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• W141294482 abstract "Altered motoneuron excitability is involved in amyotrophic lateral sclerosis pathobiology. To test the hypothesis that inhibitory interneuron innervation of spinal motoneurons is abnormal in an amyotrophic lateral sclerosis mouse model, we measured GABAergic, glycinergic, and cholinergic immunoreactive terminals on spinal motoneurons in mice expressing a mutant form of human superoxide dismutase-1 with a Gly93→Ala substitution (G93A-SOD1) and in controls at different ages. Glutamic acid decarboxylase, glycine transporter-2, and choline acetyltransferase were used as markers for GABAergic, glycinergic, and cholinergic terminals, respectively. Triple immunofluorescent labeling of boutons contacting motoneurons was visualized by confocal microscopy and analyzed quantitatively. Glycine transporter-2-bouton density on lateral motoneurons was decreased significantly in G93A-SOD1 mice compared with controls. This reduction was absent at 6 weeks of age but present in asymptomatic 8-week-old mice and worsened with disease progression from 12 to 14 weeks of age. Motoneurons lost most glycinergic innervation by 16 weeks of age (end-stage) when there was a significant decrease in the numbers of motoneurons and choline acetyltransferase-positive boutons. No significant differences in glutamic acid decarboxylase-bouton densities were found in G93A-SOD1 mice. Reduction of glycinergic innervation preceded mitochondrial swelling and vacuolization. Calbindin-positive Renshaw cell number was decreased significantly at 12 weeks of age in G93A-SOD1 mice. Thus, either the selective loss of inhibitory glycinergic regulation of motoneuron function or glycinergic interneuron degeneration contributes to motoneuron degeneration in amyotrophic lateral sclerosis. Altered motoneuron excitability is involved in amyotrophic lateral sclerosis pathobiology. To test the hypothesis that inhibitory interneuron innervation of spinal motoneurons is abnormal in an amyotrophic lateral sclerosis mouse model, we measured GABAergic, glycinergic, and cholinergic immunoreactive terminals on spinal motoneurons in mice expressing a mutant form of human superoxide dismutase-1 with a Gly93→Ala substitution (G93A-SOD1) and in controls at different ages. Glutamic acid decarboxylase, glycine transporter-2, and choline acetyltransferase were used as markers for GABAergic, glycinergic, and cholinergic terminals, respectively. Triple immunofluorescent labeling of boutons contacting motoneurons was visualized by confocal microscopy and analyzed quantitatively. Glycine transporter-2-bouton density on lateral motoneurons was decreased significantly in G93A-SOD1 mice compared with controls. This reduction was absent at 6 weeks of age but present in asymptomatic 8-week-old mice and worsened with disease progression from 12 to 14 weeks of age. Motoneurons lost most glycinergic innervation by 16 weeks of age (end-stage) when there was a significant decrease in the numbers of motoneurons and choline acetyltransferase-positive boutons. No significant differences in glutamic acid decarboxylase-bouton densities were found in G93A-SOD1 mice. Reduction of glycinergic innervation preceded mitochondrial swelling and vacuolization. Calbindin-positive Renshaw cell number was decreased significantly at 12 weeks of age in G93A-SOD1 mice. Thus, either the selective loss of inhibitory glycinergic regulation of motoneuron function or glycinergic interneuron degeneration contributes to motoneuron degeneration in amyotrophic lateral sclerosis. Amyotrophic lateral sclerosis (ALS) is a rapidly evolving adult onset neurological disease characterized by a progressive loss of motoneurons.1Rowland LP Shneider NA Amyotrophic lateral sclerosis.N Engl J Med. 2001; 344: 1688-1700Crossref PubMed Scopus (1617) Google Scholar, 2Cleveland DW Rothstein JD From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS.Nat Rev Neurosci. 