Ray Perrins

Optoactivation of Locus Ceruleus Neurons Evokes Bidirectional Changes in Thermal Nociception in Rats

Pontospinal noradrenergic neurons are thought to form part of a descending endogenous analgesic system that exerts inhibitory influences on spinal nociception. Using optogenetic targeting, we tested the hypothesis that excitation of the locus ceruleus (LC) is antinociceptive. We transduced rat LC neurons by direct injection of a lentiviral vector expressing channelrhodopsin2 under the control of the PRS promoter. Subsequent optoactivation of the LC evoked repeatable, robust, antinociceptive (+4.7°C ± 1.0, p < 0.0001) or pronociceptive (−4.4°C ± 0.7, p < 0.0001) changes in hindpaw thermal withdrawal thresholds. Post hoc anatomical characterization of the distribution of transduced somata referenced against the position of the optical fiber and subsequent further functional analysis showed that antinociceptive actions were evoked from a distinct, ventral subpopulation of LC neurons. Therefore, the LC is capable of exerting potent, discrete, bidirectional influences on thermal nociception that are produced by specific subpopulations of noradrenergic neurons. This reflects an underlying functional heterogeneity of the influence of the LC on the processing of nociceptive information.

Retrograde optogenetic characterization of the pontospinal module of the locus coeruleus with a canine adenoviral vector.

Noradrenergic neurons of the brainstem extend projections throughout the neuraxis to modulate a wide range of processes including attention, arousal, autonomic control and sensory processing. A spinal projection from the locus coeruleus (LC) is thought to regulate nociceptive processing. To characterize and selectively manipulate the pontospinal noradrenergic neurons in rats, we implemented a retrograde targeting strategy using a canine adenoviral vector to express channelrhodopsin2 (CAV2-PRS-ChR2-mCherry). LC microinjection of CAV2-PRS-ChR2-mCherry produced selective, stable, transduction of noradrenergic neurons allowing reliable opto-activation in vitro. The ChR2-transduced LC neurons were opto-identifiable in vivo and functional control was demonstrated for >6 months by evoked sleep-wake transitions. Spinal injection of CAV2-PRS-ChR2-mCherry retrogradely transduced pontine noradrenergic neurons, predominantly in the LC but also in A5 and A7. A pontospinal LC (ps:LC) module was identifiable, with somata located more ventrally within the nucleus and with a discrete subset of projection targets. These ps:LC neurons had distinct electrophysiological properties with shorter action potentials and smaller afterhyperpolarizations compared to neurons located in the core of the LC. In vivo recordings of ps:LC neurons showed a lower spontaneous firing frequency than those in the core and they were all excited by noxious stimuli. Using this CAV2-based approach we have demonstrated the ability to retrogradely target, characterise and optogenetically manipulate a central noradrenergic circuit and show that the ps:LC module forms a discrete unit. This article is part of a Special Issue entitled SI: Noradrenergic System.

The Roles of Central Cholinergic and Electrical Synapses made by Spinal Motoneurones in Xenopus Embryos

The hatchling tadpole of Xenopus laevis has been used successfully as a simple model system in which to study the spinal neural circuits that control locomotion (Roberts, 1990). It is therefore an ideal simple system in which to study possible functions for central synapses made by spinal motoneurones. Such synapses are known to exist, being made on premotor interneurones (for example Renshaw cells) as well as on other motoneurones, in a variety of vertebrates from fish to adult mammals. However, very little is known of their role during locomotion, which is probably in part due to the complexity of the systems in which they have been found (e.g. Noga, Shefchyk, Jamal & Jordan, 1987). We have recently shown that motoneurones are integral parts of the simple Xenopus circuit which produces the drive during swimming, rather than just being output devices to the muscles, as was previously supposed. By making simultaneous intracellular recordings from pairs of spinal motoneurones we have directly demonstrated the presence of cholinergic synapses between motoneurones, the activation of which gave rise to fast EPSPs which were blocked by nicotinic antagonists (Perrins & Roberts, 1995; Figure 1A). A more local electrical coupling between motoneurones was also sometimes observed (Perrins & Roberts, 1995). Both these types of interaction were only found between motoneurones on the same side of the spinal cord. For these pairs about 50% were chemically coupled and 10% electrically coupled. Since motoneurones on the same side of the spinal cord spike roughly in phase, excitation from these two types of connection would be expected to occur on-cycle during swimming. We therefore investigated the composition of the fast on-cycle excitation which underlies spiking activity in spinal neurones, which had previously been thought to be due entirely to kainate/AMPA receptor activation by an excitatory amino acid. By the local application of the nicotinic antagonists dihydro-β-erythroidine and d-tubocurarine (both 10 µM), we showed that 20% of the on-cycle excitation received by motoneurones during swimming is provided by cholinergic EPSPs. The local application of 100 µM Cd2+, which blocks all chemical neurotransmission, demonstrated that 50% of the fast on-cycle excitation was due to electrotonic coupling with other, spiking neurones. The anatomical simplicity of the Xenopus spinal cord (which contains just eight morphological classes of neurone; Roberts & Clarke, 1982), along with the results of the paired recording study mean that the most likely source for these two types of excitation is other motoneurones. The local application of CNQX showed that the remaining 30% of the fast excitation was due to kainate/AMPA receptor activation.