To gain better insight into the ontogenic function of enkephalin (ENK) in the spinal cord, it is necessary to have a clear picture of the developing pattern of the ENKergic neurons. To address this question, we used transgenic mice which reveal ENKergic neurons easily by expressing enhanced green fluorescent protein (GFP) under the specific transcriptional control of the preproenkephalin (PPE) gene. GFP-positive neurons first appeared at embryonic day (E) 11.5 in the ventromedial part of the cervical ventral gray matter. At E13, they were mainly present in the intermediate zone. Thereafter, GFP-positive neurons increased progressively in number and extended from ventral to dorsal regions. Quantitative analysis showed that GFP-positive neurons peaked in number at postnatal day (P) 7 at the cervical level. The number of GFP-positive neurons reached a peak at P3 at the lumbar level. At P21, the distribution pattern of GFP immunoreactivity was similar to that in the adult spinal cord. Double-labeling results showed that about one-third of the total γ-aminobutyric acid cell population colocalized with GFP: 34.9 ± 3.5% at E16 and 32.4 ± 3.7% at P3. Double-labeled neurons accounted for nearly half of the GFP-positive neurons: 42.4 ± 2.4% at E16 and 44.1 ± 2.9% at P3. Taken together, the present results suggest that ENKergic neurons develop according to a rostrocaudal and ventrodorsal gradient. These results have broad implications for understanding the functional roles of ENKergic neurotransmission in the developing spinal cord.
- Fluorescent in situ hybridization histochemistry
- Immunofluorescence labeling
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