Neurochemical Features And Developmental Patterns Of γ-aminobutyric Acid (GABA)Ergic And Enkephalin (ENK)Ergic Neurons In The Spinal Cord

γ-aminobutyric acid (GABA) ergic neurons and enkephalin (ENK) ergic neurons are important interneurons in the spinal dorsal horn (SDH). These interneurons together with primary afferent fibers, projection neurons, decending terminals and other types of interneurons in the superficial layers comprise the complex nociceptive circuitry, which play important roles in modulating transmission of nociceptive information. The assembly of the complex neuronal circuit depends on the generation of functionally distinct types of dorsal horn neurons during development. Any disturbance of the development of these elements will strongly affect the formation of nociceptive circuitry in the SDH. So it is necessary to examine them during the development course. However, the neurochemical features, temperal and spatial distribution, origin, migration and transcriptional regulation of GABAergic and ENKergic neurons in the spinal cord remain largely unknown.In the present study, we used glutamic acid decarboxylase (GAD)67 -green fluorescence protein (GFP) knock-in mouse to characterize GABAergic neurons and preproenkephalin (PPE)-GFP transegenic mouse to characterize ENKergic neurons. We performed the following three parts of the experiment by using morphological and molecular biological methods.1. The neurochemical features of GABAergic and ENKergic neurons in the SDH.By using double immunofluorescence labeling, fluorescent in situ hybridization combined with immunofluorescence labeling, single-cell reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR methods, we confirmed the validity of GAD67-GFP knock-in mouse and PPE-GFP transgenic mouse. And then we observed the neurochemical features, the expression of 5-hydroxytryptamine (5-HT)3A receptor and the co-existence of GABAergic and ENKergic neurons in the SDH. The results were as followings: (1) Double immunofluorescence labeling results showed that all the GFP-positive neurons were co-localized with neuronal nuclei protein (NeuN). More than 98% of the GFP-positive neurons were positive for GAD67 and GABA. The double labeled staining for GFP and in situ hybridization for GAD67 mRNA showed that GFP immunoreactive neurons expressed GAD67 mRNA in the spinal cord. GFP-positive neurons constituted 31.51%, 33.34%, and 44.70% of the NeuN-positive neurons in laminae I, II, and III, respectively. The expression pattern of GFP-positive neurons in the PPE-GFP transgenic mouse paralleled with that of PPE mRNA expression in different brain regions. All the GFP-positive neurons in the spinal cord of the PPE-GFP transgenic mouse co-localized with NeuN and PPE mRNA. ENKergic neurons constituted 16.95%, 40.68%, and 12.45% of the NeuN-positive neurons in laminae I, II, and III, respectively. Thus, the above double-lebeling study convinced us of the usefulness of the mice for the studies of GABAergic and ENKergic neurons in the spinal cord. (2) The proportions of calretinin (CR)-, parvalbumin (PV)- and calbinding DK28 (CB)-positive cells among GABAergic neurons in the SDH were 58.38%, 19.63%, and 2.81%, respectively. The proportions of CR-, PV- and CB-positive cells among ENKergic neurons in the SDH were 61.15%, 5.05% and 24.74%, respectively. About 12.44% of ENKergic neurons in the SDH were immunoreactive for nitric oxide synthase (NOS). We found that 20.61% of ENKergic neurons in the SDH expressed VGLUT1 and 21.21% of ENKergic neurons were positive for VGLUT2. Quantitative analysis indicated that more than 44.41% of GABAergic neurons showed signals for PPE mRNA in the SDH. While 53.93% of PPE mRNA-expressing neurons were immunoreactive for GABA. Single-cell RT-PCR results showed that 5-HT3A receptor subunit was detected in 28.07% of GABAergic neurons and 22.58% of ENKergic neurons. These detailed results have broad implications for understanding the functional roles of GABAergic and ENKergic neurotransmission in the SDH.2. The developmental pattern and transcriptional regulation of GABAergic neurons in the spinal cord.By using GAD67-GFP knock-in mouse and early B factor 2 (Ebf2) knock out mouse, we observed the temporal and spatial distribution, origin, migration and transcriptional regulation of GABAergic neurons in the spinal cord. Double immunofluorescent labeling, 5-bromo-2-deoxyuridine (BrdU) labeling, spinal cord slice culture, Time-lapse observation and single-cell RT-PCR were used in this part. The results were as followings:(1) GFP-positive GABAergic neurons appeared at embryonic day (E) 11.5 in the ventral region of the spinal cord and became abundant in the whole future gray matter at E12. Thereafter, GFP-positive neurons increased progressively in number and extended from ventral to dorsal regions. The intensity of GFP-positive neurons in the dorsal horn peaked at E17. At postnatal day 14, the distribution pattern of GFP immunoreactivity was similar to that of GABAergic neurons in adult spinal cord. Taken together, the present results suggest that the GFP immunoreactivity, and thus the expression of GABA, undergoes a ventral to dorsal shift in the spinal cord during development.