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Synaptic organization and ionic basis of on and off channels in mudpuppy retina. I. Intracellular analysis of chloride-sensitive electrogenic properties of receptors, horizontal cells, bipolar cells, and amacrine cells.

Intracellular recordings from receptors, horizontal cells, bipolars, and amacrines have been carried out in the perfused mudpuppy eyecup. The introduction of a chloride-free (c-f) medium results in initial transient potential changes in many cells followed by a slow loss of light-evoked activity of the depolarizing bipolar, the horizontal cell, and the on depolarization of amacrine cells. The hyperpolarizing bipolar remains responsive to light stimulation in a c-f medium, but the antagonistic surround mechanism is abolished. These effects are reversible after returning to a normal ionic medium. The results of this study provide insight into the retinal connections which underlie ganglion cell receptive field organization. It is concluded that the depolarizing bipolar is excitatory to on ganglion cells and is also the pathway for on-excitation of on-off cells. The hyperpolarizing bipolar mediates the off discharge of off and on-off cells. Amacrine cells receive input from both depolarizing and hyperpolarizing bipolar cells. These findings raise the possibility that transmembrane movements of chloride ions are critical for the light responsiveness of horizontal and depolarizing bipolar cell activity.

Animals

Feedforward lateral inhibition in retinal bipolar cells: input-output relation of the horizontal cell-depolarizing bipolar cell synapse.

Lateral inhibition is the ubiquitous strategy used by visual neurons for spatial resolution throughout the animal kingdom. It has been a puzzle whether lateral inputs in retinal bipolar cells are mediated by the horizontal cell (HC)-cone feedback synapse, by the HC-bipolar cell feedforward synapse, or by both. By blocking the central inputs of the depolarizing bipolar cells (DBCs) with L-2-amino-4-phosphonobutyrate, we were able to eliminate the contribution of the feedback synapse and to demonstrate the postsynaptic light response in DBCs mediated by the HC-DBC feedforward synapse. The HC-DBC feedforward synapse contributes roughly one-third of the surround response whereas the HC-cone-DBC feedback synapse probably contributes the rest.

Ambystoma

Retinal bipolar cells receive negative feedback input from GABAergic amacrine cells.

Bipolar cells make reciprocal synapses with amacrine cells in the inner plexiform layer; both feedforward connections and feedback connections are present. The physiological properties of the feedback synapse have not been well described. Since some amacrine cells are thought to be GABAergic, we examined bipolar cells for feedback input from gamma-aminobtyric acid (GABA)ergic amacrine cells. Solitary bipolar cells were dissociated enzymatically from the goldfish retina. Cells were voltage clamped with a patch pipette and their GABA sensitivity was examined. GABA evoked responses in all bipolar cells with a large axon terminal, which were identified to be the rod dominant ON type, and in some bipolar cells with a small axon terminal. The highest GABA sensitivity was located at the axon terminal. The least effective dose was as low as 100 nM. A small insignificant response of high threshold was evoked when GABA was applied to the dendrite and soma. GABA increased the Cl conductance and caused membrane hyperpolarization. The bipolar cells had the GABAA receptor coupled with a benzodiazepine receptor. The GABA-evoked response was not susceptible to Co ions, which suppressed the GABA-induced responses in turtle cones by 50% at 5 microM concentration. Incomplete desensitization was observed, suggesting that the GABAergic pathway seems capable of transmitting signals tonically. The present results strongly indicate that the rod-dominant ON-type bipolar cells and some bipolar cells with a small axon terminal receive negative feedback inputs from GABAergic amacrine cells.

Action Potentials

Localization of immunoreactive cholecystokinin precursor to amacrine cells and bipolar cells of the macaque monkey retina.

