Immunocytochemical localization of the glutamate transporter GLT-1 in goldfish (Carassius auratus) retina.

Glutamate is the major excitatory neurotransmitter in the retina of vertebrates. Electrophysiological experiments in goldfish and salamander have shown that neuronal glutamate transporters play an important role in the clearance of glutamate from cone synaptic clefts. In this study, the localization of the glutamate transporter GLT-1 has been investigated immunocytochemically at the light and electron microscopical levels in the goldfish retina using a GLT-1-specific antibody. GLT immunoreactivity (IR) was observed at the light microscopical level in Müller cells, bipolar cells, the outer plexiform layer (OPL), and the inner plexiform layer (IPL). At the electron microscopical level, membrane-bound and cytoplasmic GLT-IR in the OPL was located in finger-like protrusions of the cone terminal located near the invaginating postsynaptic processes of bipolar and horizontal cells. GLT-IR was not observed in the vicinity of synaptic ribbons. This location of GLT-1 allows modulation of the glutamate concentration in the synaptic cleft, thereby shaping the dynamics of synaptic transmission between cones and second-order neurons. In the inner IPL, GLT-IR was observed in the cytoplasm and was membrane bound in mixed rod/cone bipolar cell terminals and cone bipolar cell terminals. The membrane-bound GLT-1 was generally observed at some distance from the synaptic ribbon. The morphology of the bipolar cell terminal together with the localization of GLT-1 suggests that at least these glutamate transporters are not primarily involved in rapid uptake of glutamate release by the bipolar cells. The GLT-IR in the cytoplasm of Müller cells was located throughout the entire goldfish retina from the outer limiting membrane to the inner limiting membrane. The location of GLT-1 in Müller cells is consistent with the role of Müller cells in converting glutamate to glutamine.

Clearance of neurotransmitter from the cone synaptic cleft in goldfish retina.

Dendrites of bipolar and horizontal cells protrude deeply into the synaptic terminals of cones in goldfish retina. This arrangement gives the impression that the cone synaptic terminal surrounds a morphologically shielded compartment, the cone synaptic cleft, from which clearance of neurotransmitter by diffusion is limited. In this study the time constant of this clearance has been approached in two ways: (1) the morphological parameters determining the clearance (extracellular synaptic volume and leak area), were estimated using morphometric methods. These data were introduced into a diffusion model of the cone pedicle, yielding a time constant for the clearance of < 1 msec; (2) the time constant of the light onset response when the glutamate transporter in the cone was blocked with DL-threo-beta-hydroxyaspartate or dihydrokainate, was interpreted as the time constant of the clearance, yielding values of almost 650 msec compared to around 90 msec in control conditions. The decay time-constant of the Ca-dependent tail-currents in cones was used, as an approximation of the dynamics of the intracellular Ca-concentration and thus of the glutamate release by the cones. The decay time constant was about 800 msec. This suggests that the intracellular Ca-concentration in the synaptic terminal and hence the glutamate release by the cones drops with a similar large time constant. These results indicate that the cone pedicle in goldfish does not limit the clearance of neurotransmitters from the synaptic cleft and that the fast light onset response of horizontal cells under control conditions is due to activation of glutamate transporters by hyperpolarization of the cone membrane potential while the glutamate release drops slowly. The slow horizontal cell light onset response in beta-hydroxyaspartate or dihydrokainate may be due to a slow reduction of the glutamate release by the cones at light onset.

Expression and localization of ionotropic glutamate receptor subunits in the goldfish retina--an in situ hybridization and immunocytochemical study.

The expression and distribution of AMPA, kainate and NMDA glutamate receptor subunits was studied in the goldfish retina. For the immunocytochemical localization of the AMPA receptor antisera against GluR2, GluR2/3 and GluR4 were used, and for in situ hybridization rat specific probes for GluR1 and GluR2 and goldfish specific probes for GluR3 and GluR4 were used. The localization of the low affinity kainate receptor and NMDA receptor was studied using antisera against GluR5-7 and NR1. All AMPA receptor subtypes were demonstrated to be present in the goldfish retina both by immunocytochemistry and in situ hybridization. In situ hybridization revealed expression of all AMPA receptors subunit at the inner border of the INL. Only GluR3 was also strongly expressed in the outer border of the INL. Some of the ganglion cells displayed a strong signal for GluR1, GluR3 and GluR4. GluR1-immunoreactivity was present in subsets of bipolar, amacrine, and ganglion cells. GluR2 and GluR2/3-immunoreactivity was mainly localized in the outer plexiform layer. GluR2 and GluR2/3-immunoreactivity are associated with the photoreceptor synaptic terminals. GluR4-immunoreactivity is present on Müller cells in the inner retina and on dendrites of bipolar cells in the OPL, whereas GluR5-7-immunoreactivity was prominently present on horizontal cell axon terminals. Finally, NR1-immunoreactivity was confined to amacrine cells, the inner plexiform layer and ganglion cells. This study shows that there is a strong heterogeneity of glutamate receptor subunit expression in the various layers of the retina. Of the AMPA receptor subunits GluR3 seems to be expressed the most widely in all layers with strong glutamatergic synaptic interactions whereas all the other subunits seem to have a more restricted expressed pattern.