Enkephalinergic (ENKergic) neurons have been proposed to play crucial roles in pain modulation in the trigeminal subnucleus caudalis (Vc). To assist an advance in the research of ENKergic neurons, here we used preproenkephalin-green fluorescent protein (PPE-GFP) transgenic mice, in which all ENKergic neurons were fluorescent. We first performed fluorescent in situ hybridization combined with immunofluorescent histochemistry to confirm the specificity of this transgenic mouse and its advantages in showing ENKergic neurons in the Vc. Then based on this useful transgenic mouse, we examined the phenotypic diversity of PPE-GFP neurons by immunostaining for several markers that characterize ENKergic neuron subtypes. About 25.9±1.9% of GFP-positive neurons were regarded as immunoreactive for glutamic acid decarboxylase (GAD)(67) mRNA and 14.7±1.4% of GFP-positive neurons were positive for γ-aminobutyric acid. The proportions of calbindin-, calretinin-positive cells among the ENKergic neurons were 8.4±1.2% and 7.3±1.7%, respectively. Only 1.1±0.1% of GFP-positive neurons colocalized with parvalbumin and no GFP-positive neurons were found to co-express neuronal nitric oxide synthase. We then injected retrograde tracer into the thalamic regions and observed that a small number of ENKergic neurons in the Vc were retrogradely labeled with the tracer. The present results provide a detailed morphological evidence of the neurochemical features of ENKergic neurons. These results have broad implications for understanding the functional roles of ENKergic neurotransmission in the Vc.
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