We used antisera that recognized precursors of the neuropeptide cholecystokinin extended at the carboxyl terminus in an immunocytochemical study of the macaque retina. A subpopulation of bipolar cells with long, obliquely oriented dendrites was labeled. Their axons terminated exclusively in the fifth stratum of the inner plexiform layer, where they contacted processes of amacrine and ganglion cells. Based on their morphology, these cells appeared to be the type that contacts short-wavelength cones selectively. Two types of amacrine cells were also labeled, and processes from both types formed dense plexuses in the second and fourth strata of the inner plexiform layer. The majority of their synaptic connections were with other amacrine cells, but they had more contacts with bipolar cell axons and retinal ganglion cell dendrites than any other peptidergic cells in the macaque retina. We studied extracts of macaque retina with gel-filtration chromatography and radioimmunoassays to confirm our immunohistochemical results. We found cholecystokinin octapeptide and other immunoreactive forms that were amidated at their carboxyl termini and were therefore likely to be biologically active. Unlike most other regions of the CNS, however, the retina had relatively low concentrations of amidated forms, and forms with extended carboxyl termini that are presumably their precursors were far more abundant. These findings suggest that the rate of cholecystokinin synthesis in the retina is quite high, as we would expect if the peptide were found in tonically active neurons.

Amino Acid Sequence

Properties of GABA-activated whole-cell currents in bipolar cells of the rat retina.

This paper describes experiments on GABA-activated whole-cell membrane currents in bipolar cells freshly isolated from the adult rat retina. The main goal was to determine whether bipolar cell responses to GABA could be resolved in terms of mediation by the GABAA receptor, the GABAB receptor, or both. Bipolar cells were isolated by gentle enzymatic dissociation and identified by their distinct morphology. GABA agonists and antagonists were applied focally by pressure and the resultant currents were recorded under whole-cell voltage clamp. In all bipolar cells tested, GABA (0.1-100 microM) induced a monophasic response associated with a conductance increase (IGABA). The shift in reversal potential for IGABA as a function of pipet [Cl-] paralleled that predicted based on the Nernst equation for Cl-. IGABA was mimicked by muscimol (5-20 microM) and antagonized by bicuculline (20-100 microM). Baclofen (0.1-1.0 mM) produced no apparent conductance change. "Hot spots" of sensitivity to GABA which might be associated with regions of synaptic contact were not found; both the soma and processes of all bipolar cells were responsive to focally applied GABA. Furthermore, all bipolar cells tested responded to glycine. In conclusion, we have established the presence of GABAA receptors on rat retinal bipolar cells. Our data suggest further that these cells lack GABAB receptors. Finally, our observation that bipolar cells in the rat retina are relatively homogeneous in terms of their sensitivity to GABA and glycine lead us to postulate that the functional significance of the presence of receptors and their distribution on a neuron may be dictated more by the topography of the presynaptic inputs than by its inherent chemosensitivity.

Animals

Dopamine cells and rod bipolar cells contain protein kinase C-like immunoreactivity in some vertebrate retinas.

The localization of cells immunoreactive to a monoclonal antibody against protein kinase C (PKC) and to polyclonal antibodies against tyrosine hydroxylase (TH) was investigated in the retina of fish (carp, goldfish, dace and catfish), frog, turtle, chick and some mammalians (guinea pig, rat, cat and rabbit) by means of fluorescence microscopy. PKC-like immunoreactivity was found in dopamine (DA) or TH-like immunoreactive (IR) cells in all the species examined and also in rod bipolar cells in the fish (except for catfish), and in presumed rod bipolar cells in the other animals (except for frog and turtle). In the catfish, frog and turtle retinas, no PKC-like IR bipolar cells were found. In the rat retina, some other amacrine cells in addition to TH-like IR amacrine cells were reactive to the anti-PKC antibody. It is of interest that PKC-like immunoreactivity is commonly found in DA cells and probably in rod bipolar cells in most animal species, although the functional significance is unknown at present.

Animals

Spatial organization of the bipolar cell's receptive field in the retina of the tiger salamander.

