Survey of retinal ganglion cell morphology in marmoset.

In primate retina, the midget, parasol, and small bistratified cell populations form the large majority of ganglion cells. In addition, there is a variety of low-density wide-field ganglion cell types that are less well characterized. Here we studied retinal ganglion cells in the common marmoset, Callithrix jacchus, using particle-mediated gene transfer. Ganglion cells were transfected with an expression plasmid for the postsynaptic density 95-green fluorescent protein. The retinas were processed with established immunohistochemical markers for bipolar and/or amacrine cells to determine ganglion cell dendritic stratification. In total over 500 ganglion cells were classified based on their dendritic field size, morphology, and stratification in the inner plexiform layer. Over 17 types were distinguished, including midget, parasol, broad thorny, small bistratified, large bistratified, recursive bistratified, recursive monostratified, narrow thorny, smooth monostratified, large sparse, giant sparse (melanopsin) ganglion cells, and a group that may contain several as yet uncharacterized types. Assuming each characterized type forms a hexagonal mosaic, the midget and parasol cells account for over 80% of all ganglion cells in the central retina but only ∼50% of cells in the peripheral (>2 mm) retina. We conclude that the fovea is dominated by midget and parasol cells, but outside the fovea the ganglion cell diversity in marmoset is likely as great as that reported for nonprimate retinas. Taken together, the ganglion cell types in marmoset retina resemble those described previously in macaque retina with respect to morphology, stratification, and change in proportion across the retina.

Does heterogeneity of intracellular Ca[Formula: see text] dynamics underlie speed tuning of direction-selective responses in starburst amacrine cells?

The starburst amacrine cell (SAC) plays a fundamental role in retinal motion perception. In the vertebrate retina, SAC dendrites have been shown to be directionally selective in terms of their Ca[Formula: see text] responses for stimuli that move centrifugally from the soma. The mechanism by which SACs show Ca[Formula: see text] bias for centrifugal motion is yet to be determined with precision. Recent morphological studies support a presynaptic delay in glutamate receptor activation induced Ca[Formula: see text] release from bipolar cells preferentially contacting SACs. However, bipolar cells are known to be electrotonically coupled so time delays between the bipolar cells that provide input to SACs seem unlikely. Using fluorescent microscopy and imunnostaining, we found that the endoplasmic reticulum (ER) is omnipresent in the soma extending to the distal processes of SACs. Consequently, a working hypothesis on heterogeneity of intracellular Ca[Formula: see text] dynamics from ER is proposed as a possible explanation for the cause of speed tuning of direction-selective Ca[Formula: see text] responses in dendrites of SACs.

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.

[Morphological and functional diversity of retinal ganglion cells in the common marmoset].

Retinal ganglion cells are projecting neurons that send visual information from the retina to the brain. They generate different patterns of action potentials in response to different kinds of visual information. In the retinas of mammals such as mice or rabbits, there are functionally and morphologically diverse retinal ganglion cells. However, in the primate retina, midget and parasol cells are believed to be dominant, and show less diversity. In this study, we performed organotypic culture of retinas and acute gene transfection of GFPs by gene-gun. We found more diverse retinal ganglion cells, including directional selective ganglion cells, than we expected, even in the retinas of primates such as common marmosets. Further, we found a third pathway from the retina to the brain via the thalamus, in addition to the magnocellular and parvocellular pathways.

Identification of a pathway from the retina to koniocellular layer K1 in the lateral geniculate nucleus of marmoset.

