Early signs of motoneuron vulnerability in a disease model system: Characterization of transverse slice cultures of spinal cord isolated from embryonic ALS mice

Neurobiology Sector and Istituto Nazionale di Fisica della Materia Unit, International School for Advanced Studies, via Beirut 2-4, 34014 Trieste, Italy.
Neuroscience (Impact Factor: 3.36). 02/2006; 138(4):1179-94. DOI: 10.1016/j.neuroscience.2005.12.009
Source: PubMed


Mutations in the SOD1 gene are associated with familial amyotrophic lateral sclerosis. The mechanisms by which these mutations lead to cell loss within the spinal cord ventral horns are unknown. In the present report we used the G93A transgenic mouse model of amyotrophic lateral sclerosis to develop and characterize an in vitro tool for the investigation of subtle alterations of spinal tissue prior to frank neuronal degeneration. To this aim, we developed organotypic slice cultures from wild type and G93A embryonic spinal cords. We combined immunocytochemistry and electron microscopy techniques to compare wild type and G93A spinal cord tissues after 14 days of growth under standard in vitro conditions. By SMI32 and choline acetyl transferase immunostaining, the distribution and morphology of motoneurons were compared in the two culture groups. Wild type and mutant cultures displayed no differences in the analyzed parameters as well as in the number of motoneurons. Similar results were observed when glial fibrillary acidic protein and myelin basic protein-positive cells were examined. Cell types within the G93A slice underwent maturation and slices could be maintained in culture for at least 3 weeks when prepared from embryos. Electron microscopy investigation confirmed the absence of early signs of mitochondria vacuolization or protein aggregate formation in G93A ventral horns. However, a significantly different ratio between inhibitory and excitatory synapses was present in G93A cultures, when compared with wild type ones, suggesting the expression of subtle synaptic dysfunction in G93A cultured tissue. When compared with controls, G93A motoneurons exhibited increased vulnerability to AMPA glutamate receptor-mediated excitotoxic stress prior to clear disease appearance. This in vitro disease model may thus represent a valuable tool to test early mechanisms contributing to motoneuron degeneration and potential therapeutic molecular interventions.

5 Reads
  • Source
    • "These data suggest that it would be feasible to use MEA to investigate the properties of spinal networks after SCI and how chemical or electrical stimulation can facilitate their oscillatory discharges. It is noteworthy that, using spinal organotypic cultures from mice expressing the genetic phenotype G93A of hereditary lateral amyotrophic sclerosis, a selective dysregulation of synaptic transmission was demonstrated (Avossa et al., 2006). Likewise, MEAs were used to study the electrophysiological activity of motoneurons in spinal cord slices from mouse models of spinal muscular atrophy (Zhang et al., 2010). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Microelectrode arrays (MEAs) represent an important tool to study the basic characteristics of spinal networks that control locomotion in physiological conditions. Fundamental properties of this neuronal rhythmicity like burst origin, propagation, coordination, and resilience can, thus, be investigated at multiple sites within a certain spinal topography and neighboring circuits. A novel challenge will be to apply this technology to unveil the mechanisms underlying pathological processes evoked by spinal cord injury (SCI). To achieve this goal, it is necessary to fully identify spinal networks that make up the locomotor central pattern generator (CPG) and to understand their operational rules. In this review, the use of isolated spinal cord preparations from rodents, or organotypic spinal slice cultures is discussed to study rhythmic activity. In particular, this review surveys our recently developed models of SCI by evoking excitotoxic (or even hypoxic/dysmetabolic) damage to spinal networks and assessing the impact on rhythmic activity and cell survival. These pathological processes which evolve via different cell death mechanisms are discussed as a paradigm to apply MEA recording for detailed mapping of the functional damage and its time-dependent evolution.
    Frontiers in Neuroengineering 03/2013; 6:2. DOI:10.3389/fneng.2013.00002
  • Source
    • "Wild type and mutant cultures displayed no differences in the analyzed parameters as well as in the number of motor neurons, and there were no signs of mitochondria vacuolization or protein aggregate formation in G93A ventral horns. Together, the results of Panov et al. (2011b) and Avossa et al. (2006) suggest that in tgSOD1 rats the BM and SCM are primarily normal but suffer from some pathological events, which rapidly deteriorate mitochondria and cause death of neurons. "
    Amyotrophic Lateral Sclerosis, 01/2012; , ISBN: 978-953-307-806-9
  • Source
    • "Excessive synaptic excitation might mediate some hyperexcitability (Pieri et al., 2003a; Pieri et al., 2003b; van Zundert et al., 2008); however, insufficient synaptic inhibition could be important too. Spinal cord slice cultures from mutant SOD1 transgenic mice showed an imbalance between excitatory and inhibitory innervation (Avossa et al., 2006). Glycine and GABA are the two main inhibitory neurotransmitters in the central nervous system that activate different ionotropic receptors permeable to chloride ions. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a rapidly evolving and fatal adult-onset neurological disease characterized by progressive degeneration of motoneurons. Our previous study showed that glycinergic innervation of spinal motoneurons is deficient in an ALS mouse model expressing a mutant form of human superoxide dismutase-1 with a Gly93→Ala substitution (G93A-SOD1). In this study, we have examined, using whole-cell patch-clamp recordings, glycine receptor (GlyR)-mediated currents in spinal motoneurons from these transgenic mice. We developed a dissociated spinal cord culture model using embryonic transgenic mice expressing enhanced green fluorescent protein (eGFP) driven by the Hb9 promoter. Motoneurons were identified as Hb9-eGFP-expressing (Hb9-eGFP(+)) neurons with a characteristic morphology. To examine GlyRs in ALS motoneurons, we bred G93A-SOD1 mice to Hb9-eGFP mice and compared glycine-evoked currents in cultured Hb9-eGFP(+) motoneurons prepared from G93A-SOD1 embryos and from their nontransgenic littermates. Glycine-evoked current density was significantly smaller in the G93A-SOD1 motoneurons compared with control. Furthermore, the averaged current densities of spontaneous glycinergic miniature IPSCs (mIPSCs) were significantly smaller in the G93A-SOD1 motoneurons than in control motoneurons. No significant differences in GABA-induced currents and GABAergic mIPSCs were observed between G93A-SOD1 and control motoneurons. Quantitative single-cell reverse transcription-PCR found lower GlyRα1 subunit mRNA expression in G93A-SOD1 motoneurons, indicating that the reduction of GlyR current may result from the downregulation of GlyR mRNA expression in motoneurons. Immunocytochemistry demonstrated a decrease of surface postsynaptic GlyR on G93A-SOD1 motoneurons. Our study suggests that selective alterations in GlyR function contribute to inhibitory insufficiency in motoneurons early in the disease process of ALS.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 02/2011; 31(8):2815-27. DOI:10.1523/JNEUROSCI.2475-10.2011 · 6.34 Impact Factor
Show more