The midget-parvocellular pathway of marmoset retina: a quantitative light microscopic study

The midget-parvocellular pathway in foveal retina of primates shows a "private line" (one-to-one) connectivity with cone photoreceptors. The connectivity of this pathway outside the fovea is not well understood. Here, we studied the population of OFF midget bipolar cells across the retinae of marmoset monkeys (Callithrix jacchus) by using light microscopy. Cone pedicles were labeled with peanut agglutinin, OFF midget bipolar cells were labeled with antibodies against CD15, and midget ganglion cells were retrogradely labeled from the lateral geniculate nucleus and subsequently photofilled. Each midget bipolar cell contacts a single cone in foveal retina, but outside the fovea midget bipolar cells contact multiple cones: one to two cones at 1 mm ( approximately 8 degrees); three to four cones at 3-4 mm ( approximately 25 degrees); and five or more cones beyond 6 mm (>50 degrees). Throughout this eccentricity range, all medium (M) and long (L) wavelength sensitive cones make similar number of contacts with midget bipolar cells, but short wavelength sensitive (S) cones make little or no contact. By calculating the numerical convergence between midget bipolar and midget ganglion cells, we estimate that midget ganglion cells receive input from up to 25 cones at approximately 5 degrees, and from more than 65 cones at approximately 50 degrees. No obvious differences were seen between the retinae of animals with di- or trichromatic color vision. The finding that the one-to-one connectivity is restricted to the fovea predicts that in marmosets spectral mixing of M/L cone inputs will occur peripheral to 10 degrees of visual angle.     

Cone bipolar cells in the retina of the microbat Carollia perspicillata

We studied the retinal cone bipolar cells of Carollia perspicillata, a microchiropteran bat of the phyllostomid family. Microchiroptera are strongly nocturnal, with small eyes and rod‐dominated retinae. However, they also possess a significant cone population (2–4%) comprising two spectral types, which are hence the basis for daylight and color vision. We used antibodies against the calcium‐binding protein recoverin and the carbohydrate epitope 15 (CD15) as reliable markers for certain cone bipolar cells. Dye injections of recoverin‐ or CD15‐prelabeled cone bipolar cells in vertical slices revealed the morphology of the axon terminal system of individual bipolar cells. Seven distinct cone bipolar cell types were identified. They differed in the morphology and stratification level of their axon terminal system in the inner plexiform layer and in immunoreactivity for recoverin and/or CD15. Additional immunocytochemical markers were used to assess the functional ON/OFF subdivision of the inner plexiform layer. In line with the extended thickness of the ON sublayer of the inner plexiform layer in the microbat retina, more ON than OFF cone bipolar cell types were found, namely, four versus three. Most likely, in the bats' predominantly dark environment, ON signals have greater importance for contrast perception. We conclude that the microbat retina conforms to the general mammalian blueprint, in which light signals of intensities above rod sensitivity are detected by cones and transmitted to various types of ON and OFF cone bipolar cells. J. Comp. Neurol. 523:963–981, 2015. © 2014 Wiley Periodicals, Inc.     

Morphological Classification of Bipolar Cells of the Primate Retina

Bipolar cells were studied in Golgi - Colonnier-stained whole mounts of macaque monkey retinae. A piece of retina, at 6 - 7 mm eccentricity, was particularly well stained for the analysis of the different bipolar cell types. Many midget bipolar cells were encountered and the dichotomy into flat and invaginating midget bipolars was confirmed. Six types of diffuse cone bipolar cell are distinguished. They differ in their dendritic branching pattern, in the number of cones contacted-usually between five and ten-and in the shape and branching level of their axons. The size, shape and stratification of the axons were found to be the most reliable distinguishing features for classifying diffuse cone bipolar cells. The stratification of the axons in the inner plexiform layer (IPL), whether closer to the amacrine or ganglion cells, was used to name diffuse cone bipolar cells in the order DB1 to DB6. Blue cone and rod bipolar cells were confirmed as distinct types. Axon terminals of diffuse cone bipolars were found to tile their sublamina of the IPL in a territorial manner. From this the density of each type could be estimated, and it is shown that a single cone is likely to be in contact with as many as 15 individual diffuse bipolar cells, as well as two midget bipolars. The diffuse bipolar cells observed contact all the cone pedicles in their dendritic fields. It is, therefore, unlikely that they carry a chromatic signal into the inner retina. The presence of many midget bipolar cells, which make contact with one cone pedicle only, suggests that midget bipolars provide chromatic input to ganglion cells in peripheral retina as well as in the fovea. The data show that the P- and M-cell pathways of the primate visual system are, to a significant extent, already anatomically discrete at the photoreceptor synapse.     