Three well characterized pathways in primate vision (midget-parvocellular, parasol-magnocellular, bistratified-koniocellular) have been traced from the first synapse in the retina, through the visual thalamus (lateral geniculate nucleus, LGN), to the visual cortex. Here we identify a pathway from the first synapse in the retina to koniocellular layer K1 in marmoset monkeys (Callithrix jacchus). Particle-mediated gene transfer of an expression plasmid for the postsynaptic density 95-green fluorescent protein (PSD95-GFP) was used to label excitatory synapses on retinal ganglion cells and combined with immunofluorescence to identify the presynaptic bipolar cells. We found that axon terminals of one type of diffuse bipolar cell (DB6) provide dominant synaptic input to the dendrites of narrow thorny ganglion cells. Retrograde tracer injections into the LGN and photofilling of retinal ganglion cells showed that narrow thorny cells were preferentially labeled when koniocellular layer K1 was targeted. Layer K1 contains cells with high sensitivity for rapid movement, and layer K1 sends projections to association visual areas as well as to primary visual cortex. We hypothesize that the DB6-narrow thorny-koniocellular pathway contributes to residual visual functions ("blindsight") that survive injury to primary visual cortex in adult or early life.

The Muscle Sensor for on-site neuroscience lectures to pave the way for a better understanding of brain-machine-interface research.

Neuroscience is an expanding field of science to investigate enigmas of brain and human body function. However, the majority of the public have never had the chance to learn the basics of neuroscience and new knowledge from advanced neuroscience research through hands-on experience. Here, we report that we produced the Muscle Sensor, a simplified electromyography, to promote educational understanding in neuroscience. The Muscle Sensor can detect myoelectric potentials which are filtered and processed as 3-V pulse signals to shine a light bulb and emit beep sounds. With this educational tool, we delivered "On-Site Neuroscience Lectures" in Japanese junior-high schools to facilitate hands-on experience of neuroscientific electrophysiology and to connect their text-book knowledge to advanced neuroscience researches. On-site neuroscience lectures with the Muscle Sensor pave the way for a better understanding of the basics of neuroscience and the latest topics such as how brain-machine-interface technology could help patients with disabilities such as spinal cord injuries.

Diversity of retinal ganglion cells identified by transient GFP transfection in organotypic tissue culture of adult marmoset monkey retina.

The mammalian retina has more diversity of neurons than scientists had once believed in order to establish complicated vision processing. In the monkey retina, morphological diversity of retinal ganglion cells (RGCs) besides dominant midget and parasol cells has been suggested. However, characteristic subtypes of RGCs in other species such as bistratified direction-selective ganglion cells (DSGC) have not yet been identified. Increasing interest has been shown in the common marmoset (Callithrix jacchus) monkey as a "super-model" of neuroscientific research. Here, we established organotypic tissue culture of the adult marmoset monkey retina with particle-mediated gene transfer of GFP to survey the morphological diversity of RGCs. We successfully incubated adult marmoset monkey retinas for 2 to 4 days ex vivo for transient expression of GFP. We morphologically examined 121 RGCs out of more than 3240 GFP-transfected cells in 5 retinas. Among them, we identified monostratified or broadly stratified ganglion cells (midget, parasol, sparse, recursive, thorny, and broad thorny ganglion cells), and bistratified ganglion cells (recursive, large, and small bistratified ganglion cells [blue-ON/yellow-OFF-like]). By this survey, we also found a candidate for bistratified DSGC whose dendrites were well cofasciculated with ChAT-positive starburst dendrites, costratified with ON and OFF ChAT bands, and had honeycomb-shaped dendritic arbors morphologically similar to those in rabbits. Our genetic engineering method provides a new approach to future investigation for morphological and functional diversity of RGCs in the monkey retina.

The manipulation of neural and cellular activities by ectopic expression of melanopsin.

