Biomedical subjects
Publications and source records attributed to P Sterling.
The synaptic ribbon in neurons that release transmitter via graded potentials has been considered as a conveyor belt that actively moves vesicles toward their release sites. But evidence has accumulated to the contrary, and it now seems plausible that the ribbon serves instead as a safety belt to tether vesicles stably in mutual contact and thus facilitate multivesicular release by compound exocytosis.
We perceive motion when presented with spatiotemporal changes in contrast (second-order cue). This requires linear signals to be rectified and then summed in temporal order to compute direction. Although both operations have been attributed to cortex, rectification might occur in retina, prior to the ganglion cell. Here we show that the Y ganglion cell does indeed respond to spatiotemporal contrast modulations of a second-order motion stimulus. Responses in an OFF ganglion cell are caused by an EPSP/IPSP sequence evoked from within the dendritic field; in ON cells inhibition is indirect. Inhibitory effects, which are blocked by tetrodotoxin, clamp the response near resting potential thus preventing saturation. Apparently the computation for second-order motion can be initiated by Y cells and completed by cortical cells that sum outputs of multiple Y cells in a directionally selective manner.
Because the mouse retina has become an important model system, we have begun to identify its specific neuron types and their synaptic connections. Here, based on electron micrographs of serial sections, we report that the wild-type mouse retina expresses the standard rod pathways known in other mammals: (1) rod --> cone (via gap junctions) to inject rod signals into the cone bipolar circuit; and (2) rod --> rod bipolar --> AII amacrine --> cone bipolar --> ganglion cell. The mouse also expresses another rod circuit: a bipolar cell with cone input also receives rod input at symmetrical contacts that express ionotropic glutamate receptors (Hack et al., 1999, 2001). We show that this rod-cone bipolar cell sends an axon to the outer (OFF) strata of the inner plexiform layer to form ribbon synapses with ganglion and amacrine cells. This rod-cone bipolar cell receives direct contacts from only 20% of all rod terminals. However, we also found that rod terminals form gap junctions with each other and thus establish partial syncytia that could pool rod signals for direct chemical transmission to the OFF bipolar cell. This third rod pathway probably explains the rod responses that persist in OFF ganglion cells after the well known rod pathways are blocked (Soucy et al., 1998).
The receptive field of the Y-ganglion cell comprises two excitatory mechanisms: one integrates linearly over a narrow field, and the other integrates nonlinearly over a wide field. The linear mechanism has been attributed to input from bipolar cells, and the nonlinear mechanism has been attributed to input from a class of amacrine cells whose nonlinear "subunits" extend across the linear receptive field and beyond. However, the central component of the nonlinear mechanism could in theory be driven by bipolar input if that input were rectified. Recording intracellularly from the Y-cell in guinea pig retina, we blocked the peripheral component of the nonlinear mechanism with tetrodotoxin and found the remaining nonlinear receptive field to be precisely co-spatial with the central component of the linear receptive field. Both linear and nonlinear mechanisms were caused by an excitatory postsynaptic potential that reversed near 0 mV. The nonlinear mechanism depended neither on acetylcholine nor on feedback involving GABA or glycine. Thus the central components of the ganglion cell's linear and nonlinear mechanisms are apparently driven by synapses from the same rectifying bipolar cell.
ON bipolar neurons in retina detect the glutamate released by rods and cones via metabotropic glutamate receptor 6 (mGluR6), whose cascade is unknown. The trimeric G-protein G(o) might mediate this cascade because it colocalizes with mGluR6. To test this, we studied the retina in mice negative for the alpha subunit of G(o) (Galpha(o)-/-). Retinal layering, key cell types, synaptic structure, and mGluR6 expression were all normal, as was the a-wave of the electroretinogram, which represents the rod and cone photocurrents. However, the b-wave of the electroretinogram, both rod- and cone-driven components, was entirely missing. Because the b-wave represents the massed response of ON bipolar cells, its loss in the Galpha(o) null mouse establishes that the light response of the ON bipolar cell requires G(o). This represents the first function to be defined in vivo for the alpha subunit of the most abundant G-protein of the brain.
