We have previously shown that a subset of axon terminals in the locus coeruleus (LC) containing methionine 5 -enkephalin (ENK) forms type I (asymmetric-type) synaptic specializations that are characteristic of excitatory-type transmitters. In addition, we previously provided ultrastructural evidence showing that ENK is colocalized with glutamate using a combination of preand postembedding immunohistochemistry. To examine cellular substrates for interactions between glutamate and other endogenous opioid peptides in the LC, we examined the localization of the vesicular glutamate transporter 1 (VGLUT1), a transporter protein involved in the accumulation of the transmitter glutamate into synaptic vesicles, with either ENK or preprodynorphin (ppDYN). Dual-immunofluorescence and electron microscopy showed prominent coexistence of VGLUT1 and ENK in varicose processes of the LC, confirming our previous report using postembedding immunolabeling for glutamate. Likewise, VGLUT1 and ppDYN were identified in common varicose processes in the LC using confocal fluorescence microscopy. Immunoelectron microscopy using gold-silver labeling for VGLUT1 and peroxidase labeling for ppDYN established that this endogenous opioid peptide also colocalizes with glutamate transporters. The majority of these formed asymmetric-type synapses. Taken together, these results demonstrate that excitatory LC afferents are enriched with endogenous opioid peptides and are positioned to modulate LC neuronal activity dually. © 2005 Wiley-Liss, Inc.
Key words: norepinephrine; excitatory amino acid; dynorphin; encephalin; glutamateThe nucleus locus coeruleus (LC), located on the floor of the fourth ventricle in the rostral pons, provides extensive noradrenergic projections throughout the neuraxis. The collection and processing of salient sensory information having effects on memory, attention and arousal, cardiovascular regulation, antinociception, regulation of anxiety states and stress response have been associated with these neurons (Berridge and Waterhouse, 2003). The LC receives a major glutamatergic input from the medullary nucleus paragigantocellularis (PGi) (Ennis and AstonJones, 1988; Aston-Jones et al., 1991). These afferents also express endogenous opioid peptides, including methionine 5 -enkephalin (ENK) (Drolet et al., 1992).Modulation of postsynaptic glutamate receptors activates LC neurons (Oleskevich et al., 1993;Van Bockstaele et al., 2000), while the activity of LC neurons is inhibited by opioid peptides via activation of an inward rectifying