Glutamate and glutamate receptors in the vertebrate retina by Victoria Connaughton

Victoria Connaughton

1. General overview of synaptic transmission.

Cells communicate with each other electrically, through gap junctions, and chemically, using neurotransmitters. Chemical synaptic transmission allows nerve signals to be exchanged between cells which are electrically isolated from each other. The chemical messenger, or neurotransmitter, provides a way to send the signal across the extracellular space, from the presynaptic neuron to the postsynaptic cell. The space is called a cleft and is typically more than 10 nanometers across. Neurotransmitters are synthesized in the presynaptic cell and stored in vesicles in presynaptic processes, such as the axon terminal. When the presynaptic neuron is stimulated, calcium channels open and the influx of calcium ions into the axon terminal triggers a cascade of events leading to the release of neurotransmitter. Once released, the neurotransmitter diffuses across the cleft and binds to receptors on the postsynaptic cell, allowing the signal to propagate. Neurotransmitter molecules can also bind onto presynaptic autoreceptors and transporters, regulating subsequent release and clearing excess neurotransmitter from the cleft. Compounds classified as neurotransmitters have several characteristics in common (reviewed in Massey, 1990, Erulkar, 1994). Briefly, (1) the neurotransmitter is synthesized, stored, and released from the presynaptic terminal. (2) Specific neurotransmitter receptors are localized on the postsynaptic cells, and (3) there exists a mechanism to stop neurotransmitter release and clear molecules from the cleft. Common neurotransmitters in the retina are glutamate, GABA, glycine, dopamine, and acetylcholine. Neurotransmitter compounds can be small molecules, such as glutamate and glycine, or large peptides, such as vasoactive intestinal peptide (VIP). Some neuroactive compounds are amino acids, which also have metabolic functions in the presynaptic cell.

Fig. 1. Structure of the glutamate molecule

Glutamate (Fig. 1) is believed to be the major excitatory neurotransmitter in the retina. In general, glutamate is synthesized from ammonium and alpha-ketoglutarate (a component of the Krebs Cycle) and is used in the synthesis of proteins, other amino acids, and even other neurotransmitters (such as GABA; Stryer, 1988). Though glutamate is present in all neurons, only a few are glutamatergic, releasing glutamate as their neurotransmitter. Neuroactive glutamate is stored in synaptic vesicles in presynaptic axon terminals (Fykse and Fonnum, 1996). Glutamate is incorporated into the vesicles by a glutamate transporter located in the vesicular membrane. This transporter selectively accumulates glutamate through a sodium-independent, ATP-dependent process (Naito and Ueda, 1983, Tabb and Ueda, 1991, Fykse and Fonnum, 1996), resulting in a high concentration of glutamate in each vesicle. Neuroactive glutamate is classified as an excitatory amino acid (EAA) because glutamate binding onto postsynaptic receptors typically stimulates, or depolarizes, the postsynaptic cells.

2. Histological techniques identify glutamatergic neurons.

Fig. 2. Glutamate immunoreactivity

Using immunocytochemical techniques, neurons containing glutamate are identified and labeled with a glutamate antibody. In the retina, photoreceptors, bipolar cells, and ganglion cells are glutamate immunoreactive (Ehinger et al, 1988, Marc et al., 1990, Van Haesendonck and Missotten, 1990, Kalloniatis and Fletcher, 1993, Yang and Yazulla, 1994, Jojich and Pourcho, 1996) (Fig. 2). Some horizontal and/or amacrine cells can also display weak labeling with glutamate antibodies (Ehinger et al., 1988, Marc et al., 1990, Jojich and Pourcho, 1996; Yang, 1996). These neurons are believed to release GABA, not glutamate, as their neurotransmitter (Yazulla, 1986), suggesting the weak glutamate labeling reflects the pool of metabolic glutamate used in the synthesis of GABA. This has been supported by the results from double-labeling studies using antibodies to both GABA and glutamate: glutamate-positive amacrine cells also label with the GABA antibodies (Jojich and Pourcho, 1996, Yang, 1996).

Fig. 3. Autoradiogram of glutamate uptake through glutamate transporters

Photoreceptors, which contain glutamate, actively take up radiolabeled glutamate from the extracellular space, as do Muller cells (Fig. 3) (Marc and Lam, 1981; Yang and Wu, 1997). Glutamate is incorporated into these cell types through a high affinity glutamate transporter located in the plasma membrane. Glutamate transporters maintain the concentration of glutamate within the synaptic cleft at low levels, preventing glutamate-induced cell death (Kanai et al., 1994). Though Muller cells take up glutamate, they do not label with glutamate antibodies (Jojich and Pourcho, 1996). Glutamate incorporated into Muller cells is rapidly broken down into glutamine, which is then exported from glial cells and incorporated into surrounding neurons (Pow and Crook, 1996). Neurons can then synthesize glutamate from glutamine (Hertz, 1979, Pow and Crook, 1996).