1. The spatial properties of rods, horizontal cells and bipolar cells were studied by intracellular recording in the isolated, perfused retina of the tiger salamander, Ambystoma tigrinum. Low stimulus intensities were used in order to keep cell responses close to, or within, their linear intensity/response range. 2. Spatial properties of bipolar cell receptive fields, measured while perfusing with normal Ringer solution, were compared with those measured during exposure to agents that eliminated the bipolar cells' receptive field surround (RFS). In this way, the spatial properties of the receptive field centre (RFC) and those of the RFS could be characterized independently. 3. To a good approximation, the contribution to the horizontal cell's response of unit area of its receptive field declined exponentially with distance from the centre of the receptive field. The (apparent) length constant describing this decay was 200 microns. The one-dimensional length constant of the horizontal cell syncytium was thus 248 microns. The variation of response amplitude with the radius of a centred circular stimulus was consistent with this finding. 4. This was true also of the RFCs of bipolar cells. The one-dimensional length constant of the RFC of off-centre bipolar cells averaged 124 microns. That of the RFC of on-centre cells averaged 62 microns though values were more variable, the RFCs of some on-centre cells being comparable to those of off-centre cells. These values were independent of the class of photoreceptor driving the bipolar cell. 5. The large size of the RFCs of off-centre cells and many on-centre cells cannot by explained by light scatter within the retina or by voltage spread within the rod syncytium. We proposed that off-centre cells are tightly coupled in a syncytium. On-centre cells, on average, are less tightly coupled. 6. The spatial properties of the bipolar cell's RFS were consistent with the notion that the RFS represents a convolution of the horizontal cell's receptive field and the bipolar cell's RFC. 7. The spatial properties of bipolar cell receptive fields were reconstructed from the measured properties of their RFCs and the measured properties of horizontal cell receptive fields. Under the conditions of our experiments, the bipolar cell's response could be described by a linear difference between a component generated by the RFC and a component generated by the RFS. 8. The spatial filtering characteristics of the bipolar cells were calculated from our data.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials

Cone synapses with Golgi-stained bipolar cells that are morphologically similar to a center-hyperpolarizing and a center-depolarizing bipolar cell type in the turtle retina.

Two types of Golgi-stained bipolar cells have been examined by light and electron microscopy to determine the ultrastructure of their synapses with photoreceptors and the spectral type of photoreceptor with which they connect in the retina of the turtle (Pseudemys scripta elegans). We have chosen bipolar cells that correspond in morphology to a center-hyperpolarizing type (H-bipolar) and a center-depolarizing type (D-bipolar) shown in Marchiafava and Weiler's paper (Proc. R. Soc. B 208:103-113, '80). The latter authors recorded intracellular responses and marked their cells with the fluorescent dye Procion yellow so that we have a clear picture of their morphology. These bipolars have been called B4 and B6, respectively, according to our recent classification scheme of Golgi-stained cells. Serial section electron microscopy of two B4 bipolars shows that they made wide-cleft, striated basal junctions with red and green cone pedicles. They connected to six or seven different cone pedicles. The three B6 bipolar cells studied made narrow-cleft, semi-invaginating basal junctions with cone pedicles. The dendrites of B6 bipolars did not become central elements at ribbon synapses although they invaginated toward the synaptic ribbon. Serial section electron microscopy indicated that B6 bipolars contacted three to five cone pedicles also of a red or green cone type. We suggest that in the case of these two varieties of bipolar cell in turtle retina, sign-conserving synapses with cones are wide-cleft basal junctions while sign-inverting synapses are narrow-cleft, semi-invaginating in morphology.

Animals

Simulated bipolar cells in fovea of human retina. II. Spectral responses of bipolar cells.

The static model developed in Part I is used to study spectral responses of C-type bipolar cells (BC). Once unique loci are adjusted to their proper wavelengths, and with a specified set of absorption spectra for cones, spectral responses of C-type BCs are dependent on only the balance between BC receptive field center and surround responses. This is true regardless of cone mosaic or BC receptive field organization. The unique yellow loci for the r-g channel is set at 576.7 nm while the unique green locus for the blue-center BC is set at 517.7 nm. A unique orange locus for a combined r-g and bl-y channels is set at 600 nm by multiplying the blue-center BC response spectra by a factor of six before adding to the r-g channel.

Adaptation, Ocular

Simulated bipolar cells in fovea of human retina. III. Effects of chromatic adaptation in bipolar cell spectral responses.