GABA gating an anion channel primarily permeable to chloride can hyperpolarize or depolarize, depending on whether the chloride equilibrium potential (E(Cl)) is negative or positive, respectively, to the resting membrane potential (E(rest)). If the transmembrane Cl(-) gradient is set by active transport, those neurons or neuronal regions that exhibit opposite responses to GABA should express different chloride transporters. To test this, we immunostained retina for the K-Cl cotransporter (KCC2) that normally extrudes chloride and for the Na-K-Cl cotransporter (NKCC) that normally accumulates chloride. KCC2 was expressed wherever E(Cl) is either known or predicted to be negative to E(rest) (ganglion cells, bipolar axons, and OFF bipolar dendrites), whereas NKCC was expressed wherever E(Cl) is either known or predicted to be positive to E(rest) (horizontal cells and ON bipolar dendrites). Thus, in the retina, the opposite effects of GABA on different cell types and on different cellular regions are probably primarily determined by the differential targeting of these two chloride transporters.
We prepared antibodies selective for the C-terminus of the human mGluR6 receptor and used confocal and electron microscopy to study the patterns of immunostaining in retina of monkey, cat, and rabbit. In all three species punctate stain was restricted to the outer plexiform layer. In monkey, stain was always observed in the central element of the postsynaptic "triad" of rod and cone terminals. In monkey peripheral retina, stain was seen only in central elements, but in the fovea, stain was also observed in some dendrites contacting the base of the cone terminal. S-cone terminals, identified by staining for S opsin, showed staining of postsynaptic dendrites. These were identified as dendrites of the ON S-cone bipolar cell by immunostaining for the marker cholecystokinin precursor. The staining pattern suggests that all types of ON bipolar cells, despite their marked differences in function, express a single isoform of mGluR6. Ultrastructurally, mGluR6 was located not on the tip of the central element, near the site of vesicle release, but on its base at the mouth of the invagination, 400-800 nm from the release site. Thus, the mGluR6 receptors of ON bipolar cells lie at about the same distance from sites of vesicle release as the iGluR receptors of OFF bipolar cells at the basal contacts.
Explore the source record for details and available documents.
Cone synaptic terminals couple electrically to their neighbors. This reduces the amplitude of temporally uncorrelated voltage differences between neighbors. For an achromatic stimulus coarser than the cone mosaic, the uncorrelated voltage difference between neighbors represents mostly noise; so noise is reduced more than the signal. Here coupling improves signal-to-noise ratio and enhances contrast sensitivity. But for a chromatic stimulus the uncorrelated voltage difference between neighbors of different spectral type represents mostly signal; so signal would be reduced more than the noise. This cost of cone coupling to encoding chromatic signals was evaluated using a compartmental model of the foveal cone array. When cones sensitive to middle (M) and long (L) wavelengths alternated regularly, and the conductance between a cone and all of its immediate neighbors was 1,000 pS (approximately 2 connexons/cone pair), coupling reduced the difference between the L and M action spectra by nearly fivefold, from about 38% to 8%. However, L and M cones distribute randomly in the mosaic, forming small patches of like type, and within a patch the responses to a chromatic stimulus are correlated. In such a mosaic, coupling still reduced the difference between the L and M action spectra, but only by 2.4-fold, to about 18%. This result is independent of the L/M ratio. Thus "patchiness" of the L/M mosaic allows cone coupling to improve achromatic contrast sensitivity while minimizing the cost to chromatic sensitivity.
A retinal ganglion cell commonly expresses two spatially overlapping receptive field mechanisms. One is the familiar "center/surround," which sums excitation and inhibition across a region somewhat broader than the ganglion cell's dendritic field. This mechanism responds to a drifting grating by modulating firing at the drift frequency (linear response). Less familiar is the "nonlinear" mechanism, which sums the rectified output of many small subunits that extend for millimeters beyond the dendritic field. This mechanism responds to a contrast-reversing grating by modulating firing at twice the reversal frequency (nonlinear response). We investigated this nonlinear mechanism by presenting visual stimuli to the intact guinea pig retina in vitro while recording intracellularly from large brisk and sluggish ganglion cells. A contrast-reversing grating modulated the membrane potential (in addition to the firing rate) at twice the reversal frequency. This response was initially hyperpolarizing for some cells (either ON or OFF center) and initially depolarizing for others. Experiments in which responses to bars were summed in-phase or out-of-phase suggested that the single class of bipolar cells (either ON or OFF) that drives the center/surround response also drives the nonlinear response. Consistent with this, nonlinear responses persisted in OFF ganglion cells when ON bipolar cell responses were blocked by L-AP-4. Nonlinear responses evoked from millimeters beyond the ganglion cell were eliminated by tetrodotoxin. Thus, to relay the response from distant regions of the receptive field requires a spiking interneuron. Nonlinear responses from different regions of the receptive field added linearly.
