Immunocytochemical Localization of Choline Acetyltransferase in the Microbat Visual Cortex.

The purpose of the present study was to investigate the organization of choline acetyltransferase (ChAT)-immunoreactive (IR) fibers in the visual cortex of the microbat, using standard immunocytochemistry and confocal microscopy. ChAT-IR fibers were distributed throughout all layers of the visual cortex, with the highest density in layer III and the lowest density in layer I. However, no ChAT-IR cells were found in the microbat visual cortex. ChAT-IR fibers were classified into two types: small and large varicose fibers. Previously identified sources of cholinergic fibers in the mammalian visual cortex, the nucleus of the diagonal band, the substantia innominata, and the nucleus basalis magnocellularis, all contained strongly labeled ChAT-IR cells in the microbat. The average diameter of ChAT-IR cells in the nucleus of the diagonal band, the substantia innominata, and the nucleus basalis magnocellularis was 16.12 µm, 13.37 µm, and 13.90 µm, respectively. Our double-labeling study with ChAT and gamma-aminobutyric acid (GABA), and triple labeling with ChAT, GABA, and post synaptic density 95 (PSD-95), suggest that some ChAT-IR fibers make contact with GABAergic cells in the microbat visual cortex. Our results should provide a better understanding of the nocturnal bat visual system.

The organization of melanopsin-immunoreactive cells in microbat retina.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) respond to light and play roles in non-image forming vision, such as circadian rhythms, pupil responses, and sleep regulation, or image forming vision, such as processing visual information and directing eye movements in response to visual clues. The purpose of the present study was to identify the distribution, types, and proportion of melanopsin-immunoreactive (IR) cells in the retina of a nocturnal animal, i.e., the microbat (Rhinolophus ferrumequinum). Three types of melanopsin-IR cells were observed in the present study. The M1 type had dendritic arbors that extended into the OFF sublayer of the inner plexiform layer (IPL). M1 soma locations were identified either in the ganglion cell layer (GCL, M1c; 21.00%) or in the inner nuclear layer (INL, M1d; 5.15%). The M2 type had monostratified dendrites in the ON sublayer of the IPL and their cell bodies lay in the GCL (M2; 5.79%). The M3 type was bistratified cells with dendrites in both the ON and OFF sublayers of the IPL. M3 soma locations were either in the GCL (M3c; 26.66%) or INL (M3d; 4.69%). Additionally, some M3c cells had curved dendrites leading up towards the OFF sublayer of the IPL and down to the ON sublayer of the IPL (M3c-crv; 7.67%). Melanopsin-IR cells displayed a medium soma size and medium dendritic field diameters. There were 2-5 primary dendrites and sparsely branched dendrites with varicosities. The total number of the neurons in the GCL was 12,254.17 ± 660.39 and that of the optic nerve axons was 5,179.04 ± 208.00 in the R. ferrumequinum retina. The total number of melanopsin-IR cells was 819.74 ± 52.03. The ipRGCs constituted approximately 15.83% of the total RGC population. This study demonstrated that the nocturnal microbat, R. ferrumequinum, has a much higher density of melanopsin-IR cells than documented in diurnal animals.

Identification of synaptic pattern of NMDA receptor subunits upon direction-selective retinal ganglion cells in developing and adult mouse retina.

Direction selectivity of the retina is a unique mechanism and critical function of eyes for surviving. Direction-selective retinal ganglion cells (DS RGCs) strongly respond to preferred directional stimuli, but rarely respond to the opposite or null directional stimuli. These DS RGCs are sensitive to glutamate, which is secreted from bipolar cells. Using immunocytochemistry, we studied with the distributions of N-methyl-d-aspartate (NMDA) receptor subunits on the dendrites of DS RGCs in the developing and adult mouse retina. DS RGCs were injected with Lucifer yellow for identification of dendritic morphology. The triple-labeled images of dendrites, kinesin II, and NMDA receptor subunits were visualized using confocal microscopy and were reconstructed from high-resolution confocal images. Although our results revealed that the synaptic pattern of NMDA receptor subunits on dendrites of DS RGCs was not asymmetric in developing and adult mouse retina, they showed the anatomical connectivity of NMDA glutamatergic synapses onto DS RGCs and the developmental formation of the direction selectivity in the mouse retina. Through the comprehensive interpretation of the direction-selective neural circuit, this study, therefore, implies that the direction selectivity may be generated by the asymmetry of the excitatory glutamatergic inputs and the inhibitory inputs onto DS RGCs.

