Glycinergic Interplexiform Cells Research Articles

Glycinergic feedback enhances synaptic gain in the distal retina

Glycine input originates with interplexiform cells, a group of neurons situated within the inner retina that transmit signals centrifugally to the distal retina. The effect on visual function of this novel mechanism is largely unknown. Using gramicidin-perforated patch whole cell recordings, intracellular recordings and specific antibody labelling techniques, we examined the effects of the synaptic connections between glycinergic interplexiform cells, photoreceptors and bipolar cells. To confirm that interplexiform cells make centrifugal feedback on bipolar cell dendrites, we recorded the postsynaptic glycine currents from axon-detached bipolar cells while stimulating presynaptic interplexiform cells. The results show that glycinergic interplexiform cells activate bipolar cell dendrites that express the α3 subunit of the glycine receptor, as well as a subclass of unidentified receptors on photoreceptors. By virtue of their synaptic contacts, glycine centrifugal feedback increases glutamate release from photoreceptors and suppresses the uptake of glutamate by the type 2A excitatory amino acid transporter on photoreceptors. The net effect is a significant increase in synaptic gain between photoreceptors and their second-order neurons.

Glycine receptors are functionally expressed on bullfrog retinal cone photoreceptors

Using immunocytochemical and whole cell recording techniques, we examined expression of glycine receptors on bullfrog retinal cone photoreceptors. Immunofluorescence double labeling experiments conducted on retinal sections and isolated cell preparations showed that terminals and inner segments of cones were immunoreactive to both α1 and β subunits of glycine receptors. Moreover, application of glycine induced a sustained inward current from isolated cones, which increased in amplitude in a dose-dependent manner, with an EC 50 (concentration of glycine producing half-maximal response) of 67.3±4.9 μM, and the current was blocked by the glycine receptor antagonist strychnine, but not 5,7-dichlorokynurenic acid (DCKA) of 200 μM, a blocker of the glycine recognition site at the N-methyl- d-aspartate (NMDA) receptor. The glycine-induced current reversed in polarity at a potential close to the calculated chloride equilibrium potential, and the reversal potential was changed as a function of the extracellular chloride concentration. These results suggest that strychnine-sensitive glycine receptors are functionally expressed in bullfrog cones, which may mediate signal feedback from glycinergic interplexiform cells to cones in the outer retina.

Differential distribution of glycine transporters in Müller cells and neurons in amphibian retinas

Amphibian retinas are commonly used for electrophysiological studies on neural function and transduction because they share the same general properties as higher vertebrate retinas. Glycinergic synapses have been well described in amphibian retinas. However, the role of glycine transporters in the synapses is largely unknown. We studied the distribution and function of glycine transporters in the retinas from tiger salamanders, mudpuppies, and leopard frogs by immunofluorescence labeling and whole-cell recording methods. Our results indicated that GlyT1- and GlyT2-like transporters were present in Müller cells and neurons, respectively. GlyT1 labeling was present in Müller glial cells and co-localized with Glial fibrillary acidic protein (GFAP), a Müller cell marker, whereas the GlyT2 immunoreactivity was present in the somas of amacrine cells (ACs) and processes in the inner plexiform layer (IPL) and the outer plexiform layer (OPL). Because the axon processes of glycinergic interplexiform cells (IPCs) are the only source of glycine input in the OPL, GlyT2 staining revealed a spatial pattern of the axon processes of IPCs in the OPL. The function of GlyT2 in the IPCs was studied in tiger salamander retinal horizontal cells (HCs) by whole-cell gramicidin perforated recording. The results demonstrated that inhibition of GlyT2 by a specific inhibitor, amoxapine, increased a tonic glycine input to HCs. Thus, the GlyT2 transporter is responsible for uptake of synaptic glycine in the outer retina. We also compared the distribution of glycine transporters in other amphibian species: salamander, mudpuppy, and frog. The results are consistent with the general pattern that GlyT1-like transporters are present in Müller cells and GlyT2-like transporters in neurons in amphibian retinas.