Bipolar cell-photoreceptor connectivity in the zebrafish (Danio rerio) retina

Bipolar cells convey luminance, spatial, and color information from photoreceptors to amacrine and ganglion cells. We studied the photoreceptor connectivity of 321 bipolar cells in the adult zebrafish retina. 1,1′‐Dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI) was inserted into whole‐mounted transgenic zebrafish retinas to label bipolar cells. The photoreceptors that connect to these DiI‐labeled cells were identified by transgenic fluorescence or their positions relative to the fluorescent cones, as cones are arranged in a highly ordered mosaic: rows of alternating blue‐ (B) and ultraviolet‐sensitive (UV) single cones alternate with rows of red‐ (R) and green‐sensitive (G) double cones. Rod terminals intersperse among cone terminals. As many as 18 connectivity subtypes were observed, 9 of which—G, GBUV, RG, RGB, RGBUV, RGRod, RGBRod, RGBUVRod, and RRod bipolar cells—accounted for 96% of the population. Based on their axon terminal stratification, these bipolar cells could be further subdivided into ON, OFF, and ON–OFF cells. The dendritic spread size, soma depth and size, and photoreceptor connections of the 308 bipolar cells within the nine common connectivity subtypes were determined, and their dendritic tree morphologies and axonal stratification patterns compared. We found that bipolar cells with the same axonal stratification patterns could have heterogeneous photoreceptor connectivity whereas bipolar cells with the same dendritic tree morphology usually had the same photoreceptor connectivity, although their axons might stratify on different levels. J. Comp. Neurol. 520:3786–3802, 2012. © 2012 Wiley Periodicals, Inc.     

Synaptic inputs onto small bistratified (blue‐ON/yellow‐OFF) ganglion cells in marmoset retina

The inner plexiform layer of the retina contains functional subdivisions, which segregate ON and OFF type light responses. Here, we studied quantitatively the ON and OFF synaptic input to small bistratified (blue‐ON/yellow‐OFF) ganglion cells in marmosets (Callithrix jacchus). Small bistratified cells display an extensive inner dendritic tier that receives blue‐ON input from short‐wavelength‐sensitive (S) cones via blue cone bipolar cells. The outer dendritic tier is sparse and is thought to receive yellow‐OFF input from medium (M)‐ and long (L)‐wavelength‐sensitive cones via OFF diffuse bipolar cells. In total, 14 small bistratified cells from different eccentricities were analyzed. The cells were retrogradely labeled from the koniocellular layers of the lateral geniculate nucleus and subsequently photofilled. Retinal preparations were processed with antibodies against the C‐terminal binding protein 2, the AMPA receptor subunit GluR4, and/or gephyrin to identify bipolar and/or amacrine input. The results show that the synaptic input is evenly distributed across the dendritic tree, with a density similar to that reported previously for other ganglion cell types. The population of cells showed a consistent pattern, where bipolar input to the inner tier is about fourfold greater than bipolar input to the outer tier. This structural asymmetry of bipolar input may help to balance the weight of cone signals from the sparse S cone array against inputs from the much denser M/L cone array. J. Comp. Neurol. 517:655–669, 2009. © 2009 Wiley‐Liss, Inc.     

Comparison of photoreceptor spatial density and ganglion cell morphology in the retina of human,macaque monkey,cat, and the marmoset Callithrix jacchus