Thus, histological techniques are used to identify potential glutamatergic neurons by labeling neurons containing glutamate (through immunocytochemistry) and neurons that take up glutamate (through autoradiography). To determine if these cell types actually release glutamate as their neurotransmitter, however, the receptors on postsynaptic cells have to be examined.

3. Glutamate receptors.

Once released from the presynaptic terminal, glutamate diffuses across the cleft and binds onto receptors located on the dendrites of the postsynaptic cell(s). Multiple glutamate receptor types have been identified. Though glutamate will bind onto all glutamate receptors, each receptor is characterized by its sensitivity to specific glutamate analogues and by the features of the glutamate-elicited current. Glutamate receptor agonists and antagonists are structurally similar to glutamate (Fig. 4), which allows them to bind onto glutamate receptors. These compounds are highly specific and, even in intact tissue, can be used in very low concentrations because they are poor substrates for glutamate uptake systems (Tachibana and Kaneko, 1988, Schwartz and Tachibana, 1990).

Fig. 4. Glutamate receptor agonists and antagonists

Two classes of glutamate receptors (Fig. 5) have been identified: (1) ionotropic glutamate receptors, which directly gate ion channels, and (2) metabotropic glutamate receptors, which may be coupled to an ion channel or other cellular functions via an intracellular second messenger cascade. These receptor types are similar in that they both bind glutamate and glutamate binding can influence the permeability of ion channels. However, there are several differences between the two classes.

Fig. 5. Ionotropic and metabotropic glutamate receptors and channels

4. Ionotropic glutamate receptors.

Glutamate binding onto an ionotropic receptor directly influences ion channel activity because the receptor and the ion channel form one complex (Fig. 5a). These receptors mediate fast synaptic transmission between neurons. Each ionotropic glutamate receptor, or iGluR, is formed from the co-assembly of individual subunits. The assembled subunits may or may not be homologous, with the different combinations of subunits resulting in channels with different characteristics (Keinanen et al., 1990, Verdoorn et al., 1991, Moyner et al., 1992; Nakanishi, 1992, Ozawa and Rossier, 1996).

Fig. 6. Comparison between NMDA and non-NMDA receptors

Two iGluR types (see Fig. 6) have been identified: (1) NMDA receptors, which bind glutamate and the glutamate analogue N-Methyl-D-Aspartate (NMDA) and (2) non-NMDA receptors, which are selectively agonized by kainate, AMPA, and quisqualate, but not NMDA.

Non-NMDA receptors. Glutamate binding onto a non-NMDA receptor opens non-selective cation channels more permeable to sodium (Na+) and potassium (K+) ions than calcium (Ca+2) (Mayer and Westbrook, 1987). Glutamate binding elicits a rapidly activating inward current at membrane potentials negative to 0 mV, and an outward current at potentials positive to 0 mV. Kainate, quisqualate, and AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) are the specific agonists at these receptors; CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dione-benzo[f]quinoxaline-7-sulfonamide), and DNQX (6,7-dinitroquinoxaline-2,3-dione) are the antagonists.

Fig. 7. Whole-cell patch clamp to show quisqualate and kainate gated currents

In retina, non-NMDA receptors have been identified on horizontal cells, OFF-bipolar cells, amacrine cells, and ganglion cells (see below). Patch clamp recordings (Gilbertson et al., 1991, Zhou et al., 1993, Boos et al., 1993, Cohen and Miller, 1994, Yu and Miller, 1995) indicate that AMPA, quisqualate, and/or kainate application can evoke currents in these cells. However, the kinetics of the ligand-gated currents differ. AMPA – and quisqualate-elicited currents rapidly desensitize; whereas, kainate-gated currents do not (Fig. 7a). The desensitization at AMPA/quisqualate receptors can be reduced (Fig. 7b) by adding cyclothiazide (Yamada and Tang, 1993), which stabilizes the receptor in an active (or non-desensitized) state (Yamada and Tang, 1993, Kessler et al., 1996).

Each non-NMDA receptor is formed from the co-assembly of several subunits (Boulter et al., 1990, Nakanishi et al., 1990, Nakanishi, 1992). To date, seven subunits (named GluR1 through GluR7) have been cloned (Hollmann et al., 1989, Boulter et al., 1990, Keinanen et al., 1990, Nakanishi et al., 1990, Bettler et al., 1990, 1992, Egebjerg et al., 1991). Expression of subunit clones in Xenopus oocytes revealed that GluR5, GluR6, and GluR7 (along with subunits KA1 and KA2) co-assemble to form kainate(-preferring) receptors; whereas, GluR1, GluR2, GluR3, and GluR4 are assembled into AMPA(-preferring) receptors (Nakanishi, 1992).

