Abstract The single pellet reaching task is commonly used in rodents to assess the acquisition of fine motor skill and recovery of function following nervous system injury. Although this task is useful for gauging skilled forelimb use in rodents, the process of training animals is labor intensive and variable across studies and labs. To address these limitations, we developed a single pellet reaching paradigm for training and testing group housed mice within their home cage. Mice enter a training compartment attached to the outside of the cage and retrieve millet seeds presented on a motorized pedestal that can be individually positioned to present seeds to either forelimb. To identify optimal training parameters, we compared task participation and success rates between groups of animals that were presented seeds at two different heights (floor vs mouth height) and at different intervals (fixed-time vs trial-based). The mouth height/fixed interval presentation style was most effective at promoting reaching behavior as all mice reached for seeds within 5 d. Using this paradigm, we assessed stroke-induced deficits in home-cage reaching. Following three weeks of baseline training, reaching success rate was ∼40%, with most trials performed during the dark cycle. A forelimb motor cortex stroke significantly decreased interaction with presented seeds within the first 2 d and impaired reaching success rates for the first 7 d. Our data demonstrate that group-housed mice can be efficiently trained on a single pellet reaching task in the home cage and that this assay is sensitive to stroke induced motor impairments.
Ischemic stroke is the second leading cause of death worldwide. Following an ischemic event, neuronal death is triggered by uncontrolled glutamate release leading to overactivation of glutamate sensitive N-methyl-d-aspartate receptor (NMDAR). For gating, NMDARs require not only the binding of glutamate, but also of glycine or a glycine-like compound as a co-agonist. Low glycine doses enhance NMDAR function, whereas high doses trigger glycine-induced NMDAR internalization (GINI) in vitro. Here, we report that following an ischemic event, in vivo, GINI also occurs and provides neuroprotection in the presence of a GlyT1 antagonist (GlyT1-A). Mice pretreated with a GlyT1-A, which increases synaptic glycine levels, exhibited smaller stroke volume, reduced cell death, and minimized behavioral deficits following stroke induction by either photothrombosis or endothelin-1. Moreover, we show evidence that in ischemic conditions, GlyT1-As preserve the vasculature in the peri-infarct area. Therefore, GlyT1 could be a new target for the treatment of ischemic stroke.
Abstract Glycine fulfills several roles in biology including protein synthesis, inhibitory transmission via glycine receptor activation and excitatory transmission through glutamate-sensitive N-methyl-D-aspartate receptors (NMDARs). Low glycine doses enhance NMDAR function while high doses trigger glycine-induced NMDAR internalization (GINI) in vitro. The physiological relevance of GINI has been questioned given that the high-affinity glycine transporter type 1 (GlyT1), located on astrocytes and neurons, maintains synaptic glycine concentrations far below the level that would saturate the glycine binding site (GBS) on NMDARs. Here, we report evidence that GINI occurs also in vivo and is neuroprotective following ischemic insult. Mice pre-treated with a GlyT1 antagonist (GlyT1-A), which increased glycine levels, exhibited smaller stroke volume, reduced cell death, and minimized behavioural deficits following stroke induction by either photothrombosis or endothelin-1. We demonstrate that in a modified in vitro ischemic paradigm, glycine is released at levels surpassing what occurs during ischemia alone. Therefore, glycine accumulates in the synaptic cleft, enhances occupancy of GBS and reaches the set point to trigger GINI. We report that GINI is observed during stroke, in vivo, only in the presence of a GlyT1-A. Moreover, we show evidence of a protective effect on the vasculature in the peri-infarct area. Therefore, these data strongly suggest that GlyT1 is a therapeutic target to prevent cell death following an ischemic event.
Abstract Ischemic stroke is the second leading cause of death worldwide. Compelling evidence demonstrates that following an ischemic event, neuronal death is triggered by uncontrolled glutamate release leading to overactivation of glutamate sensitive N-methyl-D-aspartate receptor (NMDAR). For gating, NMDARs require not only the binding of glutamate, but also the binding of glycine or a glycine-like compound as a co-agonist. Glycine fulfills several roles in biology including protein synthesis, inhibitory transmission via glycine receptor activation and excitatory transmission through NMDARs. Low glycine doses enhance NMDAR function while high doses trigger glycine-induced NMDAR internalization (GINI) in vitro. The physiological relevance of GINI has been questioned given that the high-affinity glycine transporter type 1 (GlyT1), located on astrocytes and neurons, maintains synaptic glycine concentrations far below the level that would saturate the glycine binding site (GBS). Here, we report that following an ischemic event, in vivo, GINI also occurs and provides neuroprotection. Mice pre-treated with a GlyT1 antagonist (GlyT1-A), which increases glycine synaptic levels, exhibited smaller stroke volume, reduced cell death, and minimized behavioural deficits following stroke induction by either photothrombosis or endothelin-1. Therefore, increasing glycine levels in the synaptic cleft will enhance GBS occupancy and will reach the set point to trigger GINI. However, we report that GINI is triggered during ischemic stroke, in vivo, only in the presence of GlyT1-As. Moreover, we show evidence that in ischemic conditions, GlyT1-As preserve the vasculature in the peri-infarct area. Therefore, the clinical efficacy of GlyT1-As should be tested for the treatment of ischemic stroke.
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