We studied the relationship between the morphology of ganglion cells and the spatial density of photoreceptors in the retina of two Old World primates, human and macaque monkey; the diurnal New World marmoset Callithrix jacchus; and the cat. Ganglion cells in macaque and marmoset were labelled by intracellular injection with Neurobiotin or by Dil diffusion labelling in fixed tissue. Cone photoreceptor densities were measured from the same retinas. Supplemental data for macaque and data for human and cat were taken from published studies. For the primates studied, the central retina is characterised by a constant numerical convergence of cones to ganglion cells. Midget ganglion cells derive their input, via a midget bipolar cell, from a single cone. Parasol cells derive their input, via a midget bipolar cell, from a single cone. Parasol cells derive their input from 40–140 cones. Outside the central retina, the convergence increases with eccentricity. The convergence to beta cells in the cat retina is very close to that for parasol cells in primate retina. The convergence of rod photoreceptors to ganglion cells is similar in human, macaque, and marmoset, with parasol cells receiving input from 10–15 times more rods than midget cells. The low convergence of cones to midget cells in human and macaque retinas is associated with distinctive “clusters” in midget cells' dendritic fields. Convergence in marmoset is higher, and the clusters are absent. We conclude that the complementary changes in photoreceptor density and ganglion cell morphology should be considered when forming linking hypotheses between dendritic field, receptive field, and psychophysical properties of primate vision. © 1996 Wiley-Liss, Inc.     

Color vision and retinal chromatic information processing in teleost: A review

Teleosts exist under conditions where the intensity and spectral composition of available light is a function of media transmissivity, season, and geographic location. Moreover, the composition of a stimulus is dependent upon the object's direction and distance from the subject. Even should the same stimulus be presented repeatedly, the animal's ability to perceive it will be a function of temperature and season. Such diverse visual conditions have been dealt with (evolutionarily) by the development of specialized features such as a reflective tapetum, area and temperature dependent distribution of visual pigments, and an area-specific distribution of photoreceptor types.The cyprinid retina has a least 7 distinct photoreceptor types. Each of the photoreceptor types make synaptic contacts with bipolar and horizontal cells of one or more classes. There are at least 4 horizontal cell classes, 3 of which are thought to make color-specific feedback connections on photoreceptors. The receptors are not only the first neural retinal element, but also act as interneurons and display the first indication of antagonistic spectral and spatial response properties. Indeed, the complexity of the spectral response patterns observed within horizontal and receptor cells makes one wonder how information is sorted out in order to produce the high spectral and spatial resolution at the ganglion cell level. Two points must be kept in mind: (1) a photoreceptor's response is due primarily to quantum capture in that receptor's outer segment and is not due to horizontal cell feedback or inter-receptor connections; (2) bipolar cells form direct contacts with receptors and ganglion cells. The bipolar cells therefore provide a direct — straight-through — information transfer pathway.The importance of primary, direct neural pathways should not be underestimated. In general, the direct pathways, RECEPTOR→BIPOLAR→GANGLION CELL, provide the primary and most direct means of chromatic information transfer (Fig. 19). The secondary, internal, pathways, which include the horizontal and amacrine cells, are important only to the extent that they modify and contribute to both spectral and spatial contrast. The horizontal cells do contribute to the bipolar cell's surround properties, but the bipolar cell's greatest sensitivity and most dominant responses are within its central receptive field. The same can be said of the ganglion cells, their dominant response properties are to central field stimulation. The surround responses of both bipolars and ganglions are weak and temporally slow.The direct neural pathways can be subdivided into two systems which shall be termed the ON and OFF channels. Each of these channels consist of receptor, bipolar and ganglion cell combinations. These direct pathways are functionally defined by the response polarity of the bipolar and ganglion cell pair to a step increase in illumination. The ON pathways or channels consist of bipolar/ganglion cell combinations, each of which depolarizes to an increase in stimulus energy (+ΔI stimulus). Each component of the OFF pathways hyperpolarizes to the same stimulus. The ON and OFF pathways can also be identified anatomically. The ON pathways obviously make sign-inverting synapses between photoreceptors and bipolar cells, in order to transform the receptor's hyperpolarization into a depolarization: an ON response. The ON bipolar also terminates in only one lamina of the inner plexiform layer (layer b). The OFF pathways conserve the photoreceptors' hyperpolarizing response and the OFF bipolar cells terminate in sublamina ‘a’ of the IPL where they contact OFF ganglion cells. The ON and OFF pathways are believed to have different spectral sensitivities, with the ON channels representing the shorter wavelengths and the OFF channels favoring the longer wavelengths. The transfer of chromatic information is obviously more complex than simply ON and OFF responses.In addition to the independent ON and OFF pathways, some amacrine and ganglion cells receive input from both ON and OFF bipolars, forming the ON-OFF (transient) amacrine and ganglion cell classes. The ON-OFF cells terminate in both sublaminae of the IPL (Fig. 19). This combined input produces transient depolarizations to stimulus onset and cessation, due to the dominate depolarizing phase of the individual bipolar cell's response. The 3 identified ganglion cell types, ON, OFF, and ON-OFF may represent 3 distinct cell classes, or simply be components of a continuum with different ganglion cells contacting ON and OFF bipolar cells with different ratios. The continuum theory is supported by the observation that individual ganglion cells may produce ON, OFF, or ON-OFF responses depending on stimulus conditions, i.e., a ganglion cell's response may be altered by selecting stimulus conditions which favor ON or OFF bipolar cell types.The chromatic response properties of individual retinal neurons can best be described be defining which photoreceptor systems supply which interneurons. However, of all the bipolar cell types, only one or two have been adequately described in terms of receptor inputs. Moreover, no attempts have been made to define which bipolar cell types contact which ganglion cell classes. Therefore, it remains impossible to describe discrete (cell class to cell class) retinal chromatic pathways. It is impossible to determine the general response properties of such pathways by recording from ganglion cells. A spectrally/spatially defined ganglion cell permits one to deduce the spectral properties of the participating bipolar cells and whether they are ON or OFF units. With this information, the participating horizontal and receptor cell types can be described.The optic nerve may also affect chromatic information transfer. It is suggested that different size ganglion cells convey different chromatic information. Since there is a direct correlation between cell size and axon diameter and between axon diameter and conduction velocity, short versus long wavelength information may be transmitted to the CNS at different rates.Retinal ganglion cells project to and innervate numerous CNS structures. The optic tectum receives the majority of the projections. Even though numerous histological and functional studies on optic tectum have been reported, information on the response properties of individual cell types is non-existent. For example, the cytoarchitecture of the optic tectum is known, but no information is available on neural pathways or cellular interactions. We also do not know which CNS structures are involved in particular behavioral patterns.It is clear that different behavioral patterns are associated with different spectral sensitivities, i.e., spectral sensitivity is task dependent. Reflex type behaviors are apparently mediated by long wavelength stimuli, whereas more complex behaviors require differential integration across CNS structures and tend to be associated with medium and short wavelength stimuli. Data have been presented which suggest that spectral sensitivity is also a function of previous experience and may be altered during critical developmental periods. The major voids in our knowledge of chromatic information processing exist in CNS pathways, degree of task dependency and neural plasticity.     