2001; 2: 806-819Crossref PubMed Scopus (1188) Google Scholar About 10% of ALS cases are familial ALS with inheritance patterns, and 90% are sporadic ALS with no known genetic component. Autosomal dominant mutations in the Cu/Zn superoxide dismutase-1 (SOD1, ALS1) gene occur in ∼20% of familial ALS cases.3Rosen DR Siddique T Patterson D Figlewicz DA Sapp P Hentati A Donaldson D Goto J O'Regan JP Deng HX Rahman Z Krizus A McKenna-Yasek D Cayabyab A Gaston SM Berger R Tanzi RE Halperin JJ Herzfeldt B Van Den Bergh R Hung W-Y Bird T Deng G Mulder DW Smyth C Laing NG Soriano E Pericak-Vance MA Haines J Rouleau GA Gusella JS Horvitz HR Brown RH Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis.Nature. 1993; 362: 59-62Crossref PubMed Scopus (5568) Google Scholar Transgenic mice overexpressing the human mutated SOD1 gene with a glycine/alanine substitution at codon 93 (G93A) develop a fatal motoneuron disease resembling ALS in humans.4Gurney ME Pu H Chiu AY Dal Canto MC Polchow CY Alexander DD Caliendo J Hentati A Kwon YW Deng HX Chen W Zhai P Sufit RL Siddique T Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.Science. 1994; 264: 1772-1775Crossref PubMed Scopus (3494) Google Scholar Many theories have been communicated that implicate perturbations in axonal transport, protein integrity, mitochondria, antioxidant status, and inflammation in the mechanisms of ALS pathogenesis.5Bruijn LI Miller TM Cleveland DW Unraveling the mechanisms involved in motor neuron degeneration in ALS.Annu Rev Neurosci. 2004; 27: 723-749Crossref PubMed Scopus (1202) Google Scholar, 6Boillee S Vande Velde C Cleveland DW ALS: a disease of motor neurons and their nonneuronal neighbors.Neuron. 2006; 52: 39-59Abstract Full Text Full Text PDF PubMed Scopus (1153) Google Scholar Considerable data supported the contributions of glutamate-mediated excitotoxicity in ALS.7Plaitakis A Glutamate dysfunction and selective motor neuron degeneration in amyotrophic lateral sclerosis: a hypothesis.Ann Neurol. 1990; 28: 3-8Crossref PubMed Scopus (167) Google Scholar, 8Rothstein JD Martin LJ Kuncl RW Decreased glutamate transport by the brain and spinal cord in amyotrophic lateral sclerosis.N Engl J Med. 1992; 326: 1464-1468Crossref PubMed Scopus (1058) Google Scholar, 9Heath PR Shaw PJ Update on the glutamatergic neurotransmitter system and the role of excitotoxicity in amyotrophic lateral sclerosis.Muscle Nerve. 2002; 26: 438-458Crossref PubMed Scopus (275) Google Scholar Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in spinal motoneurons were altered in human ALS10Kawahara Y Kwak S Sun H Ito K Hashida H Aizawa H Jeong SY Kanazawa I Human spinal motoneurons express low relative abundance of GluR2 mRNA: an implication for excitotoxicity in ALS.J Neurochem. 2003; 85: 680-689Crossref PubMed Scopus (108) Google Scholar, 11Kawahara Y Ito K Sun H Aizawa H Kanazawa I Kwak S Glutamate receptors: rNA editing and death of motor neurons.Nature. 2004; 427: 801Crossref PubMed Scopus (458) Google Scholar and G93A-SOD1 mice.12Zhao P Ignacio S Beattie EC Abood ME Altered presymptomatic AMPA and cannabinoid receptor trafficking in motor neurons of ALS model mice: implications for excitotoxicity.Eur J Neurosci. 2008; 27: 572-579Crossref PubMed Scopus (61) Google Scholar, 13Pieri M Gaetti C Spalloni A Cavalcanti S Mercuri N Bernardi G Longone P Zona C alpha-Amino-3-hydroxy-5-methyl-isoxazole-4-propionate receptors in spinal cord motor neurons are altered in transgenic mice overexpressing human Cu,Zn superoxide dismutase (Gly93–>Ala) mutation.Neuroscience. 