Abstracts

Rationale: Glutamate reuptake is critical to brain development and function. Decreased glutamate reuptake by reactive astrocytes is thought to contribute to pathological network activity by increasing extracellular glutamate levels. We have investigated the maturation of glutamate reuptake in the developing rat neocortex and hippocampus and in a model of cortical brain malformation associated with network hyperexcitability. We hypothesize that loss of astrocytic glutamate reuptake during neonatal cortical development may directly contribute to pathological synaptogenesis and may be an important component of neonatal epileptogenesis. Methods: Cortical malformations were created by briefly placing a freezing probe on the skulls of neonatal rat pups. Neocortical brain slices from sham operated and freeze lesioned rats were prepared 3-60 days later. Brain slices were then loaded with glutamate FRET biosensor and MNI-glutamate was photolysed using UV laser light. Glutamate biosensor images were collected using a high-speed RedShirt NeuroCCD camera. Glutamate transporter currents were recorded by patch-clamping astrocytes at -80mV and photolysing MNI-glutamate. Results: We have found that protein expression of GLT-1, GLAST, and glutamine synthetase reach adult levels earlier in the hippocampus as compared to neocortex. We next used a combination of FRET-based glutamate biosensor imaging and UV laser photolysis of MNI-glutamate to assay glutamate reuptake in acute brain slices. Recovery from an exogenous glutamate challenge was faster in the immature hippocampus as compared to neocortex. We patch clamped astrocytes in the developing neocortex and hippocampus and recorded glutamate transporter currents evoked by UV laser photolysis of MNI-glutamate. Glutamate transporter decay kinetics were significantly slower in the neocortex as compared to the hippocampus until P21. We next examined how neonatal brain insult affected glutamate transport by astrocytes. Rat pups were anesthetized and a cortical freeze lesion was performed. We examined glutamate reuptake in freeze lesioned animals at P7, a time when reactive astrocytosis is high. We found that in the area surrounding the freeze lesion GLT-1 protein expression was decreased. Combined FRET-based glutamate biosensor imaging and UV laser photolysis of MNI-glutamate revealed a decreased ability to recover from exogenous glutamate challenges. Surprisingly, however, glutamate transporter currents recorded form individual astrocytes had faster decay kinetics in the freeze lesioned neocortex. Conclusions: Our results suggest that while global glutamate transport capacity may be compromised, individual astrocytes have robust glutamate transport in the neonatal brain following injury. Future studies will examine this apparent disconnect between individual astrocyte function and global glutamate handling and its role in increasing synaptogenesis. Overall, these studies indicate that there is a developmental delay in the maturation of neocortical glutamate reuptake compared to hippocampus and that insult has complex effects on glutamate reuptake.