Identification of Synaptic Patterns of NMDA Receptor Subtypes Upon Direction-Selective Rabbit Retinal Ganglion Cells.

PURPOSE: The objective of this study was to identify anisotropies that contribute to the directional preference of direction-selective retinal ganglion cells (DS RGCs) in the rabbit retina. We investigated the distributions of N-methyl-d-aspartate receptor 1 (NMDAR1), NMDAR2A and NMDAR2B receptor subunits in the dendritic arbors of rabbit DS RGCs. METHODS: The distributions of the NMDAR subunits on the DS RGCs were determined using immunocytochemistry. DS RGCs were injected with Lucifer yellow, and the cells were identified by their characteristic morphology. The triple-labeled images of dendrites, kinesin II and NMDARs were visualized using confocal microscopy and were reconstructed from high-resolution confocal images. RESULTS: We found no evidence of asymmetry in any of the NMDAR subunits examined on the dendritic arbors of both the ON and OFF layers of DS RGCs. CONCLUSIONS: Our results indicate that direction selectivity appears to lie in the neuronal circuitry afferent to the DS RGCs.

Immunocytochemical localization of the calcium-binding proteins calbindin D28K, calretinin, and parvalbumin in bat visual cortex.

It is a common misconception that bats are blind, and various studies have suggested that bats have visual abilities. The purpose of this study was to investigate the cytoarchitecture of calbindin D28K (CB)-, calretinin (CR)-, and parvalbumin (PV)-immunoreactive (IR) neurons in the bat visual cortex using immunocytochemistry. The highest density of CB- and PV-IR neurons was located in layer IV of the visual cortex. The majority of CB- and PV-IR neurons were characterized by a stellate or round/oval shape. CR-IR neurons were predominantly located in layers II/III, and the cells were principally round/oval in shape. Two-color immunofluorescence revealed that 65.96%, 24.24%, and 77.00% of the CB-, CR-, and PV-IR neurons, respectively, contained gamma-aminobutyric acid (GABA). We observed calcium-binding protein (CBP)-IR neurons in specific layers of the bat visual cortex and in specific cell types. Many of the CBP-IR neurons were GABAergic interneurons. These data provide useful clues to aid in understanding the functional aspects of the bat visual system.

Localization of Nitric Oxide Synthase-containing Neurons in the Bat Visual Cortex and Co-localization with Calcium-binding Proteins.

Microchiroptera (microbats) is a suborder of bats thought to have degenerated vision. However, many recent studies have shown that they have visual ability. In this study, we labeled neuronal nitric oxide synthase (nNOS)-the synthesizing enzyme of the gaseous non-synaptic neurotransmitter nitric oxide-and co-localized it with calbindin D28K (CB), calretinin (CR), and parvalbumin (PV) in the visual cortex of the greater horseshoe bat (Rhinolophus ferrumequinum, a species of microbats). nNOS-immunoreactive (IR) neurons were found in all layers of the visual cortex. Intensely labeled neurons were most common in layer IV, and weakly labeled neurons were most common in layer VI. Majority of the nNOS-IR neurons were round- or oval-type neurons; no pyramidal-type neurons were found. None of these neurons co-localized with CB, CR, or PV. However, the synthesis of nitric oxide in the bat visual cortex by nNOS does not depend on CB, CR, or PV.