NMDA receptors. Glutamate binding onto an NMDA receptor also opens non-selective cation channels, resulting in a conductance increase. However, the high conductance channel associated with these receptors is more permeable to Ca+2 than Na+ ions (Mayer and Westbrook, 1987) and NMDA-gated currents typically have slower kinetics than kainate- and AMPA-gated channels. As the name suggests, NMDA is the selective agonist at these receptors. The compounds MK-801, AP-5 (2-amino-5-phosphonopentanoic acid), and AP-7 (2-amino-7-phosphoheptanoic acid) are NMDA receptor antagonists.

NMDA receptors are structurally complex, with separate binding sites for glutamate, glycine, magnesium ions (Mg+2), zinc ions (Zn+2), and a polyamine recognition site (Fig. 6b). There is also an antagonist binding site for PCP and MK-801 (Lodge, 1997). The glutamate, glycine, and magnesium binding sites are important for receptor activation and gating of the ion channel. In contrast, the zinc and polyamine sites are not needed for receptor activation, but affect the efficacy of the channel. Zinc blocks the channel in a voltage-independent manner (Westbrook and Mayer, 1987). The polyamine site (Ransom and Stec, 1988, Williams et al., 1994) binds compounds such as spermine or spermidine, either potentiating (Ranson and Stec, 1988; Williams et al., 1994) or inhibiting (Williams et al., 1994) the activity of the receptor, depending on the combination of subunits forming each NMDA receptor (Williams et al., 1994).

To date, five subunits (NR1, NR2a, N2b, N2c, and N2d) of NMDA receptors have been cloned (Moriyoshi et al., 1991, Ikeda et al., 1992, Katsuwada et al., 1992, Meguro et al., 1992, Ishii et al., 1993). As with non-NMDA receptors, NMDA receptor subunits can co-assemble as homomers (i.e., five NR1 subunits; Moyner et al., 1992, Moriyoshi et al., 1992) or heteromers (one NR1 + four NR2 subunits; Meguro et al., 1992, Katsuwada et al., 1992, Moyner et al., 1992, Ishii et al., 1993). However, all functional NMDA receptors express the NR1 subunit (Moyner et al., 1992, Nakanishi, 1992, Ishii et al., 1993).

Fig. 8. NMDA receptor activation

The glutamate, glycine, and Mg+2 binding sites confer both ligand-gated and voltage-gated properties onto NMDA receptors. NMDA receptors are ligand-gated because the binding of glutamate (ligand) is required to activate the channel. In addition, micromolar concentrations of glycine must also be present (Fig. 8) (Johnson and Ascher, 1987, Kleckner and Dingledine, 1988). The requirement for both glutamate and glycine makes them co-agonists (Kleckner and Dingledine, 1988) at NMDA receptors.

Mg+2 ions provide a voltage-dependent block of NMDA-gated channels (Nowak et al., 1984). This can be seen in the current-voltage (I-V) relationship presented in Fig. 9 (from Nowak et al., 1984).

Fig. 9. Mg+2 ions block NMDA receptor channels

I-V curves plotted from currents recorded in the presence of Mg+2 have a characteristic J-shape (dotted line); whereas, a linear relationship is calculated in Mg+2-free solutions (solid line). At negative membrane potentials, Mg+2 ions occupy the binding site causing less current to flow through the channel. As the membrane depolarizes, the Mg+2 block is removed (Nowak et al., 1984).

Retinal ganglion cells and some amacrine cell types express functional NMDA receptors in addition to non-NMDA receptors (i.e., Massey and Miller, 1988, 1990, Mittman et al., 1990, Dixon and Copenhagen 1992, Diamond and Copenhagen, 1993, Cohen and Miller, 1994). The currents elicited through these different iGluR types can be distinguished pharmacologically. Non-NMDA receptor antagonists block a transient component of the ganglion cell light response; whereas, NMDA receptor antagonists block a more sustained component (Mittman et al., 1990, Diamond and Copenhagen, 1993, Hensley et al., 1993, Cohen and Miller, 1994). These findings suggest the currents elicited through co-localized NMDA and non-NMDA receptors mediate differential contributions to the ON- and OFF-light responses observed in ganglion cells (i.e., Diamond and Copenhagen, 1993).

5. Metabotropic glutamate receptors.

Unlike ionotropic receptors, which are directly linked to an ion channel, metabotropic receptors are coupled to their associated ion channel through a second messenger pathway. Ligand (glutamate) binding activates a G-protein and initiates an intracellular cascade (Nestler and Duman, 1994). Metabotropic glutamate receptors (mGluRs) are not co-assembled from multiple subunits, but are one polypeptide (Fig. 5b). To date, eight mGluRs (mGluR1-mGluR8) have been cloned (Houamed et al., 1991, Masu et al., 1991, Abe et al., 1992, Tanabe et al., 1992, Nakajima et al., 1993, Saugstad et al., 1994, Duvoisin et al., 1995). These receptors are classified into three groups (I, II, and III) based on structural homology, agonist selectivity, and their associated second messenger cascade (Table 1, end of chapter) (reviewed in Nakanishi, 1994, Knopfel et al., 1995, Pin and Duvoisin, 1995, Pin and Bockaert, 1995).

