Phenotypic diversity and expression of GABAergic inhibitory interneurons during postnatal development in lumbar spinal cord of glutamic acid decarboxylase 67–green fluorescent protein mice

Object Inhibition of remyelination is part of the complex problem of persistent dysfunction after spinal cord injury (SCI), and residual myelin debris may be a factor that inhibits remyelination. Phagocytosis by microglial cells and by macrophages that migrate from blood vessels plays a major role in the clearance of myelin debris. The object of this study was to investigate the mechanisms underlying the failure of significant remyelination after SCI. Methods The authors investigated macrophage recruitment and related factors in rats by comparing a contusion model (representing contusive SCI with residual myelin debris and failure of remyelination) with a model consisting of chemical demyelination by lysophosphatidylcholine (representing multiple sclerosis with early clearance of myelin debris and remyelination). The origin of infiltrating macrophages was investigated using mice transplanted with bone marrow cells from green fluorescent protein–transfected mice. The changes in levels of residual myelin debris and the infiltration of activated macrophages in demyelinated lesions were investigated by immunostaining at 2, 4, and 7 days postinjury. To investigate various factors that might be involved, the authors also investigated gene expression of macrophage chemotactic factors and adhesion factors. Results Activated macrophages coexpressing green fluorescent protein constituted the major cell population in the lesions, indicating that the macrophages in both models were mainly derived from the bone marrow, and that very few were derived from the intrinsic microglia. Immunostaining showed that in the contusion model, myelin debris persisted for a long period, and the infiltration of macrophages was significantly delayed. Among the chemotactic factors, the levels of monocyte chemoattractant protein–1 and granulocyte–macrophage colony-stimulating factor were lower in the contusion model at 2 and 4 days postinjury. Conclusions The results suggest that the delayed infiltration of activated macrophages is related to persistence of myelin debris after contusive SCI, resulting in the inhibition of remyelination.

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Inhibitory Synaptic Modulation of Renshaw Cell Activity in the Lumbar Spinal Cord of Neonatal Mice

Journal of Neurophysiology ◽

10.1152/jn.00100.2010 ◽

2010 ◽

Vol 103 (6) ◽

pp. 3437-3447 ◽

Cited By ~ 8

Author(s):

Hiroshi Nishimaru ◽

Tadachika Koganezawa ◽

Miyo Kakizaki ◽

Tatsuhiko Ebihara ◽

Yuchio Yanagawa

Keyword(s):

Spinal Cord ◽

Fluorescent Protein ◽

Cell Activity ◽

Simultaneous Recording ◽

Ventral Root ◽

Lumbar Spinal Cord ◽

Motor Behaviors ◽

Axon Collaterals ◽

Highly Correlated ◽

Green Fluorescent

In the mammalian spinal cord, Renshaw cells (RCs) are excited by axon collaterals of motoneurons (MNs), and in turn, provide recurrent inhibition of MNs. They are considered an important element in controlling the motor output. However, how RCs are modulated by spinal circuits during motor behaviors remains unclear. In this study, the physiological nature of inhibitory synaptic inputs to RCs in the lumbar segment during spontaneous motoneuronal activity was examined in the isolated spinal cord taken from glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mouse neonates. Whole cell recordings of RCs in current-clamp mode showed that they receive phasic inhibition that could modulate the RC firing evoked by excitation of MNs. In voltage-clamp recording, we observed a barrage of spontaneous inhibitory postsynaptic currents (sIPSCs) mediated by glycine and/or GABA. These sIPSCs persisted in the presence of mecamylamine, a nicotinic receptor antagonist, indicating that excitation of other RCs by MN axon collaterals may not be essential for these inhibitory actions. Simultaneous recording of RC and the ventral root in the same segment showed that the RCs received inhibitory inputs when spontaneous MN firing occurred. Paired recordings of a RC and a MN showed that during the bursting activity in the ventral root, the magnitude of the RC sIPSCs and the magnitude of the excitatory inputs that MNs receive are highly correlated. These results indicate that RCs are modulated by inhibition that matches the MN excitation in timing and amplitude during motor behaviors.

