The Distribution And Developmental Patterns Of Enkephalinergic Neurons In The Retina Of Ppe-gfp Transgenic Mice

The vertebrate retina is a complex multi-layered sensory tissue. Enkephalin(ENK) is an important neurotransmitter in the nervous system, which belongs to akind of endogenous opioid peptides and plays a variety of physiological functions,such as the neuroendocrine regulation, analgesia, nerve immune response and soon. At present, the physiological effects of ENK by acting on the correspondingreceptors in the retina has become more clear. It has also been confirmed thatENK has a close relationship with retinal photoreceptor. However, the detaileddistribution of ENKergic neurons in the retina has not been reported. Moreover,calcium-binding protein D28k (CB), calretinin (CR), parvalbumin (PV), andnitric oxide synthase (NOS) are expressed in human and mammalian retina.Previous studies have shown that CB, CR, PV, and NOS have significant effectson normal growth and development of retinal neurons. However, whether or notthese molecules locate in the ENKergic neurons in the retina has not beenobserved, which is very important for understanding the role of ENKergicneurons in the regulation of light-sensitive pathway of retina. At the same time,previous results only observed expressions of ENKergic neurons in the retinafrom late embryonic stages to the early postnatal stages. While, the exact developmental and distribution patterns of ENKergic neuron in the retina havenot been elucidated. Because of the difficulties in the staining of ENKergicneurons, preproenkephalin (preproenkephalin, PPE)-GFP transgenic mice wereused to mark ENKergic neurons. In the PPE-GFP transgenic mice, GFPexpression is regulated by the PPE promoter, so that all the ENKergic neuronscan express GFP.The present studyis divided into two parts:1. Distribution of ENKergic neurons in the retina of adult PPE-GFPtransgenic mice.The present study was designed to observe the distribution and coexistence ofENKergic neurons and CB, CR, PV and NOS in the retina of the PPE-GFPtransgenic mice. Immunohistochemistry and double immunofluorescencelabeling methods were used. GFP-positive cells were mainly distributed in theinner part of the inner nuclear layer (INL) and some of them were also located inthe ganglion cell layer (GCL). None of the GFP-positive cells wereGFAP-positive astrocytes, while all of the GFP immunoreactivities wereNSE-positive. We observed the coexistence of GFP with CB,CR and PV. Theproportion of GFP/CB double-labeled neurons accounted for 8.65% and 5.84% oftotal GFP- and CB-positive neurons respectively. The proportion of GFP/CRdouble-labeled neurons accounted for 18.18% and 14.28% of total GFP- andCR-positive neurons respectively. GFP/CR double-labeled neurons weredistributed in the INL. The proportion of GFP/PV double-labeled accounted for68.75% and 91.67% of total GFP- and PV-positive neurons respectively. GFP/PVdouble-labeled neurons were primarily distributed in the INL and some werelocated in the GCL. No GFP and NOS double-labeled neurons were observed.The present results indicate the layer specific expression pattern of GFP-positiveENKergic neurons in the retina and also provided morphological evidence forfurther functional studies.2. Developmental pattern of ENKergic neurons of the retina.In this study, PPE-GFP transgenic mice was used. Andimmunohistochemistry and immunofluorescence double staining were performed to observe the distribution of ENKergic neurons in the retina at different timepoints, including 15 days of embryonic development (E15), E16, E17 and E18, aswell as 1 day after birth (P1), P3 and P7. The results show that ENKergic neuronsfirst appeared in the neuroblastic layer at E15. Then at E16 ENKergic neuronsincrease in the number and mainly locate in the ganglion cell layer and innerlayer of neuroblastic layer. At E17, ENKergic neurons are mainly distributed onthe inside part of the neuroblastic layer, some of which stretch multiple processesinto the inner plexiform layer. At E18, ENKergic neurons have a similardistribution pattern as those at E17. At P1, ENKergic neurons can be found at theganglion cell layer and inner part of neuroblastic layer. At P3, some of theENKergic neurons extend long processes to the inner plexiform layer. At P7,most of the ENKergic neurons are in the inner part of the nuclear layer, whilesome ENKergic neurons are distributed in the ganglion cell layer. The presentresults indicate that the ENKergic neurons appear in neuroblastic layer at the E15,then shift gradually inward, concentrate in inner nuclear layer at P7, finallyconserve this distribution pattern from then on. These results provide animportant clue for understanding the role of ENKergic neurons in thedevelopment of retina.