In brief, Group I mGluRs (mGluR1 and mGluR5) are coupled to the hydrolysis of fatty acids and the release of calcium from internal stores. Quisqualate and trans-ACPD are Group I agonists. Group II (mGluR2 and mGluR3) and Group III (mGluRs 4, 6, 7, and 8) receptors are considered inhibitory because they are coupled to the downregulation of cyclic nucleotide synthesis (Pin and Duvoisin, 1995). L-CCG-1 and trans-ACPD agonize Group II receptors; L-AP4 (also called APB) selectively agonizes Group III receptors. In situ hybridization studies have revealed that the mRNAs encoding Group I, II, and III mGluRs are present in retina (see below); however, with the exception of the APB receptor, the function of all these receptor types in retina has not been characterized.

APB receptor. In contrast to non-NMDA and NMDA receptors, glutamate binding onto an APB receptor elicits a conductance decrease (Slaughter and Miller, 1981, Nawy and Copenhagen, 1987, 1990) due to the closure of cGMP-gated non-selective cation channels (Nawy and Jahr, 1990) (Fig. 10).

Fig. 10.Whole-cell current traces to show kinetics of APB receptor gated currents

APB application selectively blocks the ON-pathway in the retina (Fig. 11) (Slaughter and Miller, 1981), i.e., ON-bipolar cell responses and the ON-responses in amacrine cells (Taylor and Wassle, 1995) and ganglion cells (Cohen and Miller, 1994, Kittila and Massey, 1995, Jin and Brunken, 1996) are eliminated by APB. Experimental evidence (Slaughter and Miller 1981, Massey et al., 1983) suggests the APB receptor is localized to ON-bipolar cell dendrites. Inhibition of amacrine and ganglion cell light responses, therefore, is due to a decrease in the input from ON-bipolar cells, not a direct effect on postsynaptic receptors.

Fig. 11. Intracellular recordings to show that APB selectively antagonizes the ON-pathways

APB (2-amino-4-phosphobutyric acid, also called L-AP4) is the selective agonist for all Group III mGluRs (mGluR4, 6, 7, and 8). So, which is the APB receptor located on ON-bipolar cell dendrites? MGluR4, 7, and 8 expression has been observed in both the inner nuclear layer and the ganglion cell layer (Duvoisin et al., 1995, Hartveit et al., 1995) suggesting these mGluRs are associated with more than one cell type. In contrast, mGluR6 expression has been localized to the INL (Nakajima et al., 1993, Hartveit et al., 1995) and the OPL (Nomura et al., 1994) where bipolar cell somata and dendrites are located. Furthermore, ON-responses are abolished in mice lacking mGluR6 expression (Masu et al., 1995). These mutants also display abnormal ERG b-waves, suggesting an inhibition of the ON-retinal pathway at the level of bipolar cells (Masu et al., 1995). Taken together, these findings suggest the APB receptor on ON-bipolar cells is mGluR6.

6. Glutamate transporters and transporter-like receptors.

Glutamate transporters have been identified on photoreceptors (Marc and Lam, 1981, Tachibana and Kaneko, 1988, Eliasof and Werblin, 1993) and Muller cells (Marc and Lam, 1981, Yang and Wu, 1997). From glutamate labeling studies, the average concentration of glutamate in photoreceptors, bipolar cells, and ganglion cells is 5mM (Marc et al. 1990). Physiological studies using isolated cells indicate that only µM levels of glutamate are required to activate glutamate receptors (i.e., Aizenman et al., 1988, Zhou et al., 1993, Sasaki and Kaneko, 1996). Thus, the amount of glutamate released into the synaptic cleft is several orders of magnitude higher than the concentration required to activate most postsynaptic receptors. High affinity glutamate transporters located on adjacent neurons and surrounding glial cells rapidly remove glutamate from the synaptic cleft to prevent cell death (Kanai et al., 1994). Five glutamate transporters, EAAT-1 (or GLAST), EAAT-2 (or GLT-1), EAAT-3 (or EAAC-1), EAAT-4, and EAAT-5, have been cloned (Kanai and Hediger, 1992, Pines et al., 1992, Fairman et al., 1995, Schultz and Stell, 1996, Arriza et al., 1997, Kanai et al., 1997).