Effects of chromatic adaptation on C-type bipolar cells (BC) in human retinal fovea are studied. Adaptation of the r-g channel is linear for both central fovea and parafovea. Adaptation of the parafovea bl-y channel, on the other hand, is nonlinear, which is accounted for by the slower adaptation rate of blue-sensitive cones with white light intensity as compared to rates of red- and green-sensitive cones. Achromatic adaptation of red- and green-center BCs produces uniform response decreases but without unique yellow loci shifts. Achromatic adaptation of blue-center BCs, on the other hand, does cause shifts of the unique green locus. Shifts of the crossover points for the BC response spectra occur with chromatic adaptation; the unique yellow loci shifts to shorter wavelengths with adapting wavelengths shorter than 550 nm and longer wave-lengths with longer adapting wavelengths than 550 nm. Chromatic adaptation is sufficient to explain the Bezold-Brüke effects; but to fully account for these shifts a novel hypothesis is proposed. For the green and red spectrum regions Bezold-Brücke shifts are due to r-g channel chromatic adaptation, while for the blue spectrum region bl-y channel chromatic adaptation accounts for Bezold-Brücke shifts. The two channels function independently in an either/or manner. The bl-y channel, besides having a unique green locus at 517.7 nm, has a crossover point at about 670 nm. Chromatic adaptation of the bl-y channel produces shifts of the unique red locus, which may account for extraspectral hue shifts.

Acclimatization

Suppression by glutamate of cGMP-activated conductance in retinal bipolar cells.

Depolarizing bipolar cells (DBCs) of the retina are the only neurons in the vertebrate central nervous system known to be hyperpolarized by the neurotransmitter glutamate. Both glutamate and its analogue L-2-amino-4-phosphonobutyrate (APB) hyperpolarize DBCs by decreasing membrane conductance. Furthermore, glutamate responses in DBCs slowly decrease during whole-cell recording, suggesting that the response involves a second messenger system. Here we report that intracellular cyclic GMP or GTP activates a membrane conductance that is suppressed by APB, resulting in an enhanced APB response. In the presence of GTP-gamma-S, APB causes an irreversible suppression of the conductance. Inhibitors of G-protein activation or phosphodiesterase activity decrease the APB response. Thus, the DBC glutamate receptor seems to close ion channels by increasing the rate of cGMP hydrolysis by a G protein-mediated process that is strikingly similar to light transduction in photoreceptors.

1-Methyl-3-isobutylxanthine

Membrane currents of retinal bipolar cells in culture.

1. Retinal bipolar cells were isolated from white bass retinas and maintained in a cell culture preparation. Two morphological types of bipolar cells were observed in cell culture. These were labeled large- and small-bipolar cells based mainly on the size of their somata and primary dendrites. Two types of small-bipolar cells were observed. Isolated bass bipolar cells are very similar to those described in the intact retina. 2. Under current clamp, to depolarizing current injection, small-bipolar cells produced a spike followed by a plateau. Large-bipolar cells showed a slow depolarization to a plateau level. 3. Voltage-gated membrane currents were studied using whole-cell patch-clamp techniques. Channel blocking agents were used to define the ion channels found in the membranes of these cells. 4. The large-bipolar cells were found to possess an A-current, a calcium current, and a calcium-dependent potassium current. 5. Large bipolar cells also possessed an inward rectifier that did not correspond to any previously described. 6. The two types of small-bipolar cells were found to have very similar membrane properties to one another. They lacked a large A-current but possessed a slowly activating, outward rectifying potassium current. Similar to the large-bipolar cells, they showed a calcium current and a calcium-activated potassium current. 7. The inward rectifier of small-bipolar cells was characterized as an H-current. 8. The results suggest that the membrane currents of bipolar cells set a narrow operating range about which the cells function in the intact retina. In addition these currents help shape the responses of bipolar cells to light stimuli but do not confer ON or OFF properties.

Animals

Computational model of the on-alpha ganglion cell receptive field based on bipolar cell circuitry.

