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

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. We now 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 via 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 capable approach for the subtype-selective investigation of primary afferent nerve fibres. Overall, the pharmacological accessibility of different subtypes of sensory fibres considerably overlaps, indicating that MOR, DOR and GABABR expression is not substantially segregated between heat and mechanosensitive fibres.

Semi-intact ex vivo approach to investigate spinal somatosensory circuits

“…In particular, the typical criteria used to establish mono-synaptic connectivity using electrical simulation (absence of failure upon stimulation at 20 Hz for Aδ fibers and 2 Hz for C fibers [Nakatsuka et al, 1999]) is not applicable to optogenetic stimulation, since light-evoked action potentials have longer latency and show Cre latency jitter than those evoked by electric stimulation (Figure 5—figure supplement 1F). For this reason, we and others used a less stringent criterion (e.g., 0.1 Hz optogenetic stimulation) to differentiate between mono- and polysynaptic input in the dorsal horn (Honsek et al, 2015; Wang and Zylka, 2009; Cui et al, 2016). Verification of monosynaptic connections will likely require additional tests involving, for instance, the use of tetrodotoxin together with the potassium channel antagonist 4-aminopyridine (Nelson et al, 2014; Petreanu et al, 2009).…”

Usefulness of knockout mice to clarify the role of the opioid system in chronic pain

“…These approaches have also allowed the dissection of specific roles of serotonergic neurons of the rostral ventral medulla (Cai et al, ) and subpopulations of noradrenergic neurons in the locus coeruleus (Hickey et al, ) in pain control. An optogenetic approach has also been used to dissect the specific contribution of opioid and GABA receptors in the pre‐synaptic modulation of the nociceptive information transmitted to spinal neurons (Honsek et al, ). Interestingly, MOP pre‐synaptic activation mainly inhibited C‐fibres innervating lamina I spinal cord neurons, whereas activation of pre‐synaptic DOP did not modify these responses.…”

Beyond the brain: Optogenetic control in the spinal cord and peripheral nervous system

“…Examples include in vitro slice electrophysiology experiments that explored the role of dorsal horn parvalbumin interneurons in GABAergic inhibition (29), and the con-sequences of glycinergic interneuron ablation through recording changes in the characteristics of optogenetically evoked inhibitory synaptic transmission (21). In addition, some in vitro studies involved optical stimulation of central terminals of primary afferents (22,28,30) while recording from spinal neurons. These experiments have increased our knowledge regarding the connectivity and function of peripheral and spinal sensory neurons under both normal and pathological conditions.…”