Characterization of glycinergic synapses in vertebrate retinas

Glycine is one of the essential neurotransmitters modulating visual signals in retina. Glycine activates Cl(-) permeable receptors that conduct either inhibitory or excitatory actions, depending on the Cl(-) electrical-chemical gradient (E (Cl)) positive or negative to the resting potential in the cells. Interestingly, both glycine-induced inhibitory and excitatory responses are present in adult retinas, and the effects are confined in the inner and outer retinal neurons. Glycine inhibits glutamate synapses in the inner plexiform layer (IPL), resulting in shaping light responses in ganglion cells. In contrast, glycine excites horizontal cells and On-bipolar dendrites in the outer plexiform layer (OPL). The function of glycinergic synapse in the outer retina represents the effect of network feedback from a group of centrifugal neurons, glycinergic interplexiform cells. Moreover, immunocytochemical studies identify glycine receptor subunits (alpha1, alpha2, alpha3 and beta) in retinas, forming picrotoxin-sensitive alpha-homomeric and picrotoxin-insensitive alpha/beta-heteromeric receptors. Glycine receptors are modulated by intracellular Ca(2+) and protein kinas C and A pathways. Extracellular Zn(2+) regulates glycine receptors in a concentration-dependent manner, nanomolar Zn(2+) enhancing glycine responses, and micromolar Zn(2+) suppressing glycine responses in retinal neurons. These studies describe the function and mechanism of glycinergic synapses in retinas.

Glycine receptors in a population of adult mammalian cones

Glycinergic interplexiform cells provide a feedback signal from the inner retina to the outer retina. To determine if cones receive such a signal, glycine was applied on cultured porcine cone photoreceptors recorded with the patch clamp technique. A minor population of cone photoreceptors was found to generate large currents in response to puff application of glycine. These currents reversed close to the calculated equilibrium potential for chloride ions. These glycine-elicited currents were sensitive to strychnine but not to picrotoxin consistent with the expression of alpha-beta-heteromeric glycine receptors. Glycine receptors were also activated by taurine and beta-alanine. The glycine receptor antibody mAb4a labelled a minority of the cone photoreceptors identified by an antibody specific for cone arrestin. Finally, expression of the beta subunit of the glycine receptor was demonstrated by single cell RT-PCR in a similar proportion (approximately 13%) of cone photoreceptors freshly isolated by lectin-panning. The identity of cone photoreceptors was assessed by their specific expression of the cone arrestin mRNA. The population of cone photoreceptors expressing the glycine receptor was not correlated to a specific colour-sensitive subtype as demonstrated by single cell RT-PCR experiments using primers for S opsin, cone arrestin and glycine receptor beta subunit. This glycine receptor expression in a minority of cones defines a new cone population suggesting an unexpected role for glycine in the visual information processing in the outer retina.

Amino acid neurotransmitters in the retina: a functional overview.

Physiological and pharmacological mechanisms of glutamatergic, GABAergic and glycinergic synapses in the tiger salamander retina were studied. We used immunocytochemical and autoradiographic methods to study localizations of these neurotransmitters and their uptake transporters; and electrophysiological methods (intracellular, extracellular and whole cell patch electrode recordings) to study the light responses, miniature postsynaptic currents and neurotransmitter-induced postsynaptic currents in various retinal neurons. Our results are consistent with the following scheme: Glutamate is used by the photoreceptor and bipolar cell output synapses and the release of glutamate is largely mediated by calcium-dependent vesicular processes. The postsynaptic glutamate receptors in DBCs are L-AP4 receptors, in HBCs, HCs and ganglion cells are the kainate/AMPA and NMDA receptors. Subpopulations of HCs make GABAergic synapses on cones and gate chloride condunctance through GABAA receptors. GABAergic HCs do not make feedforward synapses on bipolar cell dendrites and the neurotransmitter identity of the HCs making feedforward synapses is unknown. Subpopulations of amacrine cells make GABAergic synapses on bipolar cell synaptic terminals, other amacrine cells and ganglion cells and GABA gates chloride conductances in theses cells. Glycinergic amacrine cells make synapses on bipolar cell synaptic terminals, other amacrine cells and ganglion cells and glycine opens postsynaptic chloride channels. Glycinergic interplexiform cells make synapses on bipolar cells in the outer retina and glycine released from these cells open chloride channels in bipolar cell dendrites.

