GLUTAMINE POTENTIATES GLUTAMATE RELEASE DURING HIGH FREQUENCY STIMULATION OF ISOLATED LAYER 1 AXONS
Abstract number :
3.356
Submission category :
13. Neuropathology of Epilepsy
Year :
2009
Submission ID :
10435
Source :
www.aesnet.org
Presentation date :
12/4/2009 12:00:00 AM
Published date :
Aug 26, 2009, 08:12 AM
Authors :
Richard Reimer, H. Tani, C. Dula and J. Huguenard
Rationale: The robust excitatory neurotransmission that occurs during seizures requires recycling of synaptically released glutamate. Although a neuronal-glial glutamate-glutamine cycle is thought to mediate this process, the system A transporters which have been implicated in neuronal glutamine uptake are not expressed in axons, raising questions about the current anatomical/molecular model of the cycle. To test whether the cycle functions locally at the synapse we have isolated layer 1 (L1) axons in neocortex. This layer is relatively acellular, but densely packed with corticocortical and thalamocortical projections that synapse on to L2/3 and L5 pyramidal neurons. We modified a prep in which L2 to white matter has been transected (Cauller and Connors, 1994) by placing an additional horizontal transection below L1 on one side of the vertical cut. This produces a panhandle containing axons that terminate on neurons in the neighboring intact cortex, but are separated from their cell bodies. With this prep we have examined whether isolated axons are capable of sustained synaptic transmission and are using pharmacological manipulations to test whether glutamate release is influenced by a synaptic glutamate-glutamine cycle. Methods: Coronal brain slices (350μm) from 3-8 week old rats were placed in an interface chamber with oxygenated aCSF and transected vertically from L2 to the subcortical white matter and then horizontally below L1 on one side of the vertical cut. The slice was then superfused with oxygenated aCSF at 2ml/min with APV (50μM). The isolated L1 fibers were stimulated with a concentric bipolar electrode with 300μA 100μsec pulses at 0.2Hz, or as indicated, and evoked field excitatory postsynaptic potentials (fEPSPs) were recorded with glass micropipetets. Results: Electrical stimulation of isolated L1 fibers at 0.2Hz resulted in fEPSPs of +0.2mV in L2/3 of the in the neighboring intact cortex with consistent field amplitudes maintained for over 1 hr. However bursts of high frequency stimulation (20Hz for 45 seconds repeated three times with 2.5 minutes between bursts) resulted in a reduction of fEPSP amplitude. Further there was a slow recovery to baseline fEPSP amplitudes after return to the 0.2Hz stimulus frequency. With the addition of glutamine, the reduction of fEPSP amplitude during the high frequency was attenuated (0.1mV compared to 0.15mV in the absence of exogenous glutamine) and the time to return to baseline amplitudes was markedly reduced (0.5 min compared to 5 min in the absence of exogenous glutamine), suggesting both acute and prolonged effects of glutamine. Conclusions: Isolated axons in L1 are capable of prolonged periods of glutamate release at low frequency, but rapidly fatigue with high frequency stimulation. The effect of glutamine on fEPSP amplitude modulation by high frequency stimulation suggests that presynaptic terminals are capable of taking up glutamine and participating in glutamine-glutamate cycle to replenish neurotransmitter pools even when separated from their cell bodies.
Neuropathology of Epilepsy