Multiple Invagination Patterns and Synaptic Efficacy in Primate and Mouse Rod Synaptic Terminals.

Purpose: Optical retina images are scaled based on eye size, which results in a linear scale ratio of 10:1 for human versus mouse and 7:1 for macaque monkey versus mouse. We examined how this scale difference correlates with the structural configuration of synaptic wiring in the rod spherule (RS) between macaque and mouse retinas compared with human data. Methods: Rod bipolar cell (BC) dendrites and horizontal cell (HC) axonal processes, which invaginate the RS to form synaptic ribbon-associated triads, were examined by serial section transmission electron microscopy. Results: The number of rod BC invaginating dendrites ranged 1∼4 in the macaque RS but only 1∼2 in the mouse. Approximately 40% of those dendrites bifurcated into two central elements in the macaque, but 2% of those dendrites did in the mouse. Both factors gave rise to 10 invagination patterns of BC and HC neurites in the macaque RS but only two in the mouse. Five morphological parameters: the lengths of arciform densities and ribbons, the area of the BC-RS contact, and the surface areas of BC and HC invaginating neurites, were all independent of the invagination patterns in the macaque RS. However, those parameters were significantly greater in the macaque than in the mouse by ratios of 1.5∼1.8. Conclusions: The primate RS provides a more expansive BC-RS interface associated with the longer arciform density and more branched invaginating neurites of BCs and HCs than the mouse RS. The resulting greater synaptic contact area may contribute to more efficient signal transfer.

Helical Fasciculation of Bipolar and Horizontal Cell Neurites for Wiring With Photoreceptors in Macaque and Mouse Retinas.

Purpose: The three-dimensional configurations of rod and cone bipolar cell (BC) dendrites and horizontal cell (HC) processes outside rod and cone synaptic terminals have not been fully elucidated. We reveal how these neurites are mutually arranged to coordinate formation and maintenance of the postsynaptic complex of ribbon synapses in mouse and monkey retinas. Methods: Serial section transmission electron microscopy was utilized to reconstruct BC and HC neurites in macaque monkey and mouse, including metabotropic glutamate receptor 6 (mGluR6)-knockout mice. Results: Starting from sporadically distributed branching points, rod BC and HC neurites (B and H, respectively) took specific paths to rod spherules by gradually adjusting their mutual positions, which resulted in a closed alternating pattern of H‒B‒H‒B neurites at the rod spherule aperture. This order corresponded to the array of elements constituting the postsynaptic complex of ribbon synapses. We identified novel helical coils of HC processes surrounding the rod BC dendrite in both mouse and macaque retinas, and these structures occurred more frequently in mGluR6-knockout than wild-type mouse retinas. Horizontal cell processes also formed hook-like protrusions that encircled cone BC and HC neurites below the cone pedicles in the macaque retina. Conclusions: Bipolar and horizontal cell neurites take specific paths to adjust their mutual positions at the rod spherule aperture. Some HC processes are helically coiled around rod BC dendrites or form hook-like protrusions around cone BC dendrites and HC processes. Loss of mGluR6 signaling may be one factor promoting unbalanced neurite growth and compensatory neurite coiling.

Classification of Mouse Retinal Bipolar Cells: Type-Specific Connectivity with Special Reference to Rod-Driven AII Amacrine Pathways.