The on-alpha ganglion cell in the area centralis of the cat retina receives approximately 450 synapses from type b1 cone bipolar cells. This bipolar type forms a closely spaced array (9 microns), which contributes from 1 to 7 synapses per b1 cell throughout the on-alpha dendritic field. Here we use a compartmental model of an on-alpha cell, based on a reconstruction from electron micrographs of serial sections, to compute the contribution of the b1 array to the on-alpha receptive field. The computation shows that, for a physiologic range of specific membrane resistance (9500-68,000 omega.cm2) and a linear synapse, inputs are equally effective at all points on the on-alpha dendritic tree. This implies that the electrotonic properties of the dendritic tree contribute very little to the domed shapes of the receptive field center and surround. Rather, these shapes arise from the domed distribution of synapses across the on-alpha dendritic field. Various sources of "jitter" in the anatomical circuit, such as variation in bipolar cell spacing and fluctuations in the number of synapses per bipolar cell, are smoothed by the overall circuit design. However, the computed center retains some minor asymmetries and lumps, due to anatomical jitter, as found in actual alpha-cell receptive fields.

Animals

Voltage- and transmitter-gated currents in isolated rod bipolar cells of rat retina.

1. Bipolar cells were isolated from adult rat retinas after enzymatic and mechanical treatment. The cells could be unequivocally identified from their morphology because of high retention of their axon and dendritic processes after isolation. 2. Protein kinase C (PKC) immunoreactivity performed on sections of the rat retina labeled rod bipolar cells and a few amacrine cells. Virtually all bipolar cells in the dissociates expressed PKC immunoreactivity and were, therefore, rod bipolar cells. 3. Rod bipolar cells were examined with the tight-seal whole-cell and excised-patch recording techniques. Resting potentials of the isolated cells recorded under current-clamp conditions showed a broad unimodal distribution around -37 mV. 4. Membrane depolarization from a holding potential of -90 mV resulted in an outward current. A fast sodium inward current was not observed. Membrane hyperpolarization from a holding potential of -40 mV activated an inwardly rectifying current. 5. gamma-Aminobutyric acid (GABA) and glycine, the putative retinal neurotransmitters that mediate the bipolar cells' receptive field surround in vivo, activated chloride conductances in almost all isolated bipolar cells. GABA- and glycine-evoked currents were both desensitizing and could be antagonized by the classical blockers bicuculline, picrotoxin, and strychnine, respectively. 6. Pressure application of the drugs from fine microcapillaries to various parts of the isolated cells suggests a high GABA sensitivity at the axonal endings compared with either the somatic or dendritic region. A similar distribution was not found for glycine. On the contrary, glycine-induced single-channel events with main conductances of 52 and 34 pS were recorded from membrane patches excised from the cells' somata. 7. Conductances induced by glutamate and several excitatory amino acid agonists were observed in a number of the cells. Application of the glutamate agonist 2-amino-4-phosphonobutyric acid (APB) induced an inward current at negative holding potentials associated with the opening of ion channels. In only 5 of 93 cells, APB closed ion channels, leading to a decrease in membrane conductance.

Animals

Single cell shape and population densities of indoleamine-accumulating and displaced bipolar cells in Reeves' turtle retina.

Two types of bipolar cell in the Geoclemys reevesii retina were studied quantitatively by means of specific cell labelling with an indoleamine derivative (5,6-dihydroxytryptamine, 5,6-DHT), a nucleic acid stain (4,6-diamidino-2-phenylindole, DAPI) and Lucifer yellow CH. Indoleamine-accumulating (IA) bipolar cells were selectively labelled with 5,6-DHT applied intraocularly. After the cells accumulated 5,6-DHT, the indoleamine fluorescence was photoconverted to diaminobenzidine products to allow observation of morphological details. Close examination of many cells (cell number; n = 120) showed that the IA bipolar cells consist of a single morphological type whose axon collaterals ramify sublaminae 1, 4 and 5 respectively. This terminal branching pattern corresponds to cells that hyperpolarize when their receptive field centres are illuminated (Weiler 1981). The density of IA bipolar cells was highest in the visual streak (4130 cells mm-2) and lowest at the peripheral margin (1970 cells mm-2). By applying a small amount of DAPI to the eye, nuclei located in the most proximal row of the outer nuclear layer were labelled selectively. By using selective intracellular dye injection into DAPI-labelled cells under fluorescence microscope (Tauchi & Masland 1984, 1985), these cells were found to have Landolt's clubs and single descending axons. Dye injections into more than fifty DAPI-labelled somata showed that they belonged exclusively to displaced bipolar cells. These comprised at least two subtypes that differ in the ramification pattern of their axon terminals within the inner plexiform layer: one was monostratified, whereas the other was bistratified. The displaced bipolar cell density was as high as 9400 cells mm-2 in the central retina, falling to 2000 cells mm-2 in the superior margin. In vitro Lucifer labelling revealed that the overall bipolar cell density in the central retina was as high as 39,300 cells mm-2. Both the conventionally located and displaced bipolar cells were included in this population. About 11% of the total bipolar cell population consisted of IA bipolar cells. Assuming that one half of the conventionally located bipolar cells are the centre-hyperpolarizing type, IA bipolar cells represent approximately 28% of the total. As displaced bipolar cells represent almost one quarter of the total bipolar population, the dislocation of their somata stands out morphologically, inviting investigation of possible functional correlates.