Calcium enters the outer segment of a vertebrate photoreceptor through a cGMP-gated channel and is extruded via a Na/Ca, K exchanger. We have identified another element in mammalian cones that might help to control cytoplasmic calcium. Reverse transcription-PCR performed on isolated photoreceptors identified mRNA for the SII- splice variant of the type I receptor for inositol 1,4,5-triphosphate (IP3), and Western blots showed that the protein also is expressed in outer segments. Immunocytochemistry showed type I IP3 receptor to be abundant in red-sensitive and green-sensitive cones of the trichromatic monkey retina, but it was negative or weakly expressed in blue-sensitive cones and rods. Similarly, the green-sensitive cones expressed the receptor in dichromatic retina (cat, rabbit, and rat), but the blue-sensitive cones did not. Immunostain was localized to disk and plasma membranes on the cytoplasmic face. To restore sensitivity after a light flash, cytoplasmic cGMP must rise to its basal level, and this requires cytoplasmic calcium to fall. Cessation of calcium release via the IP3 receptor might accelerate this fall and thus explain why the cone recovers much faster than the rod. Furthermore, because its own activity of the IP3 receptor depends partly on cytoplasmic calcium, the receptor might control the set point of cytoplasmic calcium and thus affect cone sensitivity.
The AMPA receptor, ubiquitous in brain, is termed "ionotropic" because it gates an ion channel directly. We found that an AMPA receptor can also modulate a G-protein to gate an ion channel indirectly. Glutamate applied to a retinal ganglion cell briefly suppresses the inward current through a cGMP-gated channel. AMPA and kainate also suppress the current, an effect that is blocked both by their general antagonist CNQX and also by the relatively specific AMPA receptor antagonist GYKI-52466. Neither NMDA nor agonists of metabotropic glutamate receptors are effective. The AMPA-induced suppression of the cGMP-gated current is blocked when the patch pipette includes GDP-beta-S, whereas the suppression is irreversible when the pipette contains GTP-gamma-S. This suggests a G-protein mediator, and, consistent with this, pertussis toxin blocks the current suppression. Nitric oxide (NO) donors induce the current suppressed by AMPA, and phosphodiesterase inhibitors prevent the suppression. Apparently, the AMPA receptor can exhibit a "metabotropic" activity that allows it to antagonize excitation evoked by NO.
Explore the source record for details and available documents.
By simultaneously recording from retinal ganglion cells while stimulating a single cone, Chichilnisky and Baylor demonstrate that the strength of physiological connections within a retinal microcircuit is linearly proportional to the number of anatomically defined synapses.
Perception of hue is opponent, involving the antagonistic comparison of signals from different cone types. For blue versus yellow opponency, the antagonism is first evident at a ganglion cell with firing that increases to stimulation of short wavelength-sensitive (S) cones and decreases to stimulation of middle wavelength-sensitive (M) and long wavelength-sensitive (L) cones. This ganglion cell, termed blue-yellow (B-Y), has a distinctive morphology with dendrites in both ON and OFF strata of the inner plexiform layer (Dacey and Lee, 1994). Here we report the synaptic circuitry of the cell and its spatial density. Reconstructing neurons in macaque fovea from electron micrographs of serial sections, we identified six ganglion cells that branch in both strata and have similar circuitry. In the ON stratum each cell collects approximately 33 synapses from bipolar cells traced back exclusively to invaginating contacts from S cones, and in the OFF stratum each cell collects approximately 14 synapses from bipolar cells (types DB2 and DB3) traced to basal synapses from approximately 20 M and L cones. This circuitry predicts that spatially coincident blue-yellow opponency arises at the level of the cone output via expression of different glutamate receptors. S cone stimuli suppress glutamate release onto metabotropic receptors of the S cone bipolar cell dendrite, thereby opening cation channels, whereas M and L cone stimuli suppress glutamate release onto ionotropic glutamate receptors of DB2 and DB3 cell dendrites, thereby closing cation channels. Although the B-Y cell is relatively rare (3% of foveal ganglion cells), its spatial density equals that of the S cone; thus it could support psychophysical discrimination of a blue-yellow grating down to the spatial cutoff of the S cone mosaic.