(2) Birthdating studies revealed that GABAergic neurogenesis were present since E10.5. Then the generation of GABAergic neurons significantly increased, reaching a peak at E11.5. The two waves for the co-localization of GABA and BrdU in the spinal cord were seen at E11.5 and E13.5. The vast majority of GABAergic neurons were generated before E14.5. Then, GABA-positive neuron generation decreased drastically. The birthdates of GABAergic neurons in each lamina were different.(3) Both in vivo and in vitro results indicated that a small but significant fraction of GABAergic neurons in the spinal mantle layer were double-labeled with cell-cycle markers Ki-67, BrdU and phosphorylated histone H3 (P-H3). These double-labeled neurons characterized by cell-cycle markers were proliferative GABAergic nneurons which might contribute to the production of spinal GABAergic neurons at late embryonic stages.(4) Time-lapse observation results indicated that after production, GABAergic neurons migrate to the spinal mantle layer in a radial manner and then migrate to the final location. BrdU labeling results showed that GABAergic neurons born at E10.5 migrate ventrally, and do not contribute to the formation of the superficial layer of the SDH. GABAergic neurons born at E11.5 migrate dorsally and contribute to the formation of the superficial layer of the SDH.(5) The expression of Pax2 in the SDH of the Ebf2 knock out mouse decreased compared with that in the wild type mouse. While there was no change in the expression of Lmx1b, Drg11 and Tlx3. These results suggested that Ebf2 was required for the development of GABAergic neurons in the spinal cord.3. The developmental pattern of ENKergic neurons in the spinal cord.By using PPE-GFP transgenic mouse and double immunofluorescent labeling method, we observed the temperal and spatial distribution and neurochemical characteristics of ENKergic neurons during the spinal cord development.(1) GFP-positive ENKergic neurons appeared at E11.5 in the ventral region of the spinal cord. At E13.5, GFP-positive neurons were mainly present in the intermediate zone. No matter what level was considered, the first labeled GFP-positive cells were observed in the dorsal gray matter at E14. Thereafter,GFP positive neurons increased progressively in number and extended from ventra1 to dorsa1 regions. After birth, GFP-positive neurons were mainly restricted to the dorsal gray matter and also decreased in the staining intensity. At postnatal day 21, the distribution pattern of GFP immunoreactivity was similar to that of ENKergic neurons in the adult spinal cord. Taken together, the present results suggest that ENKergic neurons develop according to a rostro-caudal and ventro-dorsal gradient.(2) Double labeling results revealed a significant population of neurons expressing both GABA and ENK. Interestingly, this proportion remained stable during the course of development. The proportions of double-labeled neurons among ENKergic neurons were 43.35% at E16, 45.02% at P3. CB, CR and PV showed a dynamic pattern of co-localization with ENK in neurons of the spinal cord throughout development. The transient expression of calcium-binding proteins in ENKergic neurons might be related to the critical period of development. (3) The proportions of Pax2 among ENKergic neurons in the SDH at E15.5 and E17.5 were 65.17% and 57.74%, respectively. At P3, the GFP/Pax2 double-labeled neurons were primarily located in the superficial layers of the SDH. We also observed a portion of ENKergic neurons co-expressed Lmx1b at E15 (3.48%) or at E18 (4.50%). Double-labeled neurons were mainly observed in laminae I. The above results provide detailed morphological evidence for the regulation of ENKergic neuron development.In summary, from the above results we can draw the following conclusions. (1) We examined the expression of neurochemical substances, 5-HT receptor subtype in the GABAergic and ENKergic neurons in the spinal cord. Such detailed information will provide morphological evidence for the neurochemical characteristics of GABAergic and ENKergic neurons in the SDH. (2) Our results showed the dynamic expression pattern of GABAergic and ENKergic neurons and the migration of GABAergic neurons in the spinal cord. (3) The present study revealed the birthdating of GABAergic neurons in the spinal cord. We confirmed the presence of GABAergic neuron progenitor in the spinal mantle layer at late embryonic stages. These GABAergic neuron progenitors might be another source of GABAergic neurons of the spinal cord. (4) Our results provide evidence that Ebf2 was required for the GABAergic neuron development in the spinal cord.GABAergic and ENKergic neurons are important elements of the neuronal circuitry in the SDH and play crucial roles in the modulation of nociception. Therefore, exploring the neurochemical features, the ontogeny, origin, migration and transcriptional regulation of GABAergic and ENKergic neurons will far improve our understanding of the functional relevance of these interneurons. Most importantly, it will helpful to comprehend the mechanism on the neural plasticity under pathological conditions. The identification of neurochemical features and developmental patterns of these interneurons will aid in the design of new strategies for spinal cord injury or some developmental disorders.