2003; 122: 47-58Crossref PubMed Scopus (32) Google Scholar, 14Tortarolo M Grignaschi G Calvaresi N Zennaro E Spaltro G Colovic M Fracasso C Guiso G Elger B Schneider H Seilheimer B Caccia S Bendotti C Glutamate AMPA receptors change in motor neurons of SOD1G93A transgenic mice and their inhibition by a noncompetitive antagonist ameliorates the progression of amytrophic lateral sclerosis-like disease.J Neurosci Res. 2006; 83: 134-146Crossref PubMed Scopus (100) Google Scholar Electrophysiological studies of ALS patients indicated widespread signs of motoneuron hyperexcitability.15Eisen A Weber M Neurophysiological evaluation of cortical function in the early diagnosis of ALS.Amyotroph Lateral Scler Other Motor Neuron Disord. 2000; 1: S47-S51PubMed Google Scholar Hyperexcitability of motoneurons was also observed in spinal cord slices16Kuo JJ Schonewille M Siddique T Schults AN Fu R Bar PR Anelli R Heckman CJ Kroese AB Hyperexcitability of cultured spinal motoneurons from presymptomatic ALS mice.J Neurophysiol. 2004; 91: 571-575Crossref PubMed Scopus (143) Google Scholar and dissociated embryonic cell cultures from G93A-SOD1 mice.17Pieri M Albo F Gaetti C Spalloni A Bengtson CP Longone P Cavalcanti S Zona C Altered excitability of motor neurons in a transgenic mouse model of familial amyotrophic lateral sclerosis.Neurosci Lett. 2003; 351: 153-156Crossref PubMed Scopus (102) Google Scholar The hyperexcitability theory emphasizes mostly the contribution of excessive synaptic excitation, while the possibility of insufficient synaptic inhibition has been largely ignored. Spinal cord slice cultures from embryonic G93A-SOD1 mice showed an imbalance between excitatory and inhibitory innervation.18Avossa D Grandolfo M Mazzarol F Zatta M Ballerini L Early signs of motoneuron vulnerability in a disease model system: characterization of transverse slice cultures of spinal cord isolated from embryonic ALS mice.Neuroscience. 2006; 138: 1179-1194Crossref PubMed Scopus (58) Google Scholar Spinal cord motoneurons receive extensive glycinergic and GABAergic innervations that regulate motoneuron excitability through various mechanisms.19Rekling JC Funk GD Bayliss DA Dong XW Feldman JL Synaptic control of motoneuronal excitability.Physiol Rev. 2000; 80: 767-852PubMed Google Scholar Abnormal gamma aminobutyric acid (GABA) and glycine levels were observed in serum or autopsy tissues of ALS patients.20Niebroj-Dobosz I Janik P Amino acids acting as transmitters in amyotrophic lateral sclerosis (ALS).Acta Neurol Scand. 1999; 100: 6-11Crossref PubMed Scopus (42) Google Scholar, 21Malessa S Leigh PN Bertel O Sluga E Hornykiewicz O Amyotrophic lateral sclerosis: glutamate dehydrogenase and transmitter amino acids in the spinal cord.J Neurol Neurosurg Psychiatry. 1991; 54: 984-988Crossref PubMed Scopus (57) Google Scholar In human ALS autopsy spinal cord, binding sites for the inhibitory neurotransmitter glycine have been reported to be reduced in anterior horn.22Hayashi H Suga M Satake M Tsubaki T Reduced glycine receptor in the spinal cord in amyotrophic lateral sclerosis.Ann Neurol. 1981; 9: 292-294Crossref PubMed Scopus (41) Google Scholar, 23Whitehouse PJ Wamsley JK Zarbin MA Price DL Tourtellotte WW Kuhar MJ Amyotrophic lateral sclerosis: alterations in neurotransmitter receptors.Ann Neurol. 1983; 14: 8-16Crossref PubMed Scopus (97) Google Scholar It therefore seems possible that inhibitory neural systems are disrupted as part of the pathogenesis in ALS. In fact, riluzole, the only drug approved by the Food and Drug Administration for the treatment of ALS, directly interacts with GABA(A) and glycine receptors24Mohammadi B Krampfl K Moschref H Dengler R Bufler J Interaction of the neuroprotective drug riluzole with GABA(A) and glycine receptor channels.Eur J Pharmacol. 