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. 2015 Feb;156(2):243-251.
doi: 10.1097/01.j.pain.0000460304.63948.40.

Presynaptic inhibition of optogenetically identified VGluT3+ sensory fibres by opioids and baclofen

Silke D Honsek  1 , Rebecca P Seal, Jürgen Sandkühler
Affiliations

Presynaptic inhibition of optogenetically identified VGluT3+ sensory fibres by opioids and baclofen

Silke D Honsek et al. Pain. 2015 Feb.

Abstract

Distinct subsets of sensory nerve fibres are involved in mediating mechanical and thermal pain hypersensitivity. They may also differentially respond to analgesics. Heat-sensitive C-fibres, for example, are thought to respond to μ-opioid receptor (MOR) activation while mechanoreceptive fibres are supposedly sensitive to δ-opioid receptor (DOR) or GABAB receptor (GABABR) activation. The suggested differential distribution of inhibitory neurotransmitter receptors on different subsets of sensory fibres is, however, heavily debated. In this study, we quantitatively compared the degree of presynaptic inhibition exerted by opioids and the GABABR agonist baclofen on (1) vesicular glutamate transporter subtype 3-positive (VGluT3) non-nociceptive primary afferent fibres and (2) putative nociceptive C-fibres. To investigate VGluT3 sensory fibres, we evoked excitatory postsynaptic currents with blue light at the level of the dorsal root ganglion (DRG) in spinal cord slices of mice, expressing channelrhodopsin-2. Putative nociceptive C-fibres were explored in VGluT3-knockout mice through electrical stimulation. The MOR agonist DAMGO strongly inhibited both VGluT3 and VGluT3 C-fibres innervating lamina I neurons but generally had less influence on fibres innervating lamina II neurons. The DOR agonist SNC80 did not have any pronounced effect on synaptic transmission in any fibre type tested. Baclofen, in striking contrast, powerfully inhibited all fibre populations investigated. In summary, we report optogenetic stimulation of DRG neurons in spinal slices as a capable approach for the subtype-selective investigation of primary afferent nerve fibres. Overall, pharmacological accessibility of different subtypes of sensory fibres considerably overlaps, indicating that MOR, DOR, and GABABR expressions are not substantially segregated between heat and mechanosensitive fibres.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Comparison of Ai27 and Ai32 mice
A–C, Maximal intensity z-projection of laser-scanning microscopy stack through DRG (A) and spinal cord (B, inset C) of an Ai27 x VGluT3-cre mouse. TdTomato-fluorescence, and correspondingly ChR2 expression, is restricted to a subpopulation of small and large diameter DRG neurons, and the spinal dorsal horn. D, E, Maximal intensity z-projections of laser-scanning microscopy stacks through DRG (D) and spinal cord (E) of an Ai32 x VGluT3-cre mouse. EYFP fluorescence, indicating ChR2 expression, closely resembled tdTomato fluorescence of Ai27 mice. F, Stimulation of the spinal cord with blue light (1 ms, 488 nm, intensity: ~ 1.7 mW), generated with a monochromator (Poly V, TILLvisION), and delivered through the microscope objective (Leica HCX APO, 20x, N.A. 1.0) revealed larger postsynaptic responses in Ai32 x VGluT3-cre mice (black) than in Ai27 x VGluT3-cre mice (grey) mice. Figure illustrates typical examples of photo-stimulated currents. Vertical black bar indicates time point of stimulation. Scale bars in A – E correspond to 50 μm; in F, scale bars indicate 20 ms and 50 pA.
Figure 2
Figure 2. Light-activation of VGluT3+ fibres
A, Scheme depicting experimental approach: EPSCs were evoked with blue light (470 nm) at the level of the DRG in spinal cord slices with dorsal roots and DRGs attached. B, EPSCs were considered of monosynaptic origin, if they 1) did not exhibit failures upon repetitive stimulation (top left and right: sample traces of A- and C-fibres (distinguished by their conduction velocities), respectively), and 2) if they exhibited a low jitter upon stimulation at 0.2 Hz (bottom, see also Table 1). Scale bars indicate 200 pA, and 10 or 5 ms, for top and bottom traces, respectively. C, Control recordings revealed that light-evoked EPSCs recorded in lamina I and II were stable over the entire recording period (mean value +/− C.I.; n = 7; top: sample traces recorded at the three indicated time points). D, Paired-pulse ratios recorded at paired-pulse intervals of either 300 ms (o) or 500 ms (x) were stable over the entire recording period (n = 6).
Figure 3
Figure 3. Pharmacology of VGluT3+ C-fibres in lamina I and II
A, Sample traces of light evoked EPSCs recorded in lamina I before, during (grey shaded area) and after the application of DAMGO (top). Normalized EPSC amplitudes of 11 lamina I neurons plotted over time, grey bar indicates DAMGO application (bottom). B, Box and whisker diagram showing normalized amplitudes of light-evoked C-fibre-mediated EPSCs recorded in lamina I and II in the presence of DAMGO (0.5 μM), SNC80 (50 μM), and Baclofen (1 μM). Number of experiments is given in brackets below each group. C, Sample paired-pulse recording in control conditions and during application of DAMGO (top). Quantification of paired-pulse ratios of lamina I and II recordings during control conditions (black outline), application of test substances (blue) and wash (grey, bottom panel). Number of experiments is given in brackets below each group. Scale bars in A and C indicate 10 ms or 200 pA. Blue rectangles in sample traces indicate flash of 470 nm light.
Figure 4
Figure 4. Pharmacology of VGluT3+ A-fibres in lamina I and II
A, Box and whisker diagram showing normalized EPSC amplitudes of light-evoked A-fibre-mediated EPSCs recorded in lamina I and II in the presence of DAMGO (0.5 μM), SNC80 (50 μM), and Baclofen (1 μM). Number of experiments is given in brackets below each group. B, Quantification of paired-pulse ratios of lamina I and II recordings during control conditions (blue outline), application of test substances (dark blue), and wash (light blue). Number of experiments is given in brackets below each group.
Figure 5
Figure 5. Pharmacology of putative nociceptive C-fibres in lamina I and II
A, Box and whisker diagram showing normalized EPSC amplitudes of electrically evoked C-fibre-mediated EPSCs recorded in lamina I and II of VGluT3−/− mice in the presence of DAMGO (0.5 μM), SNC80 (50 μM), and Baclofen (1 μM). Number of experiments is given in brackets below each group. B, Quantification of paired-pulse ratios of lamina I and II recordings during control conditions (black outline), application of test substances (black), and wash (grey). Number of experiments is given in brackets below each group.
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