Synaptic connectivity in the midget-parvocellular pathway of primate central retina

The synaptic connectivity of OFF midget bipolar cells was investigated in the central retina of two primate species, the New World common marmoset monkey, Callithrix jacchus, and the Old World macaque monkey, Macaca fascicularis. In marmosets, dichromatic and trichromatic animals were compared. Bipolar output synapses were identified with antibodies against ribbon proteins (kinesin, C-terminal binding protein 2) or with an antiserum that recognizes postsynaptic glutamate receptor clusters (GluR4). The midget bipolar cells were identified immunocytochemically with antibodies to CD15 (marmoset) or an antiserum to recoverin (macaque). In marmosets, midget ganglion cells were retrogradely labeled from the parvocellular layers of the dorsal lateral geniculate nucleus. Consistent with previous studies of Old World primates, in marmoset, midget bipolar cells contacted midget ganglion cells at a ratio of 1:1. The number of output synapses made by OFF midget bipolar cells was quantified for 104 cells in two dichromatic marmosets, 108 cells in one trichromatic marmoset, and 118 cells in one macaque. The number of output synapses was comparable for all animals, ranging from 10-71 in the dichromatic marmoset (average 29.7 +/- 12.4 SD), 12-86 in the trichromatic marmoset (average 28.6 +/- 11.7 SD) and 9-48 in the macaque (average 26.5 +/- 9.3 SD) per axon terminal. In all animals the number of output synapses per axon terminal showed a unimodal distribution. Our results suggest that the midget circuitry is comparable in dichromatic and trichromatic animals.     

