Sally N. Lawson

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.

Relationship of substance P to afferent characteristics of dorsal root ganglion neurones in guinea‐pig

1 The relationship between the afferent properties and substance P‐like immunoreactivity (SP‐LI) of L6 and SI dorsal root ganglion (DRG) neuronal somata was examined in anaesthetized guinea‐pigs. Glass pipette microelectrodes filled with fluorescent dyes were used to make intracellular recordings and to label DRG somata. The dorsal root conduction velocity (CV) and the afferent receptive properties of each unit were categorized according to criteria established in other species. Categories included a variety of low threshold mechanoreceptive classes, innocuous thermoreceptive and several nociceptive classes. Nociceptive units were further subdivided on the basis of CV and the locus of the receptive field (superficial cutaneous, deep cutaneous or subcutaneous). 2 SP‐LI was determined using the avidin–biotin complex method and the relative staining intensity determined by image analysis. The possible significance of labelling intensity is discussed. Clear SP‐LI appeared in twenty‐nine of 117 dye‐labelled neurones. All SP‐LI positive units with identified receptive properties were nociceptive but not all categories of nociceptors were positive. The intensity of SP‐LI labelling varied, often systematically, in relation to afferent properties. There was a tendency for nociceptive neurones with slower CVs and/or smaller cell bodies to show SP‐LI. 3 Nineteen of fifty‐one C fibre neurones showed SP‐LI. Fewer than half the C polymodal nociceptors (CPMs) were positive. The most intensely labelled units were the deep cutaneous nociceptors and some of the CPMs in glabrous skin. C low threshold mechanoreceptors and cooling‐sensitive units did not show SP‐LI. 4 Ten of sixty‐six A fibre neurones exhibited SP‐LI, including eight of sixteen Aδ nociceptors and two of fifteen Aα/β nociceptors. A fibre neurones exhibiting SP‐LI included seven of eight deep cutaneous mechanical nociceptors and some superficial cutaneous mechano‐heat nociceptors of hairy skin. In contrast, none of twenty superficial cutaneous A high threshold mechanoreceptor units or the thirty‐five A fibre low threshold units (D‐hair and other units) showed detectable SP‐LI. 5 We conclude that SP‐LI labelling in guinea‐pig DRG neurones is related to (a) afferent receptive properties, (b) the tissue in which the peripheral receptive terminals are located, (c) the CV and (d) the soma size.

Cell type and conduction velocity of rat primary sensory neurons with calcitonin gene-related peptide-like immunoreactivity

An immunocytochemical double labelling study of L4 dorsal root ganglia from rats aged seven to 10 weeks was made with an antibody to calcitonin gene-related peptide and with RT97, an anti-neurofilament antibody which specifically labels the light neuron population. Peptide immunoreactivity was found in an average of 46.5% of all neurons. Sixty-two per cent of the small dark (RT97-negative) and 30% of the light (RT97-positive) neuron populations contained the peptide. About one-third (32%) of the cells with peptide immunoreactivity were light cells and about two-thirds (68%) were small dark cells. Intracellular electrophysiological recordings were made in vitro from neurons in lumbar (L4, L5 and L6) dorsal root ganglia from six- to eight-week-old rats, followed by dye-injection and immunocytochemistry. This showed that conduction velocities of neurons with calcitonin gene-related peptide-like immunoreactivity ranged from 0.5 to 28.6 m/s. Seventy-three neurons were successfully processed. Of these, calcitonin gene-related peptide-like immunoreactivity was found in 46% of C-fibre neurons, 33% of A delta-fibre neurons and in 17% of the A alpha/beta-fibre neurons. The peptide-like immunoreactivity was found in approximately 25% of all A-fibre neurons sampled.

Dorsal root ganglion neurons show increased expression of the calcium channel α2δ-1 subunit following partial sciatic nerve injury

Abstract Neuropathic pain is associated with changes in the electrophysiological and neurochemical properties of injured primary afferent neurons. A mRNA differential display study in rat L4/5 dorsal root ganglia (DRGs) revealed upregulation of the calcium channel α2δ-1 subunit 2 weeks after partial sciatic nerve ligation (Seltzer model of neuropathic pain). The upregulated transcript appeared to represent previously unidentified sequence from the 3′-untranslated region of rat α2δ-1 mRNA. In situ hybridization using L5 DRGs from sham operated rats showed that 73, 40 and 19% of small ( 1100 μm2) neuronal profiles, respectively, expressed α2δ-1 mRNA. Two weeks following nerve injury there was a significant ipsilateral increase, both in the percentage of DRG neurons expressing α2δ-1 mRNA and in the intensity of the hybridization signal. Comparison of this ipsilateral expression with that in sham animals, revealed that for small, medium and large neurons, respectively, the proportion of neurons labelled increased by 1.2-, 1.8- and 2.7-fold, while the hybridization signal in α2δ-1-labelled neurons increased by 2.8-, 2.5- and 3.7-fold. The most intensely labelled neuronal profiles in ipsilateral, sham and contralateral DRGs, were generally those with small cross-sectional areas. The α2δ-1 auxiliary subunit is known to modulate calcium channel function in heterologous expression systems via its association with the pore-forming α1 calcium channel subunit. Therefore the increased levels of this subunit in the populations of primary afferents described may, via modulation of calcium-dependent processes such as neurotransmitter release and neuronal excitability, influence the processing of sensory information.

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.

Primary sensory neurones: Neurofilament, neuropeptides and conduction velocity

This paper reviews and provides new data on the relationship of the peptide content in rat dorsal root ganglion (DRG) neurons to a) the neurofilament content of the soma and b) the conduction velocities of the fibres. The latter involved intracellular recordings made in vitro followed by dye injection and immunocytochemistry. Because neurofilament-poor DRG neurones have C-fibres, and A-fibre neurones are neurofilament rich, the soma neurofilament content of peptide containing neurones allowed predictions to be made about their conduction velocity ranges. Substance P-like immunoreactive (SP-LI) neurones were mostly small, neurofilament poor, but a few (15%) were neurofilament rich. From conduction velocity measurements, about half the C-fibre neurones studied and 10% of A delta-neurones but no A alpha/beta-neurones showed SP-LI. CGRP-LI neurones were also mainly neurofilament poor neurones, but 32% were neurofilament rich, including small, medium, and large neurones. Fibres of CGRP-LI neurones conducted in the C, A delta or A alpha/beta ranges. Neurones with somatostatin-LI (SOM-LI) were all neurofilament poor; preliminary data is consistent with SOM-LI neurones having C-fibres.

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.