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

Abstract

The TTX-sensitive Na(v)1.7 (PN1) Na(+) channel alpha 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 degrees C in vivo in deeply anaesthetised young guinea-pigs. Each neurone was classified, from its dorsal root conduction velocity (CV) as a C-, Adelta- or Aalpha/beta-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 Na(v)1.7-like immunoreactivity (Na(v)1.7-LI) was examined on sections of dye-injected neurones. All C-, 90 % of Adelta- and 40 % of Aalpha/beta-fibre units, including both nociceptive and LTM units, showed Na(v)1.7-LI. Positive units included 1/1 C-LTM, 6/6 C-nociceptive, 4/4 C-unresponsive (possible silent nociceptive) units, 5/6 Adelta-LTM (D hair), 13/14 Adelta-nociceptive, 2/9 Aalpha/beta-nociceptive, 10/18 Aalpha/beta-LTM cutaneous and 0/9 Aalpha/beta-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. Na(v)1.7-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 Na(v)1.7 in nociceptive units.

Figure 1. Immunostaining of neurones to show Na_v1.7 in rat and guinea-pig DRGs

A and B compare the staining with anti-Na_v1.7 antibody of rat (A) and guinea-pig (B) DRG sections on the same slide. The small cells were the most intensely stained (asterisks), medium sized cells often had an intermediate staining intensity (arrowheads) and the palest staining profiles were usually of large neurones. These images were captured under bright field optics with interference contrast to aid visualisation of the paler staining neurons, although image analysis was always carried out on bright field images. C, D and E, sections of the same guinea-pig L6 DRG on which immunocytochemistry was carried out simultaneously with the following primary antibody: C, normal anti-Na_v1.7 antibody; D, anti-Na_v1.7 antibody was pre-absorbed for 3 h prior to use with 5 × 10⁻⁵m of the peptide against which the antibody had been generated; E, no primary antibody. F, the relationship between subjectively judged relative intensity (abscissa) and the calculated relative intensity (ordinate) (see Methods); 20 % relative intensity or above was consistently judged to be clearly positive.

Figure 3. Comparison of the percentages of neurones with different CVs and sensory properties that show Na_v1.7-LI

Graphs show the percentages of nociceptive (A) and LTM (B) units in the different CV ranges, and in nociceptive and LTM (low threshold mechanoreceptive) units in all CV groups together (C) that showed clear Na_v1.7-LI (≥ 20 % relative intensity). It can be seen in A and B that these percentages were lower in groups with higher mean CVs. C, the percentage of nociceptive units that was positive was significantly greater than the percentage of LTM units that was positive (χ² test, 2 × 2 contingency table). The number of units is shown above each column. NOCI, nociceptive, all conduction velocities; C UNR, C-fibre unresponsive units.

Figure 4. Relative intensities of Na_v1.7-LI in neurones defined by sensory properties and CV

A scattergraph showing the distribution of relative intensities of Na_v1.7-LI in the different groups of nociceptive and LTM (low threshold mechanoreceptive) neurones, and, in the last two columns, all nociceptive and all LTM neurones grouped together. •, nociceptors; ○, LTMs. Cutaneous LTMs includes Aδ (D hair) units and Aα/β (G hair, field, slowly adapting and rapidly adapting) units; MS, mainly muscle spindle, proprioceptive, afferent units. Medians are shown. The Kruskall-Wallis test with Dunn's post hoc test was used to compare all subgroups except C LTMs (only one cell). The Mann-Whitney test was used to compare medians of all nociceptive and all LTM units. Medians that were significantly different are indicated with a line above the two groups and asterisks show the level of significance *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. Graphs of Na_v1.7-LI intensity against neuronal properties

Neuronal properties plotted against Na_v1.7-LI intensity are cell size (the cross-sectional area of the largest section through the neurone; A and B); dorsal root CV (C and D); and AP duration (the duration at base of somatic APs evoked by electrical stimulation of the dorsal root; E and F). The horizontal line at 20% indicates the relative intensity above which cells are considered positive for Na_v1.7. The inset in D shows superimposition of the lines for nociceptive (NOCI) and low threshold mechanoreceptive (LTM) units when only units with CVs of 1.1-12 m s⁻¹ were included (see text). On the left, units are subdivided into those with C- (•), Aδ- (○) or Aα/β-fibres (×); in these groups nociceptive and non-nociceptive units, and cooling-sensitive units were included. Unresponsive units were excluded. Linear regression analysis was carried out on each of these CV groups separately and on all units together, and regression lines are included only where there was a significant correlation. On the right, only nociceptive (•) and LTM units (○) are included; thus fewer units are plotted. Linear regression analysis was carried out on nociceptive and LTM units separately. The keys to symbols and lines may be found in A (for A, C and E) and B (for B, D and F). For significant linear correlations (P < 0.05), regression lines are shown here and the P and r² values are shown in Table 1.

Figure 2. Photomicrographs of dye-injected, physiologically identified neurones

On the left are the fluorescence photomicrographs of sections through neurones indicated by the arrow in each image injected with Lucifer yellow (A) or ethidium bromide (B, C and D). On the right (A‘-D‘) are the same neurones after immunocytochemistry to show Na_v1.7-LI; these images were captured under interference contrast optics to increase clarity, but note that all image analysis was performed on bright field images. Sections of the dye-injected neurone in A, B and C, but not D, were through the nucleus. The C polymodal nociceptor in A shows intense labelling; both Aδ-fibre units (a D hair LTM unit in B and a nociceptive unit in C) show intermediate labelling. The muscle spindle afferent in D shows weak or absent labelling and, having no nucleus, does not indicate the full size of the cell. LTM, low threshold mechanoreceptor; HTM, high threshold mechanoreceptor.