Melanopsin (OPN4) is a photosensitive pigment originally found in a subtype of retinal ganglion cells and is a 7-transmembrane G-protein-coupled receptor (GPCR). Several previous reports showed that ectopic expression of OPN4 can be used as an optogenetic tool to control neural and cellular activities in various tissues. Compared with other optogenetic pigments, OPN4 is more sensitive to light, shows long-lasting activation, and can also control intracellular Ca(2+) dynamics. Here, we review how the ectopic expression of OPN4 enables the control of neural and cellular activities in vivo. In the retina, the ectopic expression of melanopsin in retinal ganglion cells successfully restored the vision of blind mice. It has also been reported that ectopic expression of melanopsin in orexin/hypocretin neurons enabled control of wakefulness in mice by blue light. In addition to neural activity, the ectopic expression of OPN4 has been reported to enable circuit control of the nuclear factor of activated T cells (NFAT) to enhance blood-glucose homeostasis in mice. We discuss the possibility of optogenetic control of other systems through the ectopic expression of OPN4.

Ectopic expression of melanopsin in orexin/hypocretin neurons enables control of wakefulness of mice in vivo by blue light.

Melanopsin (OPN4) is a photosensitive G-protein-coupled photopigment and its ectopic expression enables control of neural activity induced by blue light. Here we report that we successfully expressed OPN4 in hypothalamic orexin/hypocretin neurons of double-transgenic mice (orexin-tTA; Bitet-O human OPN4 [hOPN4]/mCherry mice). In the double-transgenic mice, hypothalamic orexin neurons selectively expressed hOPN4 as well as mCherry as a reporter. We conducted slice patch-clamp recordings on hOPN4/mCherry-expressing orexin neurons, which showed long-lasting activation initiated by blue light even after the light was switched off. Optical fiber-guided blue light stimulation in the hypothalamus successfully initiated the electroencephalography pattern that reflects long-lasting wakefulness in the mice in vivo. Taken together, the results indicate that ectopic expression of hOPN4 in orexin neurons enables long-lasting activation of orexin neurons by blue light to control sleep/wakefulness of the mice.

A method of horizontally sliced preparation of the retina.

Various types of retinal neurons, including amacrine, ganglion, and horizontal cells, expand neurites (dendrites or axons) in horizontal direction and make synaptic or electrical contacts with other cells to integrate the visual information. Many types of ion-channels and receptors are located along these neurites, and these horizontal connections critically contribute to the information processing in the retinal circuits. However, many of previous electrophysiological and immunocytochemical studies employed slice preparations cut by vertical direction in which most of these cells and their neurites were severely damaged and removed. This might lead to the underestimation of active and passive conductance in horizontally expanding neurites, and also missing of morphological information of horizontal structures. Here, we describe an alternative slicing method of horizontally cut preparation of the retina. The slice is made horizontally at the inner layer of the retina using a vibratome slicer after the retina is embedded in the low-temperature melting agarose gel. This horizontal slice preparation enables us to directly access cells in the inner retina by patch-clamp recording, calcium imaging, single RT-PCR, and immunocytochemistry. The method described here would offer an alternative strategy for studying the functions of neurons and neural circuits in the retina.

Localization of acetylcholine-related molecules in the retina: implication of the communication from photoreceptor to retinal pigment epithelium.

It has been long speculated that specific signals are transmitted from photoreceptors to the retinal pigment epithelium (RPE). However, such signals have not been identified. In this study, we examined the retinal expression and localization of acetylcholine-related molecules as putative candidates for these signals. Previous reports revealed that α7 nicotinic acetylcholine receptors (nAChRs) are present in the microvilli of RPE cells that envelope the tips of photoreceptor outer segments (OS). Secreted mammalian leukocyte antigen 6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP-1) is a positive allosteric modulator of the α7 nAChR. Therefore, we first focused on the expression of SLURP-1. SLURP-1 mRNA was expressed in the outer nuclear layer, which is comprised of photoreceptor cell bodies. SLURP-1 immunoreactivity co-localized with rhodopsin and S-opsin in photoreceptor OS, while choline acetyltransferase (ChAT) and high affinity choline transporter (CHT-1) were also expressed in photoreceptor OS. Immunoelectron microscopy identified that the majority of SLURP-1 was localized to the plasma membranes of photoreceptor OS. These results provide evidence that SLURP-1 is synthesized in photoreceptor cell bodies and transported to photoreceptor OS, where SLURP-1 may also be secreted. Our findings suggest that photoreceptor OS communicate via neurotransmitters such as ACh and SLURP-1, while RPE cells might receive these signals through α7 nAChRs in their microvilli.