The cone axon is nearly four times thicker than the rod axon (1.6 vs 0.45 microns diameter). To assess how signal transfer and integration at the terminal depend on cable dimensions, a transducer (cone = ohmic conductance, rod = current source) coupled via passive cable to a sphere with a chloride conductance (representing GABAA receptor) was modelled. For a small signal in peripheral cone with a short axon, steady photosignal transfers independently of axon diameter despite a significant chloride conductance at the cone terminal. Temporally varying photosignal also transfers independently of axon diameter up to 20 Hz and is attenuated only 20% at 50 Hz. Thus, to accomplish the basic electrical functions of a peripheral cone, a thin axon would suffice. For a foveal cone with a long axon steady photosignal transfers independently of axon diameter, but temporally varying photosignal is attenuated 5-fold at 50 Hz for a thick axon and 10-fold for a thin axon. This might contribute to the lower sensitivity of central retina to high temporal frequencies. The cone axon contains 14-fold more microtubules than the rod axon, and its terminal contains at least 20-fold more ribbon synapses than the rod's. Since ribbon synapses sustain high rates of exocytosis, the additional microtubules (which require a thicker axon) may be needed to support a greater flux of synaptic vesicle components.
UNLABELLED: The cone 'synaptic complex' is a unique structure in which a single presynaptic axon secretes glutamate onto processes of bipolar cells (both ON and OFF) and horizontal cells. In turn, the horizontal cell processes antagonize cone and bipolar responses to glutamate (probably by GABA). What still remains largely unknown is the molecular identity of the postsynaptic receptors and their exact locations. We identified several subunits of the glutamate receptor and the GABAA receptor expressed at the cone synaptic complex and localized them ultrastructurally. Glutamate receptors: (i) Invaginating (probably ON) bipolar dendrites in the monkey and rat express the metabotropic glutamate receptor, mGluR6. The stain is intense on the dendritic membrane where it first enters the invagination, and weak at the tip nearest to the ribbon. The cone membrane is electron-dense where it apposes the intense stain for mGluR6. Surprisingly, invaginating bipolar dendrites in the cat also express the AMPA receptor subunits, GluR2/3 and GluR4. (ii) Dendrites forming basal contacts in the cat (probably OFF) express the AMPA subunits GluR2/3, GluR4, and also the kainate subunit, GluR6/7. The stain is especially intense at the dendritic tips in apposition to electron-dense regions of cone membrane. (iii) Horizontal cells in the cat express the AMPA subunits GluR2/3, GluR4 and the kainate subunit, GluR6/7. The stain is strongest in the cytosol of somas and primary dendrites, but is also present in the invaginating terminals where it localizes to the membrane subjacent to the ribbon. GABAA receptors: (i) ON and OFF bipolar dendrites in the monkey express the alpha 1 and beta 2/3 subunits. The stain is localized to the bipolar cell membrane in apposition to horizontal cell processes. (ii) Cones did not express the GABAA subunits tested by immunocytochemistry, but beta 3 mRNA was amplified by RT-PCR from rat photoreceptors. CONCLUSIONS: (i) mGluR6 receptors concentrate on dendrites at the base of the invagination rather than at the apex. This implies that receptors at both 'invaginating' and 'basal' contacts lie roughly equidistant from the release sites and should therefore receive similar spatiotemporal concentrations of glutamate. (ii) The 'cone' membrane is electron-dense opposite to the receptor sites on both ON and OFF bipolar cells. This suggests a special role for this region in synaptic transmission. Possibly, these densities signify a transporter that would regulate glutamate concentration at sites remote (> 200 nm) from the locus of vesicle release.
Explore the source record for details and available documents.