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Neurochemical properties of enkephalinergic neurons in lumbar spinal dorsal horn revealed by preproenkephalin-green fluorescent protein transgenic mice

Journal of Neurochemistry ◽

10.1111/j.1471-4159.2010.06715.x ◽

2010 ◽

pp. no-no ◽

Cited By ~ 2

Author(s):

Jing Huang ◽

Jing Chen ◽

Wei Wang ◽

Wen Wang ◽

Yoshinori Koshimizu ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Transgenic Mice ◽

Dorsal Horn ◽

Spinal Dorsal Horn ◽

Fluorescent Protein ◽

Spinal Dorsal ◽

Green Fluorescent ◽

Lumbar Spinal

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Locomotor-related activity of GABAergic interneurons localized in the ventrolateral region in the isolated spinal cord of neonatal mice

Journal of Neurophysiology ◽

10.1152/jn.00385.2011 ◽

2011 ◽

Vol 106 (4) ◽

pp. 1782-1792 ◽

Cited By ~ 4

Author(s):

Hiroshi Nishimaru ◽

Hiroyuki Sakagami ◽

Miyo Kakizaki ◽

Yuchio Yanagawa

Keyword(s):

Spinal Cord ◽

Fluorescent Protein ◽

Essential Element ◽

Rhythmic Activity ◽

Inhibitory Neurons ◽

Lumbar Spinal Cord ◽

Gabaergic Interneurons ◽

Morphological Examination ◽

Motoneuron Pool ◽

Green Fluorescent

Inhibitory neurons are an essential element of the locomotor network in the mammalian spinal cord. However, little is known about the firing pattern and synaptic modulation during locomotion in the majority of them. In this study, we performed whole cell recording in visually identified ventrolaterally located GABAergic neurons (VL-GNs) in the rostral (L2 segment) and caudal (L5 segment) lumbar cord using isolated spinal cord preparations taken from glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mouse neonates. These neurons did not respond to electrical stimulation of the ventral root, indicating that they were not Renshaw cells. Ninety-five percent of VL-GNs in the L2 segment and fifty percent of those in the L5 segment showed significant rhythmic firing during locomotor-like rhythmic activity induced by bath application of 5-HT and NMDA. Seventy percent of these neurons fired mainly during the extensor phase, and twenty-five percent fired mainly during the flexor phase. Voltage-clamp recordings revealed that most of these neurons received rhythmic inhibition during the nonfiring phase and excitatory synaptic inputs during the firing phase. Morphological examination of recorded neurons filled with neurobiotin showed that their soma was located lateral to the motoneuron pool and that they extended their processes into the local ipsilateral ventromedial region and dorsal regions. The present study indicates that these GABAergic interneurons located in the ventrolateral region adjacent to the motoneuron pool are rhythmically active during locomotion and involved in the inhibitory modulation of local locomotor network in the lumbar spinal cord.

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Analysis of Green Fluorescent Protein Expression in Transgenic Rats for Tracking Transplanted Neural Stem/Progenitor Cells

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.5a6639.2005 ◽

2005 ◽

Vol 53 (10) ◽

pp. 1215-1226 ◽

Cited By ~ 42

Author(s):

Andrea J. Mothe ◽

Iris Kulbatski ◽

Rita L. van Bendegem ◽

Linda Lee ◽

Eiji Kobayashi ◽

...

Keyword(s):

Spinal Cord ◽

Green Fluorescent Protein ◽

Progenitor Cells ◽

Fluorescent Protein ◽

Gfp Expression ◽

Rat Strain ◽

Direct Fluorescence ◽

Green Fluorescent

Green fluorescent protein (GFP) expression was evaluated in tissues of different transgenic rodents—Sprague-Dawley (SD) rat strain [SD-Tg(GFP)Bal], W rat strain [Wistar-TgN(CAG-GFP)184ys], and M mouse strain [Tg(GFPU)5Nagy/J]—by direct fluorescence of native GFP expression and by immunohistochemistry. The constitutively expressing GFP transgenic strains showed tissue-specific differences in GFP expression, and GFP immunohistochemistry amplified the fluorescent signal. The fluorescence of stem/progenitor cells cultured as neurospheres from the ependymal region of the adult spinal cord from the GFP SD and W rat strains was assessed in vitro. After transplantation of the cells into wildtype spinal cord, the ability to track the grafted cells was evaluated in vivo. Cultured stem/progenitor cells from the SD strain required GFP immunostaining to be visualized. Likewise, after transplantation of SD cells into the spinal cord, immunohistochemical amplification of the GFP signal was required for detection. In contrast, GFP expression of stem/progenitor cells generated from the W strain was readily detected by direct fluorescence both in vitro and in vivo without the need for immunohistochemical amplification. The cultured stem/progenitor cells transplanted into the spinal cord survived for at least 49 days after transplantation, and continued to express GFP, demonstrating stable expression of the GFP transgene in vivo.

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