Glutamate transporters are pharmacologically distinct from both iGluRs and mGluRs. L-glutamate, L-aspartate, and D-aspartate are substrates for the transporters (Brew and Attwell, 1987, Tachibana and Kaneko, 1988, Eliasof and Werblin, 1993); glutamate receptor agonists (Brew and Attwell, 1987, Tachibana and Kaneko, 1988, Schwartz and Tachibana, 1990, Eliasof and Werblin, 1993) and antagonists (Barbour et al., 1991, Eliasof and Werblin, 1993) are not. Glutamate uptake can be blocked by the transporter blockers dihydrokainate (DHKA) and DL-threo-beta-hydroxyaspartate (HA) (Barbour et al., 1991, Eliasof and Werblin 1993).

Fig. 12 Glutamate transporters in Muller cells are electrogenic

Glutamate transporters incorporate glutamate into Muller cells along with the co-transport of three Na+ ions (Brew and Attwell, 1987, Barbour et al., 1988) and the antiport of one K+ ion (Barbour et al., 1988, Bouvier et al., 1992) and either one OH- or one HCO3- ion (Bouvier et al., 1992) (Fig. 12). The excess sodium ions generate a net positive inward current which drives the transporter (Brew and Attwell, 1987, Barbour et al., 1988). More recent findings indicate a glutamate-elicited chloride current is also associated with some transporters (Eliasof and Jahr, 1996, Arriza et al., 1997).

It should be noted that the glutamate transporters located in the plasma membrane of neuronal and glial cells (discussed in this section) are different from the glutamate transporters located on synaptic vesicles within presynaptic terminals (see section 1). The transporters in the plasma membrane transport glutamate in a Na+- and voltage-dependent manner independent of chloride (Brew and Attwell, 1987, Barbour et al., 1988, Kanai et al., 1994). L-glutamate, L-aspartate, and D-aspartate are substrates for these transporters (i.e., Brew and Attwell, 1987). In contrast, the vesicular transporter selectively concentrates glutamate into synaptic vesicles in a Na+-independent, ATP-dependent manner (Naito and Ueda, 1983, Tabb and Ueda, 1991, Fykse and Fonnum, 1996) that requires chloride (Tabb and Ueda, 1991, Fykse and Fonnum, 1996).

Glutamate receptors with transporter-like pharmacology have been described in photoreceptors (Picaud et al., 1995a, b, Grant and Werblin, 1996) and ON-bipolar cells (Grant and Dowling 1995, 1996). These receptors are coupled to a chloride current. The pharmacology of these receptors is similar to that described for glutamate transporters, as the glutamate-elicited current is (1) dependent upon external Na+, (2) reduced by transporter blockers, and (3) insensitive to glutamate agonists and antagonists. However, altering internal Na+ concentration does not change the reversal potential (Picaud et al., 1995b) or the amplitude (Grant and Werblin, 1995, Grant and Dowling, 1996) of the glutamate-elicited current, suggesting the receptor is distinct from glutamate transporters. At the photoreceptor terminals, the glutamate-elicited chloride current may regulate membrane potential and subsequent voltage-gated channel activity (i.e., Picaud et al., 1995a). Postsynaptically, this receptor is believed to mediate conductance changes underlying photoreceptor input to ON- cone bipolar cells (Grant and Dowling, 1995).

7. Localization of glutamate receptor types in the retina.

Fig. 13. The types of neurons in the vertebrate retina

Photoreceptor, bipolar, ganglion cells comprise the vertical transduction pathway in the retina. This pathway is modulated by lateral inputs from horizontal cells in the distal retina and amacrine cells in the proximal retina (Fig. 13). As described in the previous sections, photoreceptor, bipolar, and ganglion cells show glutamate immunoreactivity. Glutamate responses have been electrically characterized in horizontal and bipolar cells, which are postsynaptic to photoreceptors, and in amacrine and ganglion cells, which are postsynaptic to bipolar cells. Taken together, these results suggest glutamate is the neurotransmitter released by neurons in the vertical pathway. Recent in situ hybridization and immunocytochemical studies have localized the expression of iGluR subunits, mGluRs, and glutamate transporter proteins in the retina. These findings are summarized below.

8. Retinal neurons expressing ionotropic glutamate receptors.

Fig.14. Whole-cell currents in OFF bipolar cells

Fig. 15. Whole-cell currents in horizontal cells

In both higher and lower vertebrates, electrophysiological recording techniques have identified ionotropic glutamate receptors on the neurons comprising the OFF-pathway (Table 2, end of chapter). In the distal retina, OFF-bipolar cells (Fig. 14) (Euler et al., 1996, Sasaki and Kaneko, 1996, Hartveit, 1997) and horizontal cells (Fig. 15) (Yang and Wu, 1991, Zhou et al., 1993, Kriaj et al., 1994) respond to kainate, AMPA, and quisqualate application, but not NMDA nor APB. (However, NMDA receptors have been identified on catfish horizontal cells (OÍDell and Christensen, 1989, Eliasof and Jahr, 1997) and APB-induced hyperpolarizations have been reported in some fish horizontal cells (Nawy et al., 1989, Takahashi and Copenhagen, 1992, Furukawa et al., 1997)).