2001; 415: 135-140Crossref PubMed Scopus (41) Google Scholar, 25He Y Benz A Fu T Wang M Covey DF Zorumski CF Mennerick S Neuroprotective agent riluzole potentiates postsynaptic GABA(A) receptor function.Neuropharmacology. 2002; 42: 199-209Crossref PubMed Scopus (73) Google Scholar in addition to its antiglutamatergic action.26Malgouris C Bardot F Daniel M Pellis F Rataud J Uzan A Blanchard JC Laduron PM Riluzole, a novel antiglutamate, prevents memory loss and hippocampal neuronal damage in ischemic gerbils.J Neurosci. 1989; 9: 3720-3727PubMed Google Scholar In this study, we examined glycinergic and GABAergic innervations of spinal motoneurons in G93A-SOD1 mice using quantitative confocal analysis. We demonstrated that abnormalities in inhibitory interneuron innervations of spinal motoneurons emerge early in the course of disease before structural evidence for motoneuron degeneration. Adult male transgenic mice expressing human mutant SOD1 gene driven by the human SOD1 promoter4Gurney ME Pu H Chiu AY Dal Canto MC Polchow CY Alexander DD Caliendo J Hentati A Kwon YW Deng HX Chen W Zhai P Sufit RL Siddique T Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.Science. 1994; 264: 1772-1775Crossref PubMed Scopus (3494) Google Scholar were studied. A mouse line (B6SJL-TgN [SOD1-G93A] 1Gur, G1H) with a high copy number of mutant alleles (∼20 copies) and a rapid disease onset was used. The mice were studied through the entire course of the disease from asymptomatic stage to end-stage disease at 6, 8, 10, 12, 14, and 16 weeks of age. The group size for each data set was 3 to 5 mice. Controls were non-transgenic littermates of the mutants. The institutional Animal Care and Use Committee approved the animal protocols. Mice were overdosed with sodium pentobarbital (50 mg/kg) and subsequently perfused transcardially with ice-cold 100 mmol/L phosphate buffer-saline (pH 7.4) followed by 4% paraformaldehyde. All mice were perfused under identical conditions by the same individual. Spinal cords were postfixed in paraformaldehyde for 3 hours, and then were cryoprotected in 20% glycerol overnight. Transverse serial sections (40 μm-thick) through lumbar spinal cord were cut on a sliding microtome (American Optical Corporation, New York, NY) and stored individually in 96-well plates in cyroprotectant buffer (1% polyvinylpyrrolidone, 40% ethylene glycol, and 0.1 mol/L potassium acetate, pH 6.5) at −20°C until used for immunocytochemistry. Free-floating sections were rinsed in Tris buffer (TBS, pH 7.4), permeabilized with 0.4% Triton X-100, blocked with 10% donkey serum, and then incubated in a cocktail of primary antibodies diluted in TBS containing 2% donkey serum and 0.1% Triton X-100 for 48 hours at 4°C. The primary antibodies used in different combinations were: mouse anti-glutamic acid decarboxylase 67 (GAD67; monoclonal, 1:10000; Chemicon, Temecula, CA); guinea pig anti-glycine transporter-2 (GlyT2; polyclonal, 1:10000; Chemicon); goat anti-choline acetyltransferase (ChAT; polyclonal, 1:200; Chemicon); rabbit anti-manganese-containing SOD (MnSOD, SOD2; polyclonal, 1:400; Assay Designs, Ann Arbor, MI); rabbit anti-growth associated protein-43 (GAP43; polyclonal, 1:1000; Chemicon); rabbit anti-synaptophysin (polyclonal, 1:1000; Dako Denmark A/S, Glostrup, Denmark). After four washes with TBS, sections were incubated for 2 hours at room temperature in a mixture of species-specific secondary antibodies (all raised in donkey) conjugated to Alexa Fluor 488, Alexa Fluor 594, or Alexa Fluor 647 (Invitrogen Corporation, Carlsbad, CA). Sections were washed again, and mounted using Prolong anti-fade medium (Invitrogen). Spinal cord sections were imaged using a 100× oil immersion objective (1.3 numerical aperture lens) mounted on a Zeiss LSM 510 confocal microscope. All sections were imaged under identical conditions and analyzed using identical parameters. ChAT immunoreactivity was used as a marker to identify motoneurons27Eckenstein F Thoenen H Production of specific antisera and monoclonal antibodies to choline acetyltransferase: characterization and use for identification of cholinergic neurons.EMBO J. 1982; 1: 363-368PubMed Google Scholar in lateral and medial pools. Motoneurons are large cells (mean diameter >20 μm for α-motoneurons) likely to be truncated within 40 μm sections. Only motoneurons with visible nuclei and nucleolus were selected. Immunolabeled presynaptic boutons were examined for apposition to ChAT-labeled large neurons. Boutons were considered to be in apposition only if there was no visible space between the bouton and the cell membrane in any optical section. Measurements of immunopositive boutons were performed using NIH Image J software. Each confocal image obtained in triple staining experiments was split into three channels and thresholded in black and white binary images. Automatic intensity thresholding and particle sizes of above 0.18 μm2 for GAD- and GlyT2-immunostaining or 0.22 μm2 for ChAT-immunostaining were chosen for defining boutons. Standardized bouton number was determined by dividing the number of boutons contacting a motoneuron by its perimeter, which was measured using Image J software. The single optical section image for each neuron was measured at the level of the nucleolus. Lateral and medial motoneuron pools were analyzed separately. To evaluate regional selectivity, GlyT2-immunoreactivity was analyzed specifically in lamina II of dorsal horn. Confocal images were obtained at 100× from randomly selected regions within lamina II identified by location. Two different confocal images from each lumbar spinal cord section and two sections from each mouse were quantified. To better resolve the density of boutons on the entire motoneuron cell bodies, we examined the bouton density throughout one half of the cell body in the lateral motoneurons. Total numbers of boutons per neuron divided by 2 were counted across the z-series optical sections (0.7 μm separation). This was accomplished by starting at the first optical section containing a given neuron and counting the number of new boutons touching the cell surface to the last optical section at the middle of the soma. The “middle” was determined by scanning back and forth through the optical sections containing intact nuclei and nucleolus several times. The locations of labeled boutons in a single optical section were compared with labeled boutons in adjacent sections to avoid double counting of the same boutons. The bouton densities were calculated by dividing the total number of boutons by its surface area, which was determined by Image J software. Eight to ten motoneurons per spinal cord and two to three lumbar spinal cord sections per animal were quantified. For colocalization analysis, images were imported into Image J software where regions of interest were outlined, and regions outside regions of interest were cleared using “segmenting assistant” plug-in. Each channel was then subjected to background subtraction, and analyzed by “colocalization threshold” plug-in (zero-zero pixels was excluded in threshold calculation). The thresholds for each channel were then calculated and pixels below this value were ignored for colocalization quantification. This method avoids the investigator bias in the thresholds setting. A pixel was defined as having colocalization when the intensities of both labels were above their respective thresholds. Using this method Pearson's correlation coefficient for pixels where