐fibre units and > 73 % of identified Aα/β‐fibre units. Compared with LTM units, the nociceptive units had APs that were longer on average by 3 times (C‐fibre units), 1.7 times (Aδ‐fibre units) and 1.4 times (Aα/β‐fibre units). They also had significantly longer rise times (RTs) and fall times (FTs) in all CV ranges. Between Aα/β‐nociceptors and Aα/β‐LTMs there was a proportionately greater difference in RT than in FT. The duration of the afterhyperpolarization measured to 80 % recovery (AHP80) was also significantly longer in nociceptive than LTM neurones in all CV ranges: by 3 times (C‐fibre units), 6.3 times (Aδ‐fibre units) and 3.6 times (Aα/β‐fibre units). The mean values of these variables in unresponsive units were similar to those of nociceptive units in each CV range; in C‐ and Aδ‐fibre groups their mean AHP duration was even longer than in nociceptive units. 3 A‐fibre LTM neurones were divided into Aδ‐ (D hair units, n= 8), and Aα/β‐ (G hair/field units, n= 22; T (tylotrich) hair units, n= 6; rapidly adapting (RA) glabrous units, n= 6; slowly adapting (SA) hairy and glabrous units, n= 2; and muscle spindle (MS) units n= 17). MS and SA units had the shortest duration APs, FTs and AHP80s of all these groups. The mean RT in D hair units was significantly longer than in all Aα/β LTM units combined. T hair units had the longest mean FT and AHP of all the A‐LTM groups. The mean AHP was about 10 times longer in T hair units than in all other A‐LTM units combined (significant), and was similar to that of A‐fibre nociceptive neurones. 4 These differences in somatic AP shape may aid in distinguishing between LTM and nociceptive or unresponsive C‐ and Aδ‐fibre units but probably not between nociceptive and unresponsive units. The differences seen may reflect differences in expression or activation of different types of ion channel.

Electrophysiological differences between nociceptive and non-nociceptive dorsal root ganglion neurones in the rat in vivo

Intracellular recordings were made from 1022 somatic lumbar dorsal root ganglion (DRG) neurones in anaesthetized adult rats, classified from dorsal root conduction velocities (CVs) as C, Aδ or Aα/β, and according to their responses to mechanical and thermal stimuli as nociceptive (including high‐threshold mechanoreceptive (HTM) units), and non‐nociceptive (including low‐threshold mechanoreceptive (LTM) and cooling units). Of these, 463 met electrophysiological criteria for analysis of action potentials (APs) evoked by dorsal root stimulation. These included 47 C‐, 71 Aδ‐ and 102 Aα/β‐nociceptive, 10 C‐, 8 Aδ− and 178 Aα/β‐LTM, 18 C‐ and 19 Aδ‐ unresponsive, and 4 C‐cooling units. Medians of AP and afterhyperpolarization (AHP) durations and AP overshoots were significantly greater for nociceptive than LTM units in all CV groups. AP overshoots and AHP durations were similar in nociceptors of all CV groups whereas AP durations were greater in slowly conducting, especially C‐fibre, nociceptors. C‐cooling units had faster CVs, smaller AP overshoots and shorter AP durations than C‐HTM units. A subgroup of Aα/β‐HTM, moderate pressure units, had faster CVs and AP kinetics than other Aα/β‐HTM units. Of the Aα/β‐LTM units, muscle spindle afferents had the fastest CV and AP kinetics, while rapidly adapting cutaneous units had the slowest AP kinetics. AP variables in unresponsive and nociceptive units were similar in both C‐ and Aδ‐fibre CV groups. The ability of fibres to follow rapid stimulus trains (fibre maximum following frequency) was correlated with CV but not sensory modality. These findings indicate both the usefulness and limitations of using electrophysiological criteria for identifying neurones acutely in vitro as nociceptive.

trkA Is Expressed in Nociceptive Neurons and Influences Electrophysiological Properties via Nav1.8 Expression in Rapidly Conducting Nociceptors

To test the hypothesis that trkA (the high-affinity NGF receptor) is selectively expressed in nociceptive dorsal root ganglion (DRG) neurons, we examined the intensity of trkA immunoreactivity in single dye-injected rat DRG neurons, the sensory receptor properties of which were identified in vivo with mechanical and thermal stimuli. We provide the first evidence in single identified neurons that strong trkA expression in DRGs is restricted to nociceptive neurons, probably accounting for the profound influence of NGF on these neurons. Furthermore, we demonstrate that trkA expression is as high in rapidly conducting (Aα/β) as in more slowly conducting (Aδ and C) nociceptors. All Aα/β low-threshold mechanoreceptors (LTMs) are trkA negative, although weak but detectable trkA is present in some C and Aδ LTMs. NGF can influence electrophysiological properties of DRG neurons, probably by binding to trkA. We found positive correlations for single identified Aα/β (but not C or Aδ) nociceptors between trkA immunocytochemical intensity and electrophysiological properties typical of nociceptors, namely long action potential and afterhyperpolarization durations and large action potential amplitudes. Furthermore, for Aα/β (notCorAδ) nociceptors, trkA intensity is inversely correlated with conduction velocity. Similar relationships, again only in Aα/β nociceptors, between electrophysiological properties and trkA expression exist for sodium channel Nav1.8 but not Nav1.9 immunoreactivities. These findings suggest that in Aα/β nociceptors, influences of NGF on expression levels of Nav1.8 are related to, and perhaps limited by, expression levels of trkA. This view is supported by a positive correlation between immuno-intensities of trkA and Nav1.8 in A-fiber, but not C-fiber, nociceptors.