Non-NMDA agonists also stimulate both amacrine cells (Fig. 16a) (Massey and Miller, 1988, Dixon and Copenhagen, 1992, Boos et al., 1993) and ganglion cells (Fig. 16b) (Mittman et al., 1990, Diamond and Copenhagen, 1993, Hensley et al., 1993, Cohen and Miller, 1994, Yu and Miller, 1995). Ganglion cells responses to NMDA have been observed (Massey and Miller, 1988, 1990, Mittman et al., 1990, Diamond and Copenhagen, 1993, Cohen and Miller, 1994); whereas, NMDA responses have been recorded in only some types of amacrine cells (Massey and Miller, 1988, Dixon and Copenhagen, 1992, Boos et al., 1993, but see Hartveit and Veruki, 1997).

Fig. 16. Glutamate receptors on amacrine and ganglion cells

Consistent with this physiological data, antibodies to the different non-NMDA receptor subunits differentially label all retinal layers (Table 3, end of chapter; Hartveit et al., 1994, Peng et al., 1995, Hughes, 1997, Pourcho et al., 1997) and mRNAs encoding the different non-NMDA iGluR subunits are similarly expressed (Hughes et al., 1992, Hamassaki-Britto et al., 1993, Brandstatter et al., 1994). In contrast, mRNAs encoding NMDA subunits are expressed predominantly in the proximal retina, where amacrine and ganglion cells are located (INL, IPL, GCL; Table 3) (Brandstatter et al., 1994, Hartveit et al., 1994), though mRNA encoding the NR2a subunit (Hartveit et al., 1994) has been observed in the OPL and antibodies to the NR2d (Wenzel et al., 1997) and the NR1 subunits (Hughes, 1997) label rod bipolar cells.

9. Retinal neurons expressing metabotropic glutamate receptors.

All metabotropic glutamate receptors, except mGluR3, have been identified in retina either through antibody staining (Peng et al., 1995, Brandstatter et al., 1996, Koulen et al., 1997, Pourcho et al., 1997) or in situ hybridization (Nakajima et al., 1993, Duvoisin et al., 1995, Hartveit et al., 1995). MGluRs are differentially expressed throughout the retina, specifically in the outer plexiform layer, inner nuclear layer, inner plexiform layer, and the ganglion cell layer (Table 4, end of chapter). Though different patterns of mGluR expression have been observed in the retina, only the APB receptor on ON-bipolar cells has been physiologically examined.

10. Retinal neurons expressing glutamate transporters.

The glutamate transporters GLAST, EAAC1, and GLT-1have been identified in retina (Table 5, end of chapter). GLAST (L-glutamate/L-aspartate transporter) immunoreactivity is found in all retinal layers (Otori et al. 1994), but not in neuronal tissue. GLAST is localized to Muller cell membranes (Otori et al. 1994, Derouiche and Rauen, 1995, Rauen et al., 1996, Lehre et al., 1997). In contrast, EAAC-1 (excitatory amino acid carrier-1) antibodies do not label Muller cells or photoreceptors. EAAC-1 immunoreactivity is observed in ganglion and amacrine cells in chicken, rat, goldfish, and turtle retinas. In addition, bipolar cells positive labeled with EAAC-1 antibody in lower vertebrates and immunopositive horizontal cells were observed in rat (Schultz and Stell, 1996). GLT-1 (glutamate transporter-1) proteins have been identified in monkey (Grunert et al., 1994), rat (Rauen et al., 1996), and rabbit (Massey et al., 1997) bipolar cells. In addition, a few amacrine cells were weakly labeled with the GLT-1 antibody in rat (Rauen et al., 1996), as were photoreceptor terminals in rabbit (Massey et al., 1997).

11. Summary and conclusions.

Fig. 17. The ribbon glutamatergic synapse in the retina

Histological analyses of presynaptic neurons and physiological recordings from postsynaptic cells suggest photoreceptor, bipolar, and ganglion cells release glutamate as their neurotransmitter. Multiple glutamate receptor types are present in the retina. These receptors are pharmacologically distinct and differentially distributed. IGluRs directly gate ion channels and mediate rapid synaptic transmission through either kainate/AMPA or NMDA receptors. Glutamate binding onto iGluRs opens cation channels, depolarizing the postsynaptic cell membrane. Neurons within the OFF-pathway (horizontal cells, OFF-bipolar cells, amacrine cells, and ganglion cells) express functional iGluRs. MGluRs are coupled to G-proteins. Glutamate binding onto mGluRs can have a variety of effects depending on the second messenger cascade to which the receptor is coupled. The APB receptor, found on ON-bipolar cell dendrites, is coupled to the synthesis of cGMP. At these receptors, glutamate decreases cGMP formation leading to the closure of ion channels. Glutamate transporters, found on glial and photoreceptor cells, are also present at glutamatergic synapses (Fig. 17). Transporters remove excess glutamate from the synaptic cleft to prevent neurotoxicity. Thus, postsynaptic responses to glutamate are determined by the distribution of receptors and transporters at a glutamatergic synapses which, in retina, determine the conductance mechanisms underlying visual information processing within the ON- and OFF-pathways.

