Immunohistochemical evidence for synaptic release of GABA from melanin-concentrating hormone containing varicosities in the locus coeruleus

Melanin-concentrating hormone (MCH) is synthesized by neurons located in the hypothalamus and projecting to widespread regions of the brain, including the locus coeruleus (LC), through which MCH could modulate sleep-wake states. Yet MCH does not appear to exert direct postsynaptic effects on target neurons, including the noradrenergic LC neurons. Previous studies using in situ hybridization showed that MCH neurons synthesize glutamic acid decarboxylase (GAD) and could thus utilize GABA as a neurotransmitter. To determine whether MCH varicosities can release GABA, we examined by fluorescent microscopy in the LC, whether their terminals also contain the vesicular transporter for GABA (VGAT). In dual-immunostained sections, we found that approximately 6% of MCH varicosities was immunopositive for VGAT and a similar proportion for synaptophysin, the presynaptic marker for small synaptic vesicles, whereas <1% was positive for the vesicular glutamate transporter (VGluT2). Moreover, of the MCH varicosities, ∼5% abutted puncta that were immunostained for gephyrin, the postsynaptic marker for GABAergic synapses. In triple-immunostained sections viewed with confocal laser scanning microscopy, we established that MCH varicosities that also contained VGAT or abutted upon gephyrin puncta contacted the tyrosine hydroxylase-immunostained neurons of the LC. Our results suggest that although MCH neurons can influence noradrenergic LC neurons through paracrine release and indirect effects of their peptide, they can also do so through synaptic release and direct postsynaptic effects of GABA and thus serve to inhibit the LC neurons during sleep, when they are silent, and the MCH neurons discharge.

► MCH varicosities in the locus coeruleus (LC) contain the vesicular GABA transporter (VGAT).
► MCH varicosities in the LC abut gephyrin, the postsynaptic marker for GABAergic synapses.
► Thus MCH neurons can directly inhibit noradrenergic LC neurons through GABAergic synapses.