Immunocytochemical analysis of bipolar cells in the macaque monkey retina

Transfer of visual information from photoreceptors to ganglion cells within the retina is mediated by specialized groups of bipolar cells. At least 10 different morphological types of bipolar cells have been distinguished in Golgi studies of primate retina. In the present study, bipolar cell populations in the macaque monkey retina were identified by their differential immunoreactivity to a spectrum of antibody markers. This enabled their spatial density and photoreceptor connections to be analysed. An antibody against the β isozyme of protein kinase C (PKCA_β) labelled many cone bipolar cells. Invaginating (presumed ON) cone bipolar cells and rod bipolar cells were prefentially labelled with a monoclonal antibody raised against rabbit olfactory bulb. Flat (presumed OFF) bipolar cells were labelled with an antiserum against the glutamate transporter protein (GLT-1). Different populations of diffuse cone bipolar cells, which contact 5–10 cones, could be distinguished. The GLT-1 artiserum preferentially labelled the flat diffuse bipolar cell type DB2 (Boycott and Wässle, 1991, Eur. J. Neurosci. 3:1069–1088) as well as flat midget bipolar cells. Antibodies to calbindin (CaBP D-28K) labelled the flat diffuse bipolar cell type DB3 and (possibly) the invaginating diffuse bipolar cell type DB5. An antibody against the α isozyme of PKC labelled an invaginating diffuse bipolar cell type (DB4) as well as rod bipolar cells. Comparison of the spatial density of cone bipolar cell populations with that of photoreceptors suggests that each bipolar cell class provides a complete coverage of the cone array (each cone is contacted by at least one member of every bipolar cell class). These results support the classification scheme of Boycott and Wässle (1991) by showing that different diffuse bipolar cell classes express different patterns of immunoreactivity, and they reinforce the view that different spatial and temporal components of the signal from the photoreceptor array are processed in parallel within the primate retina. © 1994 Wiley-Liss, Inc.     

Double Cones and the Diverse Connectivity of Photoreceptors and Bipolar Cells in an Avian Retina

Double cones are the most common photoreceptor cell type in most avian retinas, but their precise functions remain a mystery. Among their suggested functions are luminance detection, polarized light detection, and light-dependent, radical pair-based magnetoreception. To better understand the function of double cones, it will be crucial to know how they are connected to the neural network in the avian retina. Here we use serial sectioning, multibeam scanning electron microscopy to investigate double-cone anatomy and connectivity with a particular focus on their contacts to other photoreceptor and bipolar cells in the chicken retina. We found that double cones are highly connected to neighboring double cones and with other photoreceptor cells through telodendria-to-terminal and telodendria-to-telodendria contacts. We also identified 15 bipolar cell types based on their axonal stratifications, photoreceptor contact pattern, soma position, and dendritic and axonal field mosaics. Thirteen of these 15 bipolar cell types contacted at least one or both members of the double cone. All bipolar cells were bistratified or multistratified. We also identified surprising contacts between other cone types and between rods and cones. Our data indicate a much more complex connectivity network in the outer plexiform layer of the avian retina than originally expected.SIGNIFICANCE STATEMENT Like in humans, vision is one of the most important senses for birds. Here, we present the first serial section multibeam scanning electron microscopy dataset from any bird retina. We identified many previously undescribed rod-to-cone and cone-to-cone connections. Surprisingly, of the 15 bipolar cell types we identified, 11 received input from rods and 13 of 15 received at least part of their input from double cones. Therefore, double cones seem to play many different and important roles in avian retinal processing, and the neural network and thus information processing in the outer retina are much more complex than previously expected. These fundamental findings will be very important for several fields of science, including vertebrate vision, avian magnetoreception, and comparative neuroanatomy.     

Faster thalamocortical processing for dark than light visual targets

ON and OFF visual pathways originate in the retina at the synapse between photoreceptor and bipolar cells. OFF bipolar cells are shorter in length and use receptors with faster kinetics than ON bipolar cells and, therefore, process information faster. Here, we demonstrate that this temporal advantage is maintained through thalamocortical processing, with OFF visual responses reaching cortex ~3-6 ms before ON visual responses. Faster OFF visual responses could be demonstrated in recordings from large populations of cat thalamic neurons representing the center of vision (both X and Y) and from subpopulations making connection with the same cortical orientation column. While the OFF temporal advantage diminished as visual responses reached their peak, the integral of the impulse response was greater in OFF than ON neurons. Given the stimulus preferences from OFF and ON channels, our results indicate that darks are processed faster than lights in the thalamocortical pathway.     