We confirmed the classification of 15 morphological types of mouse bipolar cells by serial section transmission electron microscopy and characterized each type by identifying chemical synapses and gap junctions at axon terminals. Although whether the previous type 5 cells consist of two or three types was uncertain, they are here clustered into three types based on the vertical distribution of axonal ribbons. Next, while two groups of rod bipolar (RB) cells, RB1, and RB2, were previously proposed, we clarify that a half of RB1 cells have the intermediate characteristics, suggesting that these two groups comprise a single RB type. After validation of bipolar cell types, we examined their relationship with amacrine cells then particularly with AII amacrine cells. We found a strong correlation between the number of amacrine cell synaptic contacts and the number of bipolar cell axonal ribbons. Formation of bipolar cell output at each ribbon synapse may be effectively regulated by a few nearby inhibitory inputs of amacrine cells which are chosen from among many amacrine cell types. We also found that almost all types of ON cone bipolar cells frequently have a minor group of midway ribbons along the axon passing through the OFF sublamina as well as a major group of terminal ribbons in the ON sublamina. AII amacrine cells are connected to five of six OFF bipolar cell types via conventional chemical synapses and seven of eight ON (cone) bipolar cell types via electrical synapses (gap junctions). However, the number of synapses is dependent on bipolar cell types. Type 2 cells have 69% of the total number of OFF bipolar chemical synaptic contacts with AII amacrine cells and type 6 cells have 46% of the total area of ON bipolar gap junctions with AII amacrine cells. Both type 2 and 6 cells gain the greatest access to AII amacrine cell signals also share those signals with other types of bipolar cells via networked gap junctions. These findings imply that the most sensitive scotopic signal may be conveyed to the center by ganglion cells that have the most numerous synapses with type 2 and 6 cells.

ON Bipolar Cells in Macaque Retina: Type-Specific Synaptic Connectivity with Special Reference to OFF Counterparts.

To date, 12 macaque bipolar cell types have been described. This list includes all morphology types first outlined by Polyak (1941) using the Golgi method in the primate retina and subsequently identified by other researchers using electron microscopy (EM) combined with the Golgi method, serial section transmission EM (SSTEM), and immunohistochemical imaging. We used SSTEM for the rod-dense perifoveal area of macaque retina, reconfirmed ON (cone) bipolar cells to be classified as invaginating midget bipolar (IMB), diffuse bipolar (DB)4, DB5, DB6, giant bipolar (GB), and blue bipolar (BB) types, and clarified their type-specific connectivity. DB4 cells made reciprocal synapses with a kind of ON-OFF lateral amacrine cell, similar to OFF DB2 cells. GB cells contacted rods and cones, similar to OFF DB3b cells. Retinal circuits formed by GB and DB3b cells are thought to substantiate the psychophysical finding of fast rod signals in mesopic vision. DB6 cell output synapses were directed to ON midget ganglion (MG) cells at 70% of ribbon contacts, similar to OFF DB1 cells that directed 60% of ribbon contacts to OFF MG cells. IMB cells contacted medium- or long-wavelength sensitive (M/L-) cones but not short-wavelength sensitive (S-) cones, while BB cells contacted S-cones but not M/L-cones. However, IMB and BB dendrites had similar morphological architectures, and a BB cell contacting a single S-cone resembled an IMB cell. Thus, both IMB and BB may be the ON bipolar counterparts of the OFF flat midget bipolar (FMB) type, likewise DB4 of DB2, DB5 of DB3a, DB6 of DB1, and GB of DB3b OFF bipolar type. The ON DB plus GB, and OFF DB cells predominantly contacted M/L-cones and their outputs were directed mainly to parasol ganglion (PG) cells but also moderately to MG cells. BB cells directed S-cone-driven outputs almost exclusively to small bistratified ganglion (SBG) cells. Some FMB cells predominantly contacted S-cones and their outputs were directed to OFF MG cells. Thus, two-step synaptic connections largely narrowed down the S-cone component to SBG and some OFF MG cells. The other OFF MG cells, ON MG cells, and ON and OFF PG cells constructed M/L-cone dominant pathways.

Corrigendum: (1) Some OFF Bipolar Cell Types Make Contact With Both Rods and Cones in Macaque and Mouse Retinas; (2) OFF Bipolar Cells in Macaque Retina: Type-specific Connectivity in the Outer and Inner Synaptic Layers.

[This corrects the article on p. 105 in vol. 8, PMID: 25309346.][This corrects the article on p. 122 in vol. 9, PMID: 26500507.].

OFF bipolar cells in macaque retina: type-specific connectivity in the outer and inner synaptic layers.

