Background & Aims
Serotonergic modulation of pain transmission in the spinal cord is exerted by recruiting several receptors , including 5-HT7 (5-HT7Rs, [1, 2]). While the relevance of 5-HT7Rs in mediating acute pain is still under debate, activation of these receptors in animal models of inflammatory and neuropathic pain seems to produce a prevalent antinociceptive effect [3]. As shown by a recent study, 5-HT7R activation in mouse spinal cord slices potentiates both excitatory and inhibitory transmission with a more powerful effect on GABA- and glycinergic transmission [4]. It is still not known, however, which are the effects of 5-HT7Rs on specific dorsal horn neuron populations. For this purpose, we have utilized a transgenic mouse model expressing channelrhodopsin 2 (ChR2) specifically in inhibitory interneurons, where the transporter VGAT is present. This approach has allowed us to test the effects of 5-HT7R activation on inhibitory synaptic transmission in identified dorsal horn neurons.
Methods
Experiments were performed on ChR2-VGAT-eYFP mice (P18-P28), purchased from The Jackson Laboratory (stock#014548). Spinal cord slices were obtained as described previously [4], and patch-clamp recordings were performed from lamina II neurons. Putatively inhibitory lamina II interneurons were identified by the presence of the ChR2-mediated current, recorded at -70 mV and evoked by a 500 ms pulse of 470 nm blue light. Current clamp recordings were performed by applying 500 ms current steps at -60 mV (close to the neuron resting potential) and at -80 mV (to unmask the presence of the potassium IA current and the delayed firing pattern). Optogenetically evoked inhibitory postsynaptic currents (oIPSCs) were generated by applying 10-20 ms blue light pulses every 10 seconds, and recorded in voltage clamp at -30 mV. The selective 5-HT7 agonist LP-211 [5] was bath-applied at the concentration of 1 microMol.
Results
Putatively inhibitory neurons expressing the transporter VGAT were identified by the presence of a ChR2-mediated current showing the characteristic plateau phase. All inhibitory interneurons (n= 11) fired action potentials with a tonic pattern at -60 mV. At -80 mV, 63.6% of inhibitory neurons fired tonically and 36.4% had a delayed pattern. Excitatory interneurons (n=14) showed a prevalent delayed pattern at both -60 mV (57.2% delayed, 21.4% tonic and 21.4% phasic) and -80 mV (85.8% delayed, 7.1% phasic, 7.1% tonic). Optogenetic stimulation evoked oIPSCs in 26 out of 28 neurons. The currents had mean amplitudes of 115.5±29.2 pA and 208.7±44.4 pA in inhibitory and excitatory neurons, respectively. Application of the 5-HT7 agonist LP-211 induced a significant potentiation of oIPSC amplitudes in 8 out 15 neurons tested (3/6 inhibitory and 5/9 excitatory neurons) (mean oIPSC amplitude in control: 197±55.3 pA; LP-211: 267.51±64.0 pA, paired t-test, p<0.001).
Conclusions
The transgenic model of ChR2-VGAT-eYFP mice is useful for identifying the neuron types in characterizing the dorsal horn circuits [6]. The firing patterns observed in this study are consistent with data reported previously [7], with inhibitory interneurons firing tonically and excitatory neurons mostly showing a delayed pattern. Inhibitory postsynaptic responses, elicited by optogenetic stimulation, were observed in the vast majority of lamina II interneurons, both excitatory and inhibitory. Activation of 5-HT7 by the selective agonist LP-211 effectively increased the inhibitory postsynaptic currents evoked by light stimulation in about half of the tested dorsal horn neurons.
References
1.Bardoni, R. (2019). Serotonergic Modulation of Nociceptive Circuits in Spinal Cord Dorsal Horn. Curr Neuropharmacol 17, 1133–1145
2.Heijmans, L. et al. (2021) A systematic review on descending serotonergic projections and modulation of spinal nociception in chronic neuropathic pain and after spinal cord stimulation. Mol. Pain 17:17448069211043965
3.Bardoni R (2023) Serotonergic 5-HT7 receptors as modulators of the nociceptive system. Curr Neuropharmacol 21, 1548-1557.
4.Comitato, A. et al. (2022) 5-HT7 Receptors Regulate Excitatory- Inhibitory Balance in Mouse Spinal Cord Dorsal Horn. Front Mol Neurosci 15:946159.
5.Leopoldo, M. et al. (2011). Serotonin 5-HT7 receptor agents: structure-activity relationships and potential therapeutic applications in central nervous system disorders. Pharmacol. Ther. 129, 120–148.
6.Adamek, P., et al. (2022) Dual PI3K?/? Inhibitor Duvelisib Prevents Development of Neuropathic Pain in Model of Paclitaxel-Induced Peripheral Neuropathy. J Neurosci 42:1864-1881.
7.Yasaka, T. et al. (2010). Populations of inhibitory and excitatory interneurons in lamina II of the adult rat spinal dorsal horn revealed by a combined electrophysiological and anatomical approach. Pain 151, 475–488.