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, 11 (9), e0162969
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A Comparative Study of Three Interneuron Types in the Rat Spinal Cord

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A Comparative Study of Three Interneuron Types in the Rat Spinal Cord

Si Chen et al. PLoS One.

Abstract

Interneurons are involved in the physiological function and the pathomechanism of the spinal cord. Present study aimed to examine and compare the characteristics of Cr+, Calb+ and Parv+ interneurons in morphology and distribution by using immunhistochemical and Western blot techniques. Results showed that 1) Cr-Calb presented a higher co-existence rate than that of Cr-Parv, and both of them were higher in the ventral horn than in the dosal horn; 2) Cr+, Calb+ and Parv+ neurons distributing zonally in the superficial dosal horn were small-sized. Parv+ neuronswere the largest, and Cr+ and Calb+ neurons were higher density among them. In the deep dorsal horn, Parv+ neurons were mainly located in nucleus thoracicus and the remaining scatteredly distributed. Cr+ neuronal size was the largest, and Calb+ neurons were the least among three interneuron types; 3) Cr+, Calb+ and Parv+ neurons of ventral horns displayed polygonal, round and fusiform, and Cr+ and Parv+ neurons were mainly distributed in the deep layer, but Calb+ neurons mainly in the superficial layer. Cr+ neurons were the largest, and distributed more in ventral horns than in dorsal horns; 4) in the dorsal horn of lumbar cords, Calb protein levels was the highest, but Parv protein level in ventral horns was the highest among the three protein types. Present results suggested that the morphological characteristics of three interneuron types imply their physiological function and pathomechanism relevance.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The detection of co-existence relation for Cr with Calb and Parv in the spinal cord.
Layers I-II(A-A″) and ventral horn (B-B″),respectively, showed Cr (green), Calb (red), and Cr-Calb co-existence neurons (yellow). Layers I-II (C-C″) and the ventral horn(D-D″) respectively, showed Cr (green), Parv (red), and Cr-Parv co-existence neurons (yellow).Cr, Calb and Parv are short for Calretinin, Calbindin-D28k, Parvalbumin, and DH and VH are short for the dorsal horn and the ventral horn, respectively. All images are the same magnification.Scale bar: 30μm.
Fig 2
Fig 2. The morphological exploration of interneurons of the spinal gray matter.
Figures (A-C) showed Cr+ neurons in the dorsal horns of cervical (A), thoracic (B) and lumbar cord (C),and A′- C′ showed Cr+ neurons in the ventral horns of cervical (A′), thoracic (B′) and lumbar cord (C′), respectively.Figures (D-F) showed Calb+ neurons in the dorsal horns of cervical (D), thoracic (E) and lumbar cord (F),and Figures (D′- F)showed Calb+ neurons in the ventral horns of cervical (D′), thoracic (E′) and lumbar cord (F′), respectively.Figures G, H and I showed Parv+ neurons in the dorsal horns of cervical (G), thoracic (H) and lumbar cord (I), and the arrows showed the nucleus thoracicus which Parv+ neurons gathered (Fig.H). Figures (G′- I′) showed Parv+ neurons in the ventral horns of cervical (G′), thoracic (H′) and lumbar cord (I′), respectively. Cr, Calb and Parv are short for Calretinin, Calbindin-D28k, Parvalbumin, and CS, TS and LS are short for the cervical, thoracic and lumbar cords, respectively, and DH and VH are short for the dorsal horn and the ventral horn. The figures of dorsal horns are the same magnification. Scale bars: 150μm. The figures of ventral horns are the same magnification. Scale bars: 120μm.
Fig 3
Fig 3. The double-labeling for interneurons with NeuN+ neurons of the spinal gray matter.
The double-labeling for Cr-NeuN (A′-A*), Calb-NeuN (B′-B*) and Parv-NeuN (C′-C*) were showed above. Panels A′- C′, A″-C″ and A*-C* showed the superficial layer and the deep layer of dorsal horn as well as the ventral horn, respectively. Double-labeling neurons(▲) and NeuN+ neurons(△) were obviously observed. DH and VH are short for the dorsal horn and the ventral horn. Scale bar: A′-C′, A″-C″, 40μm; A*-C*, 60μm.
Fig 4
Fig 4. The comparison of Cr+, Calb+ and Parv+ neurons and their protein levels.
The comparison result of the somas size of Cr+, Calb+ and Parv+ neurons in different segments and regions was showed (Histogram A), and histogram B showed the quantitative analyses of their density. HistogramC and D displayed the comparison of expression levels for Cr, Calb and Parv proteins. Values are expressed as group means±SD. P<0.05, comparison between the dorsal horn and the ventral horn;ΔP<0.05, comparison among the cervical, thoracic and lumbar cords; P<0.05, comparison among three interneuron types in the same region of different spinal segments. DH and VH are short for dorsal horn and ventral horn, and CS, TS, LS are short for cervical, thoracic and lumbar cords.

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Grant support

This research was supported by the National Science Foundation of China (No. 81471288 and 31070941) and by the Major State Basic Research Development Program of China (973 Program, No. 2010CB530004).
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