The locus coeruleus (LC) projects throughout the brain and spinal cord and is the major source of central noradrenaline. It remains unclear whether the LC acts functionally as a single global effector or as discrete modules. Specifically, while spinal-projections from LC neurons can exert analgesic actions, it is not known whether they can act independently of ascending LC projections. Using viral vectors taken up at axon terminals, we expressed chemogenetic actuators selectively in LC neurons with spinal (LC^:SC) or prefrontal cortex (LC^:PFC) projections. Activation of the LC^:SC module produced robust, lateralised anti-nociception while activation of LC^:PFC produced aversion. In a neuropathic pain model, LC^:SC activation reduced hind-limb sensitisation and induced conditioned place preference. By contrast, activation of LC^:PFC exacerbated spontaneous pain, produced aversion and increased anxiety-like behaviour. This independent, contrasting modulation of pain-related behaviours mediated by distinct noradrenergic neuronal populations provides evidence for a modular functional organisation of the LC.

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 hocanatomical 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.

A neuronal membrane protein, neuropathy target esterase (NTE), reacts with those organophosphates that initiate a syndrome of axonal degeneration. NTE has homologues inDrosophila and yeast and is detected in vitroby assays with a non-physiological ester substrate, phenyl valerate. We report that NEST, the recombinant esterase domain of NTE (residues 727–1216) purified from bacterial lysates, can catalyze hydrolysis of several naturally occurring membrane-associated lipids. The active site regions of NEST and calcium-independent phospholipase A₂(iPLA₂) share sequence similarity, and the phenyl valerate hydrolase activity of NEST is inhibited by low concentrations of iPLA₂ inhibitors. However, on incubation with NEST, fatty acid was liberated only extremely slowly from the sn-2 position of phospholipids (V _max ∼0.01 μmol/min/mg and K _m ∼0.4 mm for 1-palmitoyl, 2-oleoylphosphatidylcholine). Comparison of the NEST-mediated generation of ¹⁴C-labeled products from two differentially labeled ¹⁴C-phospholipid substrates suggested that a rate-limiting sn-2 cleavage was followed very rapidly by hydrolysis of the resulting lysophospholipid. Among the various naturally occurring lipids tested with NEST, lysophospholipids were by far the most avidly hydrolyzed substrates (V _max ∼20 μmol/min/mg andK _m ∼0.05 mm for 1-palmitoyl-lysophosphatidylcholine). NEST also catalyzed the hydrolysis of monoacylglycerols, preferring the 1-acyl to the 2-acyl isomer (V _max ∼1 μmol/min/mg andK _m ∼0.4 mm for 1-palmitoylglycerol). NEST did not catalyze hydrolysis of di- or triacylglycerols or fatty acid amides. This demonstration that membrane lipids are its putative cellular substrates raises the possibility that NTE and its homologues may be involved in intracellular membrane trafficking.

The N-terminal amino acid sequences of proteolytic fragments of neuropathy target esterase (NTE), covalently labelled on its active-site serine by a biotinylated organophosphorus ester, were determined and used to deduce the location of this serine residue and to initiate cloning of its cDNA. A putative NTE clone, isolated from a human foetal brain cDNA library, encoded a 1327 residue polypeptide with no homology to any known serine esterases or proteases. The active-site serine of NTE (Ser-966) lay in the centre of a predicted hydrophobic helix within a 200-amino-acid C-terminal domain with marked similarity to conceptual proteins in bacteria, yeast and nematodes; these proteins may comprise a novel family of potential serine hydrolases. The Swiss Cheese protein which, when mutated, leads to widespread cell death in Drosophilabrain [Kretzschmar, Hasan, Sharma, Heisenberg and Benzer (1997) J. Neurosci. 17, 7425–7432], was strikingly homologous to NTE, suggesting that genetically altered NTE may be involved in human neurodegenerative disease.

Age-dependent neurodegeneration resulting from widespread apoptosis of neurons and glia characterize the Drosophila Swiss Cheese (SWS) mutant. Neuropathy target esterase (NTE), the vertebrate homologue of SWS, reacts with organophosphates which initiate a syndrome of axonal degeneration. NTE is expressed in neurons and a variety of nonneuronal cell types in adults and fetal mice. To investigate the physiological functions of NTE, we inactivated its gene by targeted mutagenesis in embryonic stem cells. Heterozygous NTE^+/− mice displayed a 50% reduction in NTE activity but underwent normal organ development. Complete inactivation of the NTE gene resulted in embryonic lethality, which became evident after gastrulation at embryonic day 9 postcoitum (E9). As early as E7.5, mutant embryos revealed growth retardation which did not reflect impaired cell proliferation but rather resulted from failed placental development; as a consequence, massive apoptosis within the developing embryo preceded its resorption. Histological analysis indicated that NTE is essential for the formation of the labyrinth layer and survival and differentiation of secondary giant cells. Additionally, impairment of vasculogenesis in the yolk sacs and embryos of null mutant conceptuses suggested that NTE is also required for normal blood vessel development.

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.