OFF bipolar cells in the macaque retina were recently classified into five types: flat midget bipolar (FMB) and diffuse bipolar (DB) 1, 2, 3a, and 3b. We examined all parallel pathways from cone photoreceptors via OFF bipolar cells to parasol and midget ganglion cells by serial section transmission electron microscopy. Basal contacts of OFF bipolar cells to cone pedicles were previously categorized as triad-associated (TA) and non-TA (NTA). The latter was further divided into two groups located in the middle and marginal areas of the pedicle at the present eccentricity of 15°. We then mapped the distributions of all three basal contacts of the five OFF bipolar cell types in the same area of cone pedicles. TA contacts were more numerous than NTA contacts in FMB (93%), DB1 (67%), and DB3a (81%) cells, but less in DB2 (30%) and DB3b (21%) cells. Cluster analysis of these contact parameters reconfirmed five distinct OFF bipolar cell types and showed these positional configurations of basal synapses to be cell type-specific. This architecture is thought to provide a spatial framework for the interstitial diffusion and local uptake of the neurotransmitter (glutamate) that spills over from ribbon synapses. All five OFF bipolar cell types formed ribbon-synaptic contacts to both parasol and midget ganglion cells. DB2 and 3a, DB1 and 3b, and FMB predominantly, moderately, and negligibly contacted parasol ganglion cells, respectively. FMB almost exclusively contacted midget ganglion cells, to which DB1 provided dominant output (58%), and DB2, 3a, and 3b provided between 3% and 10% of their output. Consequently, the cone signal sampling routes of a midget ganglion cell consisted of two substructures: the narrow (mainly 2-3 cones) FMB pathway and the wide (mainly 10 cones) DB pathway, where connection strength was four-fold greater in the FMB than DB pathway. The narrow and strong FMB pathway may confer the highest spatial resolution and sporadically may include blue cone signals.

Some OFF bipolar cell types make contact with both rods and cones in macaque and mouse retinas.

This study compared the types of OFF bipolar cells found in the macaque retina with those found in the mouse retina and determined whether these OFF bipolar cells make direct contacts with both rods and cones by serial section transmission electron microscopy. We performed scatter plots and cluster analysis of the morphological variables of their axon terminals such as the stratification level, the arbor thickness, the arbor area, and the number of ribbons. Five OFF bipolar cell types, including the recently discovered DB3b type, were identified in the macaque retina. The macaque OFF bipolar cell types FMB, DB1, DB2, DB3a, and DB3b corresponded to the mouse OFF bipolar cell types 2, 1, 4, 3a, and 3b, respectively. In addition to contacting rod bipolar cells, ~7% of rods in the macaque retina made basal contacts exclusively with one cell type, DB3b, whereas 18% of rods in the mouse retina made basal contacts with one or two of types, 3a, 3b, and 4. Approximately 3% of mouse rods were divergently connected to two OFF bipolar cells of different types, but macaque rods were solely connected to one OFF bipolar cell. Rod-rod gap junctions were localized at rod cell bodies and axons in the outer nuclear layer in both macaque and mouse retinas. The direct rod-OFF bipolar connection system is slightly more developed in the mouse retina than in the macaque retina, possibly as a fine-tuned adaptation to nocturnal conditions. This one-step direct synaptic pathway from rods to OFF bipolar cells may enhance the response speed to OFF light stimuli compared with more indirect pathways via rod-cone gap junctions (a two-step pathway) and via rod bipolar and AII amacrine cells (a three-step pathway).

Effects of mGluR6-deficiency on photoreceptor ribbon synapse formation: comparison of electron microscopic analysis of serial sections with random sections.