Targeted disruption of an orthologue of DOMAINS REARRANGED METHYLASE 2, OsDRM2, impairs the growth of rice plants by abnormal DNA methylation.

Recent methylome analyses of the entire Arabidopsis thaliana genome using various mutants have provided detailed information about the DNA methylation pattern and its function. However, information about DNA methylation in other plants is limited, partly because of the lack of mutants. To study DNA methylation in rice (Oryza sativa) we applied homologous recombination-mediated gene targeting to generate targeted disruptants of OsDRM2, a rice orthologue of DOMAINS REARRANGED METHYLASE 1 and 2 (DRM1/2), which encode DNA methyltransferases responsible for de novo and non-CG methylation in Arabidopsis. Whereas Arabidopsis drm1 drm2 double mutants showed no morphological alterations, targeted disruptants of rice OsDRM2 displayed pleiotropic developmental phenotypes in both vegetative and reproductive stages, including growth defects, semi-dwarfed stature, reductions in tiller number, delayed heading or no heading, abnormal panicle and spikelet morphology, and complete sterility. In these osdrm2 disruptants, a 13.9% decrease in 5-methylcytosine was observed by HPLC analysis. The CG and non-CG methylation levels were reduced in RIRE7/CRR1 retrotransposons, and in 5S rDNA repeats. Associated transcriptional activation was detected in RIRE7/CRR1. Furthermore, de novo methylation by an RNA-directed DNA methylation (RdDM) process involving transgene-derived exogenous small interfering RNA (siRNA) was deficient in osdrm2-disrupted cells. Impaired growth and abnormal DNA methylation of osdrm2 disruptants were restored by the complementation of wild-type OsDRM2 cDNA. Our results suggest that OsDRM2 is responsible for de novo, CG and non-CG methylation in rice genomic sequences, and that DNA methylation regulated by OsDRM2 is essential for proper rice development in both vegetative and reproductive stages.

Endogenous arginine vasopressin-positive retinal cells in arginine vasopressin-eGFP transgenic rats identified by immunohistochemistry and reverse transcriptase-polymerase chain reaction.

PURPOSE: Recently, arginine vasopressin (AVP) has been revealed to have diverse functional roles in nervous tissues beyond that of a vasoconstrictor. Several previous studies have indicated the existence of AVP in the retina, but the source of AVP has not been determined. The objective of the present study was to address the question of whether retinal cells have the ability to synthesize endogenous AVP to act in a paracrine or autocrine manner. METHODS: We used AVP-eGFP transgenic rats to find endogenous AVP-positive cells in the retina by immunohistochemistry with an AVP antibody and a GFP antibody. We also examined AVP mRNA and AVP receptor genes by reverse transcriptase (RT)-PCR of dissociated GFP-positive cells and whole retinas. RESULTS: Endogenous AVP-positive cells were found in the ganglion cell layer and inner nuclear layer of the retina of AVP-eGFP transgenic rats by immunohistochemistry. As indicated by the results of RT-PCR of dissociated GFP-positive cells, these cells have the ability to synthesize endogenous AVP, as well as transgenic AVP-eGFP. In addition, the V1a and V1b AVP receptors were found in the wild-type rat retina by whole retina RT-PCR, but the V2 receptor was not detectable. In dissociated AVP-eGFP-positive cells, no AVP receptor was detected by RT-PCR. Moreover, AVP secretion was not detected by stimulation with a high potassium (50 mM) solution. CONCLUSIONS: In the rat retina, we found retinal cells that have the ability to synthesize endogenous AVP, and that the retina possesses V1a and V1b AVP receptors. Taken together, these results suggest that the retina has an intrinsic AVP-synthesizing and -receiving mechanism that can operate in a paracrine manner via V1a and V1b receptors.