Chronic inflammatory pain is associated with increased excitability and hyperpolarization-activated current (Ih) in C- but not Aδ-nociceptors

Summary This study shows that C‐nociceptors may play a more important role than Aδ‐nociceptors in sustaining persistent inflammatory pain and that Ih/HCN2 channels may be involved. Abstract Inflammatory pain hypersensitivity results partly from hyperexcitability of nociceptive (damage‐sensing) dorsal root ganglion (DRG) neurons innervating inflamed tissue. However, most of the evidence for this is derived from experiments using acute inflammatory states. Herein, we used several approaches to examine the impact of chronic or persistent inflammation on the excitability of nociceptive DRG neurons and on their expression of Ih and the underlying hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels, which regulate neuronal excitability. Using in vivo intracellular recordings of somatic action potentials from L4/L5 DRG neurons in normal rats and rats with hindlimb inflammation induced by complete Freund’s adjuvant (CFA), we demonstrate increased excitability of C‐ but not Aδ‐nociceptors, 5 to 7 days after CFA. This included an afterdischarge response to noxious pinch, which may contribute to inflammatory mechanohyperalgesia, and increased incidence of spontaneous activity (SA) and decreased electrical thresholds, which are likely to contribute to spontaneous pain and nociceptor sensitization, respectively. We also show, using voltage clamp in vivo, immunohistochemistry and behavioral assays that (1) the inflammation‐induced nociceptor hyperexcitability is associated, in C‐ but not Aδ‐nociceptors, with increases in the mean Ih amplitude/density and in the proportion of Ih expressing neurons, (2) increased proportion of small DRG neurons (mainly IB4‐negative) expressing HCN2 but not HCN1 or HCN3 channel protein, (3) increased HCN2‐ immunoreactivity in the spinal dorsal horn, and (4) attenuation of inflammatory mechanoallodynia with the selective Ih antagonist, ZD7288. Taken together, the findings suggest that C‐ but not Aδ‐nociceptors sustain chronic inflammatory pain and that Ih/HCN2 channels contribute to inflammation‐induced C‐nociceptor hyperexcitability.

Changes in somatic action potential shape in guinea‐pig nociceptive primary afferent neurones during inflammation in vivo

1 complete Freunds adjuvant (CFA) 1. We have examined whether there are changes during inflammation in the membrane properties of nociceptive primary afferent neurones in the guinea‐pig that might contribute to hyperalgesia. Inflammation was induced by intradermal injections of complete Freunds adjuvant (CFA) in the left leg. Intracellular voltage recordings were made from the somata of ipsilateral L6 and S1 dorsal root ganglion neurones in anaesthetised untreated guinea‐pigs at 2 or 4 days after CFA treatment. 2 Units were classified as C, Aδ or Aα/β on the basis of their dorsal root conduction velocities (CVs). Units with receptive fields on the left leg were characterized as nociceptive, low‐ threshold mechanoreceptive (LTM) or unresponsive according to their responses to mechanical and thermal stimuli. The shapes of their somatic action potentials (APs) evoked by dorsal root stimulation were recorded. 3 Comparisons of data from nociceptive neurones recorded in CFA treated animals after 2 and 4 days with data from CFA untreated (control) animals showed the following significant changes: in C‐fibre nociceptors, decreased AP duration at base, AP rise time and AP fall time, and increased maximum rates of AP rise and fall with no change in afterhyperpolarization measured to 80 % recovery (AHP80); in Aδ‐fibre nociceptors, decreased AP duration at base, AP fall time and a reduction in AHP80; and in Aα7sol;β‐fibre nociceptors, a decreased AHP80 but no change in AP duration. Apart from a more negative membrane potential and AHP depth below 0 mV in Aα/β nociceptors at 4 days compared with 2 days post‐CFA, none of the above variables differed significantly between units recorded 2 or 4 days after CFA. Therefore the two groups were pooled and called CFA2+4d. 4 The reduction in AP duration in C‐fibre nociceptors was apparent both in high threshold mechanoreceptor and polymodal nociceptors and also in units with either cutaneous or subcutaneous receptive fields. 5 No significant changes in AP duration at base or AHP80 were seen 2 or 4 days after CFA compared with control in either LTM or unresponsive neurones, although some of the latter may have become classified as nociceptors after CFA treatment. 6 The alterations in membrane properties of nociceptors should permit higher discharge frequencies, thus contributing to inflammatory hyperalgesia. They suggest active changes in the expression or activation of cation channels during peripheral inflammation.