This study examined the effects of metabotropic glutamate receptor 6 (mGluR6) deficiency on ribbon synapse formation in rod spherules and cone pedicles using serial-section electron microscopy. In a wild-type (WT) mouse, only 3% of spherules had one invaginating bipolar dendrite (1B-type) and 97% of spherules were 2B-type. In contrast, in an mGluR6-knockout (KO) mouse, 29% of spherules were 1B-type and 71% of spherules were 2B-type. Spherules without bipolar invagination were not observed in either genotype. The single invaginating dendrites in 1B-type spherules were larger and the surface areas of synaptic ribbons were 23% smaller in the mGluR6-KO mouse than in the WT mouse. In cones, the number of invaginating bipolar dendrites decreased from 12 in the WT mouse to 9.5 in the mGluR6-KO mouse. This decrease correlated with a decrease in the number of cone synaptic ribbons from 10 in the WT mouse to 8 in the mGluR6-KO mouse. The mGluR6-KO phenotype showed negative effects on ribbon synapse formation. This negativity was similar to those in mGluR6-nob4, Gß3-KO, Gß5-KO, and RGS-7:RGS-11 double-KO mice, but the detailed manners and degrees of alterations appeared to vary depending on different missing components. Two published morphological assessments of the RGS-7:RGS-11 double-KO phenotype reported conflicting data; therefore, we tested the statistical techniques used in the two analyses. One statistical evaluation measure was effective in identifying a significant difference in structure between the mutant and WT phenotypes, whereas the other measure was ineffective. Conventional random section analysis using the effective measure provided sufficient data for a statistical test of the occurrence of structural changes. However, serial section analysis was required to determine the absolute numbers of ribbons and invaginating dendrites and to estimate structural parameters such as ribbon surface area.

Functional allocation of synaptic contacts in microcircuits from rods via rod bipolar to AII amacrine cells in the mouse retina.

Retinal microcircuits for night vision at the absolute threshold are required to relay a single-photon rod signal reliably to ganglion cells via rod bipolar (RB) cells and AII amacrine cells. To assess the noise reduction of intercellular signal transmission in this rod-specific pathway, we quantified its synaptic connectivity by 3D reconstruction of a series of electron micrographs. In most cases (94%), each rod made ribbon synaptic contacts onto two adjacent RB cells. Conversely, each RB cell was contacted by 25 rods. Each RB axon terminal contacted four or five AII amacrine cells via 53 ribbon synapses. Thus, the signal from one rod may be represented as 106 replicates at two RB axons. Moreover, the two adjacent RB cells contacted two to four AII amacrine cells in common, where the signals relayed by two RB cells were reunited. In more detail, over 50% of each RB output was directed predominantly to a single, preferred AII amacrine cell, although each RB cell also separately contacted another one to three AII amacrine cells. Most of the replicate signals at two RB axons were collected on a few AII amacrine cells via reunions, dominant connections, and electrical coupling by AII-AII gap junctions. Thus the original signal may be reliably represented by signal amplification with focal accumulation without gathering unnecessary noise from a wide surrounding area. This allocation of RB-AII synaptic contacts may serve as the structural basis for the physiological properties of the AII single-photon response that include high amplification, local adaptation, and regenerative acceleration.

Formation of a disulfide protein conjugate of the SH-group-containing metabolite (M-I) of esonarimod (KE-298) and its elimination in rats.

The reactivity of the thiol moiety of the active main metabolite (M-I) of esonarimod (KE-298), a novel anti-rheumatic agent, was investigated in rats. After repeated oral administration of 14C-KE-298, the radioactivity decreased rapidly and no tendency towards accumulation was found, in marked contrast to other common SH-group-containing drugs. At 30 min after intravenous administration of 14C-M-I to rats, the concentration of the 14C-M-I plasma protein conjugate in plasma was extremely low at 0.143 nmol mL(-1) (0.66% of total plasma radioactivity). The 14C-M-I plasma protein conjugate that formed in rat plasma was mixed disulfide with plasma protein. After intravenous administration of synthetic 14C-M-I plasma protein conjugate to rats, the radioactivity in plasma decreased rapidly, with the terminal half-life at 6.90 h. In-vitro, the 14C-M-I plasma protein conjugate was readily dissociated by the endogenous thiol compounds, cysteine and glutathione. These results suggest that the reactivity of the thiol moiety of M-I is extremely low. Furthermore, the 14C-M-I plasma protein conjugate decreased rapidly in-vivo, which would be related to interaction with endogenous thiol compounds. These properties of M-I are principally responsible for the zero accumulation in rat tissues. KE-298 could therefore be expected to have reduced adverse effects compared with other SH-group-containing anti-rheumatic drugs.