Regular mosaic of synaptic contacts among three retinal neurons.

Retinal bipolar, amacrine, and ganglion cells contact each other within precisely defined synaptic laminae, but the spatial distribution of contacts between the cells is generally treated as random. Here we show that not to be the case. Excitatory inputs to inner retinal neurons were visualized by introduction of a plasmid coding for the postsynaptic protein PSD95-GFP. Our initial finding was that synapses on the dendrites of retinal ganglion cells are regularly spaced, at 2-3-µm intervals, along the dendrites. Thus, the presence of a PSD95 punctum creates a nearby zone from which other inputs appear to be excluded. Despite their great variation in size and different morphologies, the spacing is similar for the arbors of different retinal ganglion cell types. Regular spacing was also observed for the starburst amacrine cells. This regularity is mirrored in the spacing of axonal varicosities of the stratified bipolar cells, which have a regular, nonrandom interval consistent with that of the PSD95 puncta on ganglion cells. Thus, for each level of the inner plexiform layer all three cell types participate in a single 2D mosaic of synaptic contacts. These findings raise a new set of questions: How does the self-avoidance of synaptic sites along an individual dendrite arise and how is it physically maintained? Why is a regular spacing of inputs important for the computational function of the cells? Finally, which of the three players, if any, is developmentally responsible for the initial establishment of the pattern?

Asymmetric inhibitory connections enhance directional selectivity in a three-layer simulation model of retinal networks.

In this paper, we found that spatial and temporal asymmetricity of excitatory connections are able to generate directional selectivity which can be enhanced by asymmetrical inhibitory connections by reconstructing a hexagonally-arranged three-layered simulation model of retina by NEURON simulator. Asymmetric excitatory inputs to ganglion cells with randomly arborizing dendrites were able to generate weaker directional selectivity to moving stimuli whose speed was less than 10 µm/msec. By just adding asymmetric inhibitory connections via inhibitory amacrine cells, directional selectivity became stronger to respond to moving stimuli at ten times faster speed (< 100 µm/msec). In conclusion, an excitatory mechanism appeared to generate directional selectivity while asymmetric inhibitory connections enhance directional selectivity in retina.

Organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro.

BACKGROUND: Organotypic tissue culture of adult rodent retina with an acute gene transfer that enables the efficient introduction of variable transgenes would greatly facilitate studies into retinas of adult rodents as animal models. However, it has been a difficult challenge to culture adult rodent retina. The purpose of this present study was to develop organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We established an interphase organotypic tissue culture for adult rat retinas (>P35 of age) which was optimized from that used for adult rabbit retinas. We implemented three optimizations: a greater volume of Ames' medium (>26 mL) per retina, a higher speed (constant 55 rpm) of agitation by rotary shaker, and a greater concentration (10%) of horse serum in the medium. We also successfully applied this method to adult mouse retina (>P35 of age). The organotypic tissue culture allowed us to keep adult rodent retina morphologically and structurally intact for at least 4 days. However, mouse retinas showed less viability after 4-day culture. Electrophysiologically, ganglion cells in cultured rat retina were able to generate action potentials, but exhibited less reliable light responses. After transfection of EGFP plasmids by particle-mediated acute gene transfer, we observed EGFP-expressing retinal ganglion cells as early as 1 day of culture. We also introduced polarized-targeting fusion proteins such as PSD95-GFP and melanopsin-EYFP (hOPN4-EYFP) into rat retinal ganglion cells. These fusion proteins were successfully transferred into appropriate locations on individual retinal neurons. CONCLUSIONS/SIGNIFICANCE: This organotypic culture method is largely applicable to rat retinas, but it can be also applied to mouse retinas with a caveat regarding cell viability. This method is quite flexible for use in acute gene transfection in adult rodent retina, replacing molecular biological bioassays that used to be conducted in isolated cultured cells.