Immunohistochemical evidence for synaptic release of glutamate from orexin terminals in the locus coeruleus

Orexin (Orx or hypocretin) is critically important for maintaining wakefulness, since in its absence, narcolepsy with cataplexy occurs. In this role, Orx-containing neurons can exert their influence upon multiple targets through the brain by release of Orx but possibly also by release of other neurotransmitters. Indeed, evidence was previously presented to suggest that Orx terminals could utilize glutamate (Glu) in addition to Orx as a neurotransmitter. Using fluorescence and confocal laser scanning microscopy, we investigated whether Orx varicosities contain the presynaptic markers for synaptic release of Glu or GABA and come into contact with postsynaptic markers for excitatory synapses within the locus coeruleus of the rat brain. We found that a proportion of the Orx+ varicosities were immunostained for the vesicular transporter for Glu, VGluT2. None were immunostained for vesicular glutamate transporter 1 (VGluT1) or VGluT3 or for the vesicular transporter for GABA, vesicular GABA transporter (VGAT). Among the Orx+ varicosities, 4% of all and 28% of large varicosities contained VGluT2. A similar proportion of the large Orx+ varicosities contained synaptophysin (Syp), a presynaptic marker for synaptic vesicles. Orx+ varicosities also contacted elements immunostained for postsynaptic density protein-95 (PSD)-95, a postsynaptic marker for glutamatergic synapses. We thus conclude that synaptic release of Glu occurs from Orx terminals within the locus coeruleus and can thus be important for the engagement of noradrenergic neurons in stimulating and maintaining arousal.