5,6-Dihydroxytryptamine

Membrane currents in retinal bipolar cells of the axolotl.

By whole-cell patch-clamping bipolar cells isolated from enzymatically dissociated retinae, we have studied the nonsynaptic ionic currents that may play a role in shaping the bipolar cell light response and in determining the level of voltage noise in these cells. Between -30 and -70 mV, the membrane current of isolated bipolar cells is time independent, and the input resistance is 1-2 G omega. Depolarization past -30 mV activates an outward current (in less than 100 ms), which then inactivates slowly (approximately 1 s). Inactivation of this current is removed by hyperpolarization over the range -20 to -80 mV. This current is carried largely by K ions. It is not activated by internal Ca2+. The membrane current of isolated bipolar cells is noisy, and the variance of this noise has a minimum between -40 and -60 mV. At its minimum, the standard deviation of the voltage noise produced by nonsynaptic membrane currents is at least 100 microV. The membrane currents of depolarizing bipolar cells in slices of retina were investigated by whole-cell patch-clamping. Their membrane properties were similar to those of isolated bipolar cells, but with a larger membrane capacitance and a smaller input resistance. Their membrane current noise also showed a minimum near -40 to -60 mV. The time-dependent potassium current in axolotl bipolar cells is not significantly activated in the physiological potential range and can therefore play little role in shaping the bipolar cells' voltage response to light. Differences in the waveform of the light response of bipolar cells and photoreceptors must be ascribed to shaping by the synapses between these cells. The noise minimum in the bipolar membrane current is near the dark potential of these cells, and this may be advantageous for the detection of weak signals by the bipolar cells.

Adenosine Triphosphate

Inputs to bipolar cell dendrites in goldfish retina.

Goldfish mixed bipolar cells, which contact both rods and cones, comprise two types; Ma, with axon terminals in the distal sublamina(a) and Mb, with axon terminals in the proximal sublamina(b) of the inner synaptic layer. Type Ma cells make only wide-cleft junctions (WCJ), whereas type Mb cells make only narrow-cleft junctions (NCJ) with rods and cores. It follows that WCJs mediate sign-conserving synaptic transmission to the center hyperpolarizing Ma cells, whereas NCJs mediate sign-inverting transmission to the center depolarizing Mb cells. Bipolar cell (BC) surrounds may be mediated largely through horizontal cell (HC) pathways. Type Ma BCs appear to be influenced primarily through HC feedback at cones and both feedback and nonribbon feedforward contacts at rods; in contrast most Mb BCs may be influenced by both HC feedback and ribbon-associated feedforward contacts at rods and cones, accounting for the greater complexity of Mb BC surrounds. BC subtypes Mal and Mbl, which contact only red-sensitive cones (+ rods), are likely to have red-opponent (non-color-coded) surrounds mediated by H1 (L-type) cone HCs; while subtypes Ma2 and Mb2 and 3, which contact also green-sensitive cones, are likely to have red + green-opponent (color-coded) surrounds mediated by H1 and H2 (R/G C-type) cone HCs.

Animals