either channel was below their respective threshold was 0.002 ± 0.005 (n = 58), and Pearson's correlation coefficient for pixels where both channels were above their respective threshold was 0.862 ± 0.005 (n = 58), indicating the thresholds had been set appropriately. Colocalization of synaptophysin with GlyT2-, GAD- or ChAT-immunoreactivity was given as the colocalization coefficients M1 and M2. These coefficients are proportional to the number of colocalizing pixels in either channel (channel 1 or channel 2) of the composite image, relative to the total number of pixels above threshold in that channel. Colocalization coefficient using threshold takes into account the number of pixels with colocalization as well as the intensities of the two labels in each pixel. The number of pixels (divided by perimeter) that have both channel 1 and channel 2 intensities above the threshold (ie, the number of colocalized pixels) was used to quantify the colocalization of GAP43- with GlyT2- or GAD-immunoreactivity. Results are presented as mean ± SEM. Statistical significance was determined by Student's t-test. The level of significance was set of P < 0.05. Lumbar spinal cord sections from G93A-SOD1 and control mice were used to localize calbindin D28k as a marker for Renshaw cells.28Arvidsson U Ulfhake B Cullheim S Ramirez V Shupliakov O Hokfelt T Distribution of calbindin D28k-like immunoreactivity (LI) in the monkey ventral horn: do Renshaw cells contain calbindin D28k-LI?.J Neurosci. 1992; 12: 718-728PubMed Google Scholar, 29Antal M Freund TF Polgar E Calcium-binding proteins, parvalbumin- and calbindin-D 28k-immunoreactive neurons in the rat spinal cord and dorsal root ganglia: a light and electron microscopic study.J Comp Neurol. 1990; 295: 467-484Crossref PubMed Scopus (132) Google Scholar, 30Sanna PP Celio MR Bloom FE Rende M Presumptive Renshaw cells contain decreased calbindin during recovery from sciatic nerve lesions.Proc Natl Acad Sci USA. 1993; 90: 3048-3052Crossref PubMed Scopus (37) Google Scholar, 31Carr PA Alvarez FJ Leman EA Fyffe RE Calbindin D28k expression in immunohistochemically identified Renshaw cells.Neuroreport. 1998; 9: 2657-2661Crossref PubMed Scopus (67) Google Scholar Calbindin was localized using a standard immunoperoxidase method with a mouse monoclonal antibody to calbindin (1:10,000; Sigma, St. Louis, MO) and visualized with diaminobenzidine (Sigma) as chromogen. Cell counts were made by viewing sections under a 40× lens mounted on a Nikon microscope. Renshaw cells were identified by their location and characteristic morphology as described previously.31Carr PA Alvarez FJ Leman EA Fyffe RE Calbindin D28k expression in immunohistochemically identified Renshaw cells.Neuroreport. 1998; 9: 2657-2661Crossref PubMed Scopus (67) Google Scholar, 32Alvarez FJ Dewey DE Harrington DA Fyffe RE Cell-type specific organization of glycine receptor clusters in the mammalian spinal cord.J Comp Neurol. 1997; 379: 150-170Crossref PubMed Scopus (130) Google Scholar The G93A-SOD1 transgenic mice with a high copy number of mutant allele first show signs of spasticity at about 10 weeks of age, and then unilateral or bilateral hindlimb paresis at around 11 to 12 weeks of age; the disease then progresses to end-stage when the mice are quadriplegic at around 16 weeks of age.4Gurney ME Pu H Chiu AY Dal Canto MC Polchow CY Alexander DD Caliendo J Hentati A Kwon YW Deng HX Chen W Zhai P Sufit RL Siddique T Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.Science. 1994; 264: 1772-1775Crossref PubMed Scopus (3494) Google Scholar, 33Martin LJ Liu Z Chen K Price AC Pan Y Swaby JA Golden WC Motor neuron degeneration in amyotrophic lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death.J Comp Neurol. 