New aspects of dopaminergic interplexiform cell organization in the goldfish retina.

Dopaminergic interplexiform cells (DA-IPCs) in the goldfish retina have been reexamined by light and electron microscopic immunocytochemistry with antisera against dopamine (DA) or tyrosine hydroxylase (TH). Successful immunostaining with a specific anti-DA antiserum offers further direct support for DA-IPCs. Anti-DA immunocytochemistry in combination with [3H]-DA autoradiography shows 92% colocalization of the two markers, indicating that [3H]-DA autoradiography is a reliable technique for identification of DA-IPCs. Incubations with anti-TH antiserum show that immunoreactive DA-IPCs have a homogeneous distribution, with an average frequency of 71 +/- 8 cells/mm2 in retinas of 14-15 cm long goldfish. Their arrangement is distinctly nonrandom. Electron microscopy of TH-immunoreactive cell processes confirms that horizontal cell axons synapse onto DA-IPCs and adds the following junctional arrangements to the circuit diagram of the DA-IPC: 1) adjacent serial synapses between DA-IPCs, external horizontal cells, and putative glycinergic interplexiform cells, 2) junctional appositions between DA-IPCs and photoreceptor cells, 3) junctional appositions between neighbouring DA-IPCs, and 4) the "gap junctional complex," typically consisting of a DA-IPC process juxtaposed with a gap junction between horizontal cell axons. The gap junction is flanked by clusters of small, round vesicles and groups of electron-dense structures resembling intermediate filaments. These morphological results support the functional involvement of DA-IPCs in adaptive retinomotor movements and in horizontal cell gap junction modulation and/or dynamics. They also suggest particular interaction between the dopaminergic and the glycinergic IPC system in the outer plexiform layer of goldfish retina.

Glycinergic interplexiform cells make synaptic contact with amacrine cell bodies in goldfish retina.

Recent works utilizing glycine-immunoreactivity (IR) and combined Golgi impregnation and 3H-glycine uptake autoradiography indicate that glycinergic interplexiform cells (IPC) may synapse upon cell bodies in the inner nuclear and ganglion cell layers in fish retina. This possibility was investigated with immunocytochemical techniques using presynaptic and postsynaptic markers for glycinergic neurons: a monoclonal antibody (mAb 7A) against the 93 kDa subunit of the strychnine-sensitive glycine receptor and a polyclonal antiserum against a glycine/BSA conjugate. Synaptic contacts onto the lateral and proximal surfaces of amacrine cell bodies and onto the distal surface of cells in the ganglion cell layer were identified with both probes. The contacts were rare with one contacted amacrine cell/section of 500 linear micron. Serial 1-micron sections were processed alternately for glycine and GABA antisera using postembedding techniques at the light microscopic level. Glycine-IR processes + boutons were apposed to GABA-IR cell bodies in 16 of 17 examples, indicating that the dendro-somatic contacts were onto GABA-immunoreactive amacrine cell bodies. In context of other published morphological data, we suggest that the dendro-somatic synapses were derived from glycinergic IPCs. Glycinergic IPCs receive input from GABAergic horizontal cells and, via a shunt conductance produced by the dendro-somatic contacts, may be involved in controlling the sensitivity, temporal, or spatial properties of amacrine cell responses to large field illumination.