Tables

Table 1

Metabotropic glutamate receptor groups (from Pin and Duvoisin, 1995).

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group	mGluR	agonist(s)	intracellular pathway
I	mGluR1, mGluR5	quisqualate, ACPD	increase phospholipase C activity, increase cAMP levels, increase protein kinase A activity
II	mGluR2, mGluR3	L-CCG-1, ACPD	decrease cAMP levels
III	mGluR4, mGluR6. mGluR7, mGluR8	L-AP4 (APB)	decrease cAMP or cGMP levels

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Table 2

Glutamate receptor types on retinal neurons, electrophysiological measurements.

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retinal cell type	non-NMDA receptor	NMDA receptor	mGluR	Glutamate receptor with transporter-like pharmacology	species	references
photoreceptors				++ (cones)	salamander	Eliasof & Werblin, 1993; Picaud et al., 1995b
				++ (rods)	salamander	Grant & Werblin, 1996
OFF-bipolar cells	++				mudpuppy	Slaughter & Miller, 1981, 1983
	++				cat	Sasaki & Kaneko, 1996
	++				salamander	Hensley et al., 1993
	++				rat	Euler et al., 1996
	++				mudpuppy	Slaughter & Miller, 1983
ON-bipolar cells	++		++(APB)		mudpuppy	Slaughter & Miller, 1981, 1983
			++(APB)	++	white perch	Grant & Dowling, 1995, 1996
			++(APB)		salamander	Hirano & MacLeish, 1991
			++(L-AP4)		salamander	Hensley et al., 1993
			++(AP-4)		rat	Euler et al., 1996
			++(APB and cGMP)		salamander	Nawy & Jahr, 1990
			++(APB and cGMP)		cat	de la Villa et al., (1995)
horizontal cells	++				white perch	Zhou et al., 1993
	++				mudpuppy	Slaughter & Miller, 1983
	++				salamander	Yang & Wu, 1991
	++	++			catfish	O’Dell & Christensen, 1989; Eliasof & Jahr, 1997
amacrine cells	++(AII)				rat	Boos et al., 1993
	++	++			mudpuppy	Slaughter & Miller, 1983
	++	++			rabbit	Massey & Miller, 1988
	++	++			rat	Hartveit & Veruki, 1997
	++ (transient & sustained AC)	++ (transient AC)			salamander	Dixon & Copenhagen, 1992
ganglion cells	++	++			salamander	Diamond & Copenhagen, 1993; Mittman et al., 1990; Hensley et al., 1993
	++	++			primates	Cohen & Miller, 1994
	++	++			rat	Aizenman et al., 1988
	++	++			mudpuppy	Slaughter & Miller, 1983
	++	++			cat	Cohen et al., 1994
	++	++			rabbit	Massey & Miller, 1988; 1990

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Table 3

Ionotropic glutamate receptor expression in retinal neurons and retinal layers, immunocytochemistry and in situ hybridization.

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retinal cell type or layer	non-NMDA receptor subunits	NMDA receptor subunits	species	references
photoreceptors	GluR6/7 (single cone outer segments)		goldfish	Peng et al., 1995
	GluR1 (cone pedicles)		cat	Pourcho et al., 1997
OPL	GluR2, GluR2/3, GluR6/7		rat	Peng et al., 1995
		NR2A (punctate)	cat	Hartveit et al., 1994
	GluR2, GluR2/3 (photoreceptors)		goldfish	Peng et al., 1995
bipolar cells	GluR2 (Mb cells)		goldfish	Peng et al., 1995
	GluR2, GluR2/3		rat	Peng et al., 1995
		NR2D (RBC)	rat	Wenzel et al., 1997
	GluR2 and/or GluR4	NR1 (RBC)	rat	Hughes, 1997
	GluR2 (RBC)		rat	Hughes et al., 1992
horizontal cells	GluR6/7		goldfish	Peng et al., 1995
	GluR2/3		cat	Pourcho et al., 1997
INL	GluR2/3, GluR6/7		rat	Peng et al., 1995
		NR2A(inner)	rat	Hartveit et al., 1994
	GluR1, 2, 5 > GluR4 (outer third), GluR1, 2, 5 (middle third), GluR1-5 (inner third)		rat	Hughes et al. 1992
	GluR1-7		rat, cat	Hamassaki-Britto et al., 1993
	KA2 (homogenous), GluR6 (inner), GluR7 (inner two thirds)	NR1 (homogenous), NR2A-B (inner third, patchy), NR2C (inner two-thirds)	rat	Brandstatter et al., 1994
IPL	GluR1, GluR2/3, GluR6/7		rat	Peng et al., 1995
		NR2A	rat, cat, rabbit, monkey	Hartveit et al., 1994
amacrine cells	GluR6	NR2A-C	rat	Brandstatter et al., 1994
	GluR2/3		cat	Pourcho et al., 1997
	GluR1, GluR2/3		rat	Peng et al., 1995
ganglion cells	GluR1		rat	Peng et al., 1995
GCL	GluR2/3, GluR6/7		rat	Peng et al., 1995
	GluR1-5		rat	Hughes et al., 1992
	GluR1-7		rat, cat	Hamassaki-Britto et al., 1993
	GluR6-7, KA2	NR1, NR2A-C	rat	Brandstatter et al., 1994
Muller cells	GluR4		rat	Peng et al., 1995