2007; 500: 20-46Crossref PubMed Scopus (231) Google Scholar In this transgenic line of mutant SOD1 mice, the morphology and number of motoneurons changes dramatically over the time course examined by confocal microscopy. At 6 and 8 weeks of age, the cytoplasm of ChAT-positive motoneurons appeared uniform with no or minimal cytoplasmic vacuolization, and the size and shape of motoneuron perikarya appeared normal (Figure 1A). No significant differences in the number of large ChAT-positive motoneurons were observed in 8-week-old G93A-SOD1 mice compared with controls (Figure 1B). At 10 weeks of age, some motoneuron cell bodies and proximal dendrites showed cytoplasmic vacoules (Figure 1A). Swollen, degenerating axons with segmental enlargements were also seen in the ventral horn neuropil and white matter. At 12 weeks of age, cytoplasmic vacuoles in motoneurons were more apparent and more motoneurons possessed vacuoles (Figure 1A). At this time, subsets of motoneurons and axons were severely swollen, but the decrease in the number of motoneurons was not significant (Figure 1B). The number of motoneurons was decreased significantly at 14 weeks of age (Figure 1B; P < 0.05). At 16 weeks of age, when the disease developed to end-stage, the number of motoneurons was reduced further (Figure 1B; P < 0.01) and the residual motoneurons tended to be smaller than that the typical α-motoneurons. Glycinergic synapses on lumbar spinal motoneurons were identified by GlyT2-immunoreactivity.34Poyatos I Ponce J Aragon C Gimenez C Zafra F The glycine transporter GLYT2 is a reliable marker for glycine-immunoreactive neurons.Brain Res Mol Brain Res. 1997; 49: 63-70Crossref PubMed Scopus (125) Google Scholar, 35Spike RC Watt C Zafra F Todd AJ An ultrastructural study of the glycine transporter GLYT2 and its association with glycine in the superficial laminae of the rat spinal dorsal horn.Neuroscience. 1997; 77: 543-551Crossref PubMed Scopus (78) Google Scholar, 36Zafra F Gomeza J Olivares L Aragon C Gimenez C Regional distribution and developmental variation of the glycine transporters GLYT1 and GLYT2 in the rat CNS.Eur J Neurosci. 1995; 7: 1342-1352Crossref PubMed Scopus (241) Google Scholar Numerous densely-packed, flattened GlyT2-immunopositive boutons (averaged 1 μm2 in size; Figure 1A, green) were closely apposed to the soma and proximal dendrites of ChAT-positive large motoneurons. Bouton density was analyzed to determine changes in glycinergic innervation. In this analysis, the number of boutons was expressed as a ratio of total surface area. We quantified GlyT2-positive boutons through the surface of half of the motoneuron (providing they had visible nucleus and nucleolus) cell bodies (z-stack) from both wild-type and G93A-SOD1 mice. All motoneurons analyzed in wild-type mice were contacted by GlyT2-positive boutons. No significant differences in the distribution and density of boutons between the soma and proximal dendrites of motoneurons, or differences in GlyT2-bouton densities between different ages of wild-type mice were observed (data not shown). At 6 weeks of age, G93A-SOD1 mouse motoneurons had GlyT2-bouton densities similar to wild-type mice (Figure 1C). However, GlyT2-bouton densities were significantly lower in asymptomatic 8-week-old G93A-SOD1 mice than in controls (Figure 1C; P < 0.05). About 20% of GlyT2-boutons were lost on G93A-SOD1 mouse motoneurons. Individual variation was observed between 8-week-old G93A-SOD1 mice (Figure 1C, error bar), mirroring the individual variation in the onset of disease. No obvious changes were observed in the size and shape of remaining GlyT2-boutons on G93A-SOD1 mouse motoneurons. Unexpectedly, the density of GlyT2-boutons showed a slight recovery at 10 weeks of age with respect to the 