Figure 1.
NaV1.8 is distributed in clusters along the axons of small, unmyelinated DRG neurons in vitro.
Endogenous NaV1.8 was immuno-localised in cultured DRG neurons after 2 DIV. Small-diameter neurons were identified by morphology (A, right panel) and by the immuno-reactivity for Peripherin (B, right panel). The region framed by the dotted square in A is magnified in the inset below. NaV1.8 is distributed in distinct puncta along the neurites, of small-diameter neurons (A, B; arrows pinpoint example of clusters, which are distributed throughout the neurites). NaV1.8 was also found to be enriched at the level of the cell bodies (Figure 1A, asterisk). The fluorescent construct NaV1.8-DsRed2 was visualised in DRG neurons. The image shows NaV1.8-DsRed2 distributed in clusters along the axon of DRG neuron (C, left panel). The discontinuous distribution of the fluorescent construct has been mapped by quantifying pixel intensity along the neurite (C, graph; right panel). Also, the fluorescent construct NaV1.8-DsRed2 colocalises with GM1 puncta along the neurite of DRG neurons, as shown by the superimposed images of NaV1.8-DsRed2 and GM1 (merge). Scale bars are 20 μm.
Figure 2.
NaV1.8 is distributed in clusters along the axons of small, unmyelinated DRG neurons in vivo.
NaV1.8 is clustered in puncta along the unmyelinated fibres of rat sciatic nerve. Teased unmyelinated fibres were identified by morphology (A, arrow) and by immuno-reactivity for Peripherin (B). Scale bars are 20 μm.
Figure 3.
NaV1.8 clusters colocalise with GM1 along the axons of small DRG neurons in vitro.
Endogenous NaV1.8 was immuno-localised in cultured DRG neurons after 2 DIV. GM1 molecules were detected with CTB. At the level of the cell bodies there is no clear colocalisation between endogenous NaV1.8 and GM1 molecules (A). In contrast, along the neurites NaV1.8 clusters colocalise with GM1 puncta (arrows, B). Phase contrast image in B demonstrates the integrity of the neurite. Scale bars are 20 μm.
Figure 4.
NaV1.8 associates with lipid rafts in the sciatic nerve.
Lipid rafts were extracted from the sciatic nerve. After centrifugation on an Iodixanol density gradient, fractions were analysed by western blotting and dot blot analysis to assess lipid raft isolation and NaV1.8 partitioning between lipid rafts and the non-raft portions of the membrane. Flotillin1 and GM1 were used as a protein and lipid marker of lipid rafts, respectively. Transferrin receptor was used as a marker of non-raft portions. In the sciatic nerve the totality of NaV1.8 is associated with lipid rafts. M represents protein ladder, the recovered fractions are numbered from 1 (top fraction) to 9 (bottom fraction).
Figure 5.
NaV1.8 associates with lipid rafts in DRG neurons in vitro.
Lipid rafts were extracted from DRG neurons after 2 DIV. After centrifugation on an Iodixanol density gradient fractions were analysed by western blotting and dot blot analysis to assess lipid raft isolation and NaV1.8 partitioning between lipid rafts and the non-raft portions of the membrane. Flotillin1 and GM1 were used as a protein and lipid marker of lipid rafts, respectively. Transferrin receptor was used a marker of non-raft portions. NaV1.8 is associated with both lipid rafts and non-raft portions of the membrane (A). Incorporation of 7KC into the neuronal plasma-membranes impairs lipid raft stability. In this condition total NaV1.8 is associated with the non-raft portion of the membrane (B). Depletion of cholesterol from the neuronal membrane, by using MβCD, leads to lipid rafts disruption. NaV1.8 is only associated with the soluble, non-raft, portion of the membrane upon this treatment (C). M represents protein ladder, the recovered fractions are numbered from 1 (top fraction) to 9 (bottom fraction).
Figure 6.
Mechanostimulation of DRG neurons in vitro.
The figure shows a representative neuron loaded with Fluo-4 responding to a mechanical stimulus. A) Shows the Fluo-4 fluorescence and DiC image of a DRG neuron. The glass probe is visible in the DiC image, at the moment of contact with a neurite (arrow), which projects from the cell body. B) Shows the Fluo-4 fluorescence in pseudo-colour, associated with different time points during the recording. C) The graph shows the recorded fluorescence intensity of different region of interests (ROIs), visible in A. The arrow indicates the time point when the cell was stimulated; cardinal numbers refer to the time points which the images in B are associated to Scale bar is 10 µm.
Table 1.
Axonal mechano-stimulation.
Figure 7.
Effect of lipid raft depletion on the speed of propagation of Fluo-4 signals upon mechano-stimulation of the neurites.
Box plot show that upon 7KC and MβCD treatments the speed of propagation (expressed in μm/sec) of the mechanically-evoked depolarisation is lower, compared to Control (CTR)- and Cholesterol (CHOL)-treated cells. * = p<0.05 vs. CTR. Mann-Whitney U Test.
Table 2.
Somal mechano-stimulation.
Figure 8.
Chemical stimulation of DRG neurons: axonal stimulation and soma recording.
The figure shows the effect, at the level of the cell bodies, of axonal chemical stimulation. A) Shows the schematic representation of the Campenot chamber set-up with the Fluo-4 fluorescence and DiC image of the cell bodies (Soma chamber) and neurites projecting to the “Distal neurite” chamber, through the “Proximal neurite” chamber. B) The graph shows the recorded fluorescence intensity of different cell bodies visible in A (Soma chamber). Each data point is the mean fluorescence intensity ± SEM of different cell bodies (n = 58). Images in pseudo-colour represent the “Soma chamber” Fluo-4 fluorescence. The arrows indicate the time points when Vehicle and the Capsaicin, ATP, Bradykinin (CAB) cocktail have been applied; cardinal numbers refer to the time points depicted by the pseudo-colour images. Scale bar is 100 µm.
Table 3.
Chemical stimulation.