Altered development in GABA co‐release shapes glycinergic synaptic currents in cultured spinal slices of the SOD1^G93A mouse model of amyotrophic lateral sclerosis

Abstract

Key pointsr Increased environmental risk factors in conjunction with genetic susceptibility have been proposed with respect to the remarkable variations in mortality in amyotrophic lateral sclerosis (ALS).r In vitro models allow the investigation of the genetically modified counter-regulator of motoneuron toxicity and may help in addressing ALS therapy. r This altered inhibition improved spinal cord excitability, affecting motor outputs in early SOD1G93A pathogenesis.Abstract Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset neurological disease characterized by a progressive degeneration of motoneurons (MNs). In a previous study, we developed organotypic spinal cultures from an ALS mouse model expressing a mutant form of human superoxide dismutase 1 (SOD1 G93A ). We reported the presence of a significant synaptic rearrangement expressed by these embryonic cultured networks, which may lead to the altered development of spinal synaptic signalling, which is potentially linked to the adult disease phenotype. Recent studies on the same ALS mouse model reported a selective loss of glycinergic innervation in cultured MNs, suggestive of a contribution of synaptic inhibition to MN dysfunction and degeneration. In the present study, we further exploit organotypic cultures from wild-type and SOD1 G93A mice to investigate the development of glycine-receptor-mediated synaptic currents recorded from the interneurons of the premotor ventral circuits. We performed single cell electrophysiology, immunocytochemistry and confocal microscopy and suggest that GABA co-release may speed the decay of glycine responses altering both temporal precision and signal integration in SOD1G93A developing networks at the postsynaptic site. Our hypothesis is supported by the finding of an increased MN bursting activity in immature SOD1 G93A spinal cords and by immunofluorescence microscopy detection of a longer persistence of GABA in SOD1 G93A glycinergic terminals in cultured and ex vivo spinal slices.

Bridging pro-inflammatory signals, synaptic transmission and protection in spinal explants in vitro

“…Organotypic spinal slices represent a biological model useful for studying the dynamics of intra-segmental processes that evidently rely on resident neuroglial cells, propriospinal neurons and circuits [ 12 – 14 ]. To investigate the impact of inflammation on spinal synaptic networks we used co-cultured mouse DRG and spinal cord explants after 2 weeks of in vitro growth (Fig.…”

“…2a ) between control (CTRL; n = 48) and treated cultures (CKs4H and CKs6H; n = 32 and n = 34, respectively). Organotypic spinal slices display prominent spontaneous electrical activity in the ventral, premotor area [ 13 , 14 ]. To enable a meaningful comparison of the shifts in communication dynamics in networks exposed to CKs, we selected the 2 WIV stage, where neurons are known to exhibit an intense synaptic activity [ 13 , 14 ].…”

“…We recorded from interneurons to assess how the pro-inflammatory stress may regulate the ventral (pre-motor) [ 14 ] circuit activity, and this was done within a time-frame where the inflammatory pathological process did not progress to the extent of excitotoxicity. The lack of clear cell damage was supported by the absence of changes in the values of resting membrane potential, input resistance, cell capacitance and AP threshold in the treated neurons in respect to control ones [ 34 – 36 ].…”

“…Organotypic slice cultures developed from the embryonic mouse spinal cord represent a complex in vitro model where sensory-motor cytoarchitecture, synaptic properties and spinal cord resident cells are retained in a 3D–fashion [ 12 , 13 ]. By the use of this model, we have recently shown that in the SOD1 G93A mouse, a genetic ALS model, spinal synapses retain greater GABA and glycine co-release than in the wild types and these changes influenced synaptic integration [ 14 ].…”

Synaptic inputs to the inferior olive from cerebellar and vestibular nuclei have distinct release kinetics and neurotransmitters

“…VIAAT is nonspecific, and the relative concentrations of glycine and GABA in a vesicle is regulated by the cytosolic levels of GABA and glycine. Corelease of glycine and GABA also occurs at other synapses (Dufour et al, 2010;Hirrlinger et al, 2019;Jonas et al, 1998;Keller et al, 2001;Medelin et al, 2016;Nabekura et al, 2004;Polter et al, 2018;Rahman et al, 2013;Rajalu et al, 2009;Russier et al, 2002;Wu et al, 2002). For VN to IO synapses, and other synapses that corelease GABA and glycine, the contribution of GABA and glycine to inhibition could be regulated by the contents of vesicles or by the composition of postsynaptic glycine and GABA receptors.…”