8-week-old G93A-SOD1 mice (Figure 1C). At 12 weeks of age, when the mice were paretic, GlyT2-bouton densities were decreased significantly. Fewer individual variations were observed at this age compared with 8-week-old mice. The loss in GlyT2-boutons was more pronounced at 14 weeks of age. At 16 weeks of age, when the disease developed to end-stage, motoneurons lost the majority of GlyT2-boutons (Figure 1C). The number of GlyT2-positive boutons slightly recovered at 10 weeks of age compared with 8-week-old G93A-SOD1 mice (Figure 1C). Considering the possibility of compensatory recovery, we triple labeled the spinal cord with GlyT2-, ChAT-, and GAP43-antibodies. GAP43 is a neurite outgrowth marker.37Benowitz LI Routtenberg A GAP-43: an intrinsic determinant of neuronal development and plasticity.Trends Neurosci. 1997; 20: 84-91Abstract Full Text Full Text PDF PubMed Scopus (1142) Google Scholar, 38Gorgels TG Oestreicher AB de Kort EJ Gispen WH Immunocytochemical distribution of the protein kinase C substrate B-50 (GAP43) in developing rat pyramidal tract.Neurosci Lett. 1987; 83: 59-64Crossref PubMed Scopus (54) Google Scholar, 39Skene JH Axonal growth-associated proteins.Annu Rev Neurosci. 1989; 12: 127-156Crossref PubMed Scopus (1012) Google Scholar, 40Skene JH Jacobson RD Snipes GJ McGuire CB Norden JJ Freeman JA A protein induced during nerve growth (GAP-43) is a major component of growth-cone membranes.Science. 1986; 233: 783-786Crossref PubMed Scopus (393) Google Scholar GAP43-immunoreactivity was observed as bouton-like swellings in apposition to motoneurons (Figure 2A). Colocalization of GlyT2- and GAP43-immunoreactivity was detected in wild-type mice (Figure 2A). The colocalization coefficients of GlyT2 with GAP43 (M1; calculated as M1 = pixelsCh1, coloc/pixelsCh1, total, where Ch1 is GlyT2) was 0.127 ± 0.023 (n = 3). At 6 and 8 weeks of age, the average number of GAP43/GlyT2 colocalized pixels was not significantly different compared with control (Figure 2B). GAP43/GlyT2 colocalized pixel number significantly increased in 10-week-old G93A-SOD1 mice (Figure 2B; P < 0.05). The colocalization coefficients of GlyT2 with GAP43 also increased to 0.182 ± 0.026 (n = 3). Because GAP43 marks neurite sprouting,40Skene JH Jacobson RD Snipes GJ McGuire CB Norden JJ Freeman JA A protein induced during nerve growth (GAP-43) is a major component of growth-cone membranes.Science. 1986; 233: 783-786Crossref PubMed Scopus (393) Google Scholar, 41Kalil K Skene JH Elevated synthesis of an axonally transported protein correlates with axon outgrowth in normal and injured pyramidal tracts.J Neurosci. 1986; 6: 2563-2570Crossref PubMed Google Scholar the increase of its colocalization with GlyT2-immunoreactivity indicates sprouting of glycinergic terminals in 10-week-old G93A-SOD1 mice. To test the specificity of glycinergic terminal sprouting on motoneurons, we also studied the colocalization of GAD- and GAP43-immunoreactivity. The colocalization coefficients of GAD with GAP43 (M1; calculated as M1 = pixelsCh1, coloc/pixelsCh1, total, where Ch1 is GAD) was 0.115 ± 0.019 (n = 3). No significant differences were found in GAP43/GAD colocalized pixel numbers at 6, 8, and 10 weeks of age compared with controls (Figure 2B). GABAergic synapses contacting motoneurons were identified using an antibody to GAD67.42Mackie M Hughes DI Maxwell D" @default.
• W141294482 created "2016-06-24" @default.
• W141294482 creator A5024878520 @default.
• W141294482 creator A5070457572 @default.
• W141294482 date "2009-02-01" @default.
• W141294482 modified "2023-10-12" @default.
• W141294482 title "Glycinergic Innervation of Motoneurons Is Deficient in Amyotrophic Lateral Sclerosis Mice" @default.
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