Glycinergic contacts in the outer plexiform layer of the Xenopus laevis retina characterized by antibodies to glycine, GABA, and glycine receptors

Electrophysiological experiments have predicted a direct synaptic input from glycinergic interplexiform cells (IPCs) to GABAergic horizontal cells in the Xenopus retina. However, previous ultrastructural studies failed to demonstrate this input. Here, we used three immunocytochemical approaches to investigate this issue. First, double-label postembedding immunocytochemistry with GABA- and glycine-like immunoreactivity (GABA-LI and glycine-LI) was used to study possible interactions of the glycinergic IPC with GABAergic horizontal cells. Processes postsynaptic to glycine-LI IPC terminals in the outer plexiform layer (OPL) fell into two groups, small microtubule-filled processes and larger electron-lucent processes with sparse microtubules and occasional mitochondria. In no case did we find glycine-LI synapses onto GABA-LI cells or processes. Second, pre-embedding immunocytochemistry was used to label GABA-LI cells and processes in the OPL. GABA-LI was sparse in horizontal cell axons and more intense in horizontal cell somas and in small processes. In agreement with our first set of experiments, GABA-LI profiles did not receive input from conventional synapses. Third, we localized glycine-receptor-like immunoreactivity (GlyR-LI) to several types of apparent synapses in the OPL. As expected, it was found at IPC synapses. Unexpectedly, GlyR-LI was also subsynaptic at photoreceptor synapses onto second order neurons, both at ribbon and basal junction type synapses. At least some of the GlyR-LI photoreceptor synapses were from cones. Also, GlyR-LI was apposed to photoreceptors and to unidentified small diameter processes, where no other indication of synaptic input was evident. Because glycine-LI is not found in photoreceptors, we suggest that glycine receptors at photoreceptor synapses are stimulated by glycine that diffuses from other sites, possibly from IPCs. This interpretation is consistent with available physiological studies of glycinergic effects in this retina.

Interplexiform cells of the goldfish retina

Dopaminergic and glycinergic interplexiform cells (IPCs) in the goldfish retina were impregnated by using two new Golgi protocols. The two cell types have markedly different morphological characteristics: Dopaminergic IPCs have primary dendrites that descend into and stratify in the inner plexiform layer, where they give rise to processes that project to the outer plexiform layer. Conversely, glycinergic IPCs have primary dendrites that ascend to the outer plexiform layer and from this dendritic arbor, many processes then project into the inner plexiform layer. The apparent coverage of dopaminergic IPCs is almost four times that of glycinergic IPCs. Even so, the coverage of each glycinergic IPC in the outer plexiform layer allows it to provide an accurate copy of the S-space to the inner plexiform layer. Considering the known GABAergic and glycinergic synaptologies in the inner plexiform layer, the glycinergic IPC must form a major element in the retinal circuitry of the goldfish.

Horizontal cell synapses onto glycine-accumulating interplexiform cells.

Horizontal cells mediate lateral transmission of signals in the outer plexiform layer of the vertebrate retina, and are presumed to contribute to surround properties of photoreceptors and bipolar cells by chemical transmission. The cell bodies and dendrites of fish horizontal cells possess presynaptic specializations characteristic of conventional chemical synapses. Horizontal cell axon terminals have not so far been shown to contain presynaptic specializations nor have the targets of the somatic and dendritic synapses been fully characterized. Using electron microscope autoradiography of retinas labelled by high-affinity 3H-glycine uptake, we show here that goldfish horizontal cells make somatodendritic and axodendritic synapses on glycinergic interplexiform cells (Gly-IPCs) as apposed to dopaminergic interplexiform cells. Thus, horizontal cells have at least three postsynaptic targets: photoreceptors, bipolar cells and Gly-IPCs. Gly-IPCs may constitute a major alternative pathway for horizontal cell signals to reach the inner plexiform layer.

Spatial organization of neurochemically classified interneurons of the goldfish retina—I. Local patterns

Certain interneurons in the goldfish retina are uniquely specifiable by their abilities to take up exogenously supplied [ 3H]γ-aminobutyric acid (GABA), [ 3H]glycine, [ 3H]dopamine or [ 3H]serotonin at low micromolar concentrations. Each ligand labels one or two unique populations of interneurons yielding six cell types characterized by soma location, soma size, level and form of dendritic arborization, and local spatial patterning. This report summarizes these qualitative features and provides quantitative evidence on the size and spatial distributions of: (1) GABA-ergic horizontal cells. (2) GABA-ergic amacrine cells. (3) glycinergic amacrine cells. (4) glycinergic interplexiform cells. (5) dopaminergic interplexiform cells. (6) indoleaminergic amacrine cells.