Connectivity between the OFF bipolar type DB3a and six types of ganglion cell in the marmoset retina

Parallel visual pathways originate at the first synapse in the retina, where cones make connections with cone bipolar cells that in turn contact ganglion cells. There are more ganglion cell types than bipolar types, suggesting that there must be divergence from bipolar to ganglion cells. Here we analyze the contacts between an OFF bipolar type (DB3a) and six ganglion cell types in the retina of the marmoset monkey (Callithrix jacchus). Ganglion cells were transfected via particle‐mediated gene transfer of an expression plasmid for the postsynaptic density 95‐green fluorescent protein (PSD95‐GFP), and DB3a cells were labeled via immunohistochemistry. Ganglion cell types that fully or partially costratified with DB3a cells included OFF parasol, OFF midget, broad thorny, recursive bistratified, small bistratified, and large bistratified cells. On average, the number of DB3a contacts to parasol cells (18 contacts per axon terminal) is higher than that to other ganglion cell types (between four and seven contacts). We estimate that the DB3a output to OFF parasol cells accounts for at least 30% of the total DB3a output. Furthermore, we found that OFF parasol cells receive approximately 20% of their total bipolar input from DB3a cells, suggesting that other diffuse bipolar types also provide input to OFF parasol cells. We conclude that DB3a cells preferentially contact OFF parasol cells but also provide input to other ganglion cell types. J. Comp. Neurol. 524:1839–1858, 2016. © 2015 Wiley Periodicals, Inc.     

Characterization of small-field bistratified amacrine cells in macaque retina labeled by antibodies against synaptotagmin-2

Macaque retinae were immunostained with monoclonal antibodies directed against the protein synaptotagmin‐2 (Syt2). Syt2 was localized in a population of small‐field amacrine cells, whose cell bodies formed a regular mosaic within the inner nuclear layer, indicating they represent a single amacrine cell type. The labeled amacrine cells had a bistratified appearance with a dense dendritic plexus in the OFF‐layer and only a few lobular processes extending into the ON‐layer of the inner plexiform layer, similar to A8 amacrine cells described in cat and human retina. Syt2‐labeled cells were immunoreactive for glycine but lacked immunoreactivity for γ‐aminobutyric acid (GABA), suggesting they use glycine as their neurotransmitter. The density of these cells increases from ～200/mm² in peripheral retina to ～1,400/mm² in central retina. Their bipolar cell input was studied by immunolabeling experiments using various bipolar cell markers combined with CtBP2, a marker of presynaptic ribbons. Our data show that Syt2‐labeled amacrine cells receive input from both OFF and ON cone bipolar cells, as well as from rod bipolar cells. The OFF input is dominated by the diffuse bipolar cell DB1 (44%) and the OFF midget bipolar cell (38%). Here we describe a population of bistratified small‐field amacrine cells closely resembling A8 amacrine cells and their cone‐dominated bipolar cell input. J. Comp. Neurol. 521:709–724, 2013. © 2012 Wiley Periodicals, Inc.     

Expression of vesicular glutamate transporter 1 in the mouse retina reveals temporal ordering in development of rod vs. cone and ON vs. OFF circuits

Glutamatergic transmission is crucial to the segregation of ON and OFF pathways in the developing retina. The temporal sequence of maturation of vesicular glutamatergic transmission in rod and cone photoreceptor and ON and OFF bipolar cell terminals is currently unknown. Vesicular glutamate transporters (VGLUTs) that load glutamate into synaptic vesicles are necessary for vesicular glutamatergic transmission. To understand better the formation and maturation of glutamatergic transmission in the rod vs. cone and ON vs. OFF pathways of the retina, we examined the developmental expression of VGLUT1 and VGLUT2 immunocytochemically in the mouse retina. Photoreceptor and bipolar cell terminals showed only VGLUT1-immunoreactivity (-IR); no VGLUT2-IR was present in any synapses of the developing or adult retina. VGLUT1-IR was first detected in cone photoreceptor terminals at postnatal day 2 (P2), several days before initiation of ribbon synapse formation at P4-P5. Rod terminals showed VGLUT1-IR by P8, when they invade the outer plexiform layer (OPL) and initiate synaptogenesis. Developing OFF bipolar cell terminals showed VGLUT1-IR around P8, 2-3 days after bipolar terminals were first identified in the inner plexiform layer (IPL) by labeling for the photoreceptor and bipolar cell terminal marker, synaptic vesicle protein 2B. Although terminals of ON bipolar cells were present in the IPL by P6-P8, most did not show VGLUT1-IR until P8-P10 and increased dramatically from P12. These data suggest a hierarchical development of glutamatergic transmission in which cone circuits form prior to rod circuits in both the OPL and IPL, and OFF circuits form prior to ON circuits in the IPL.     

