The glutamate-aspartate transporter GLAST mediates glutamate uptake at inner hair cell afferent synapses in the mammalian cochlea.
Ribbon synapses formed between inner hair cells (IHCs) and afferent dendrites in the mammalian cochlea can sustain high rates of release, placing strong demands on glutamate clearance mechanisms. To investigate the role of transporters in glutamate removal at these synapses, we made whole-cell recordings from IHCs, afferent dendrites, and glial cells adjacent to IHCs [inner phalangeal cells (IPCs)] in whole-mount preparations of rat organ of Corti. Focal application of the transporter substrate D-aspartate elicited inward currents in IPCs, which were larger in the presence of anions that permeate the transporter-associated anion channel and blocked by the transporter antagonist D,L-threo-beta-benzyloxyaspartate. These currents were produced by glutamate-aspartate transporters (GLAST) (excitatory amino acid transporter 1) because they were weakly inhibited by dihydrokainate, an antagonist of glutamate transporter-1 (excitatory amino acid transporter 2) and were absent from IPCs in GLAST-/- cochleas. Furthermore, D-aspartate-induced currents in outside-out patches from IPCs exhibited larger steady-state currents than responses elicited by L-glutamate, a prominent feature of GLAST, and examination of cochlea from GLAST-Discosoma red (DsRed) promoter reporter mice revealed that DsRed expression was restricted to IPCs and other supporting cells surrounding IHCs. Saturation of transporters by photolysis of caged D-aspartate failed to elicit transporter currents in IHCs, as did local application of D-aspartate to afferent terminals, indicating that neither presynaptic nor postsynaptic membranes are major sites for glutamate removal. These data indicate that GLAST in supporting cells is responsible for transmitter uptake at IHC afferent synapses.
Glowatzki
Elisabeth
E
Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins University, Baltimore, Maryland 21205, USA.
Cheng
Ning
N
Hiel
Hakim
H
Yi
Eunyoung
E
Tanaka
Kohichi
K
Ellis-Davies
Graham C R
GC
Rothstein
Jeffrey D
JD
Bergles
Dwight E
DE
D027322:Amino Acid Transport System X-AG / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D003051:Cochlea / Q000378:metabolism / Q000502:physiology*; D003056:Cochlear Nerve / Q000166:cytology / Q000378:metabolism; D003712:Dendrites / Q000378:metabolism; D004553:Electric Conductivity; D018698:Glutamic Acid / Q000378:metabolism* / Q000493:pharmacokinetics; D006199:Hair Cells, Auditory, Inner / Q000378:metabolism / Q000502:physiology*; D066298:In Vitro Techniques; D051379:Mice; D008822:Mice, Transgenic; D009475:Neurons, Afferent / Q000378:metabolism / Q000648:ultrastructure; D009925:Organ of Corti / Q000166:cytology / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology; D051381:Rats; D013569:Synapses / Q000378:metabolism*
D027322:Amino Acid Transport System X-AG; D018698:Glutamic Acid