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Table 4

Metabotropic glutamate receptor expression in retinal neurons and retinal layers, immunocytochemistry and in situ hybridization.

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retinal cell type or layer	Group 1	Group II	Group III	species	references
OPL	mGluR1alpha, mGluR5a (RBC dendrites)			rat	Koulen et al., 1997
			mGluR6 (RBC dendrites)	rat	Nomura et al., 1994
INL			mGluR8	mouse	Duvoisin et al., 1995
			mGluR6	rat	Nakajima et al., 1993
	mGluR5 (BC, HC), mGluR1 (AC)	mGluR2 (AC)	mGluR6 (RBC), mGluR7 (BC), mGluR4,7 (AC)	rat	Hartveit et al., 1995
IPL	mGluR1alpha			rat	Peng et al., 1995
			mGluR7 (CBC terminals; AC dendrites; few GC dendrites)	rat	Brandstatter et al., 1996
	mGluR1alpha, mGluR5a (AC dendrites)			rat	Koulen et al., 1997
amacrine cells	mGluR1alpha			rat	Peng et al., 1995
	mGluR1alpha	mGluR2/3		cat	Pourcho et al., 1997
ganglion cells	mGluR1alpha			rat	Peng et al., 1995
GCL			mGluR8	mouse	Duvoisin et al., 1995
	mGluR1alpha	mGluR2/3		cat	Pourcho et al., 1997
	mGluR1	mGluR2	mGluR4, 7	rat	Hartveit et al., 1995

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Table 5

Glutamate transporters in retinal neurons and retinal layers, immunocytochemical localizations.

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retinal cell type	EAAC-1	GLAST	GLT-1	species	reference
photoreceptors			+ (cone soma to pedicles)	rabbit	Massey et al., 1997
OPL	++			rat	Rauen et al., 1996
			++ (rod spherules > cone pedicles)	rabbit	Massey et al., 1997
horizontal cells	++			rat	Schultz & Stell, 1996; Rauen et al., 1996
bipolar cells			++ (2 types of CBCs)	rabbit	Massey et al., 1997
	++ (faint)		++	rat	Rauen et al., 1996
	++			turtle, salamander	Schultz & Stell, 1996
			++ (DB2, flat midget bipolar cells)	monkey	Grunert et al., 1994
IPL			++ (diffuse)	rabbit	Massey et al., 1997
	++		++	rat	Rauen et al., 1996
	++			goldfish, salamander, turtle, chicken, rat	Schultz & Stell, 1996
amacrine cells	++		++	rat	Rauen et al., 1996
	++				Schultz & Stell, 1996
ganglion cells	++			chicken, rat, goldfish, turtle	Schultz & Stell, 1996
	++			rat	Rauen et al., 1996
Muller cells		++		rat	Rauen et al., 1996; Lehre et al., 1997; Deroiche & Rauen, 1995

---

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Last updated May 7, 2007.

The author Dr. Victoria Connaughton was born in Sellersville Pennsylvania. She received her B.A. from Bucknell University in Biology in 1989 and her Ph. D. in Marine Studies from The University of Delaware in 1994. She is currently an Assistant Professor in the Biology Department at American University, Washington D.C. In her thesis work under Dr. Charles Epifano, she studied the visually guided feeding behavior of larval fish. Dr. Connaughton pursued postdoctoral studies with Dr. Greg Maguire at the University of Houston, and Dr. Ralph Nelson at the National Institutes of Health. Dr. Connaughton’s current research interests include electrophysiological examination of zebrafish mutants with visual system defects and the characterization of light responses in zebrafish retinal bipolar cells.