The ON and OFF channels of the visual system.

In the vertebrate retina, all photoreceptors hyperpolarize in response to light. In the outer retina, at the bipolar cell level, a dual system is created from the cones forming the ON and OFF channels. In the rod system a similar arrangement is found, but the ON and OFF channels in many species are formed using an amacrine cell network in the inner retina. Physiological experiments in which the ON bipolar cells are pharmacologically blocked reveal that in the primate the two channels remain largely segregated in the geniculostriate system until they reach the cortex, where they converge upon single cells. Behavioral studies show that following ON channel block, notable deficits arise in the detection of light increments but not light decrements. These and related studies suggest that the ON and OFF channels optimize information transfer to the CNS by providing excitatory signals for both increases and decreases in light energy.     

Bipolar cells of the ground squirrel retina

Parallel processing of an image projected onto the retina starts at the first synapse, the cone pedicle, and each cone feeds its light signal into a minimum of eight different bipolar cell types. Hence, the morphological classification of bipolar cells is a prerequisite for analyzing retinal circuitry. Here we applied common bipolar cell markers to the cone-dominated ground squirrel retina, studied the labeling by confocal microscopy and electron microscopy, and compared the resulting bipolar cell types with those of the mouse (rod dominated) and primate retina. Eight different cone bipolar cell types (three OFF and five ON) and one rod bipolar cell were distinguished. The major criteria for classifying the cells were their immunocytochemical identity, their dendritic branching pattern, and the shape and stratification level of their axons in the inner plexiform layer (IPL). Immunostaining with antibodies against Gγ13, a marker for ON bipolar cells, made it possible to separate OFF and ON bipolars. Recoverin-positive OFF bipolar cells partly overlapped with ON bipolar axon terminals at the ON/OFF border of the IPL. Antibodies against HCN4 labeled the S-cone selective (bb) bipolar cell. The calcium-binding protein CaB5 was expressed in two OFF and two ON cone bipolar cell types, and CD15 labeled a widefield ON cone bipolar cell comparable to the DB6 in primate.     

Connections of diffuse bipolar cells in primate retina are biased against S-cones

In mammalian retina, each diffuse bipolar type stratifies in a distinct layer of the inner plexiform layer. Thus, different types of bipolar cells provide output to distinct visual pathways. Here, the question of whether diffuse bipolar cell types differ with respect to their contacts with short wavelength-sensitive (S-) cones was investigated in the retinas of a New World monkey, Callithrix jacchus, and an Old World monkey, Macaca fascicularis. Subpopulations of OFF bipolar cells were labeled with antibodies to the glutamate transporter Glt-1 and ON bipolar cells were labeled with antibodies to the alpha subunit of the Go protein (Goalpha). Two types of diffuse ON bipolar cells, DB4 and DB6, were identified with antibodies to protein kinase Calpha and CD15, respectively. Cone pedicles were labeled either with peanut agglutinin coupled to fluorescein or with antibodies to the ribbon protein, C-terminus binding protein 2. We found that immunoreactivity for Glt-1 (OFF bipolar cells) is reduced at S-cones in comparison to medium/long wavelength-sensitive (M/L-) cones. Immunoreactivity for Goalpha (ON bipolar cells) is comparable at all cone types. Nearly all M/L-cone pedicles contact the diffuse ON bipolar types DB4 and DB6, but only between 60% and 75% of the S-cone pedicles make contact. Furthermore, the number of dendritic tips of DB4 and DB6 cells at S-cone pedicles is lower than that at M/L-cone pedicles. These results suggest that there is a bias in the S-cone connectivity of diffuse bipolar cells.     

Selective synaptic connections in the retinal pathway for night vision

The mammalian retina encodes visual information in dim light using rod photoreceptors and a specialized circuit: rods→rod bipolar cells→AII amacrine cell. The AII amacrine cell uses sign-conserving electrical synapses to modulate ON cone bipolar cell terminals and sign-inverting chemical (glycinergic) synapses to modulate OFF cone cell bipolar terminals; these ON and OFF cone bipolar terminals then drive the output neurons, retinal ganglion cells (RGCs), following light increments and decrements, respectively. The AII amacrine cell also makes direct glycinergic synapses with certain RGCs, but it is not well established how many types receive this direct AII input. Here, we investigated functional AII amacrine→RGC synaptic connections in the retina of the guinea pig (Cavia porcellus) by recording inhibitory currents from RGCs in the presence of ionotropic glutamate receptor (iGluR) antagonists. This condition isolates a specific pathway through the AII amacrine cell that does not require iGluRs: cone→ON cone bipolar cell→AII amacrine cell→RGC. These recordings show that AII amacrine cells make direct synapses with OFF Alpha, OFF Delta and a smaller OFF transient RGC type that co-stratifies with OFF Alpha cells. However, AII amacrine cells avoid making synapses with numerous RGC types that co-stratify with the connected RGCs. Selective AII connections ensure that a privileged minority of RGC types receives direct input from the night-vision pathway, independent from OFF bipolar cell activity. Furthermore, these results illustrate the specificity of retinal connections, which cannot be predicted solely by co-stratification of dendrites and axons within the inner plexiform layer.     

Diffuse bipolar cells provide input to OFF parasol ganglion cells in the macaque retina

Parasol retinal ganglion cells are more sensitive to luminance contrast and respond more transiently at all levels of adaptation than midget ganglion cells. This may be due, in part, to differences between bipolar cells that provide their input, and the goal of these experiments was to study these differences. Midget bipolar cells are known to be presynaptic to midget ganglion cells. To identify the bipolar cells presynaptic to parasol cells, these ganglion cells were intracellularly injected with Neurobiotin, cone bipolar cells were immunolabeled, and the double-labeled material was analyzed. In the electron microscope, we found that DB3 diffuse bipolar cells labeled by using antiserum to calbindin D-28k were presynaptic to OFF parasol cells. In the confocal microscope, DB3 bipolars costratified with OFF parasol cell dendrites and made significantly more appositions with them than expected due to chance. Flat midget bipolar cells were labeled with antiserum to recoverin. Although they made a few appositions with parasol cells, the number was no greater than would be expected when two sets of processes have overlapping distributions in the inner plexiform layer. DB2 diffuse bipolar cells were labeled with antibodies to excitatory amino acid transporter 2, and they also made appositions with OFF parasol cells. These results suggest that DB2 bipolar cells are also presynaptic to OFF parasol ganglion cells, but midget bipolar cells are not. We estimate that midperipheral OFF parasol cells receive approximately 500 synapses from 50 DB3 bipolar cells that, in turn, receive input from 250 cones.     

Morphology and connectivity of the small bistratified A8 amacrine cell in the mouse retina

Amacrine cells comprise ～30 morphological types in the mammalian retina. The synaptic connectivity and function of a few γ‐aminobutyric acid (GABA)ergic wide‐field amacrine cells have recently been studied; however, with the exception of the rod pathway‐specific AII amacrine cell, the connectivity of glycinergic small‐field amacrine cells has not been investigated in the mouse retina. Here, we studied the morphology and connectivity pattern of the small‐field A8 amacrine cell. A8 cells in mouse retina are bistratified with lobular processes in the ON sublamina and arboreal dendrites in the OFF sublamina of the inner plexiform layer. The distinct bistratified morphology was first visible at postnatal day 8, reaching the adult shape at P13, around eye opening. The connectivity of A8 cells to bipolar cells and ganglion cells was studied by double and triple immunolabeling experiments by using various cell markers combined with synaptic markers. Our data suggest that A8 amacrine cells receive glutamatergic input from both OFF and ON cone bipolar cells. Furthermore, A8 cells are coupled to ON cone bipolar cells by gap junctions, and provide inhibitory input via glycine receptor (GlyR) subunit α1 to OFF cone bipolar cells and to ON A‐type ganglion cells. Measurements of spontaneous glycinergic postsynaptic currents and GlyR immunolabeling revealed that A8 cells express GlyRs containing the α2 subunit. The results show that the bistratified A8 cell makes very similar synaptic contacts with cone bipolar cells as the rod pathway‐specific AII amacrine cell. However, unlike AII cells, A8 amacrine cells provide glycinergic input to ON A‐type ganglion cells. J. Comp. Neurol. 523:1529–1547, 2015. © 2015 Wiley Periodicals, Inc.