Density and types of calretinin-containing retinal ganglion cells in rabbit.

Single-cell injection with lipophilic dyes following immunocytochemistry is extremely valuable for revealing the morphology of a cell expressing a protein of interest, and is a more reliable technique for cell type classification than standard morphological techniques. This study focuses on calretinin (CR), which is used as a selective marker for distinct subpopulations of neurons in the rabbit retina. The present study used single-cell injection after immunocytochemistry to describe the density and types of CR-containing retinal ganglion cells (RGCs) in rabbit. The density of CR-immunoreactive cells in the rabbit RGC cell layer was 426cells/mm(2). CR-containing RGCs were identified by immunocytochemistry and were then iontophoretically injected with a lipophilic dye, DiI. Subsequently, confocal microscope was used to characterize the morphology of CR-immunoreactive RGCs based on their dendritic field size, branching pattern, and stratification of the inner plexiform layer. Our results show that 10 morphologically different types of rabbit RGCs expressed CR. CR-containing RGCs were heterogeneous in their morphology. This approach to integrate the selective expression of a particular protein with spatial patterns of dendritic arborization will lead to a better understanding of RGCs.

In vitro endothelial potential of human UC blood-derived mesenchymal stem cells.

BACKGROUND: Human mesenchymal stem cells (MSC) possess powerful ex vivo expansion and versatile differentiation potential, placing themselves at the forefront of the field of stem cell-based therapy and transplantation. Of high clinical relevance is the endothelial differentiation potential of MSC, which can be used to treat various forms of ischemic vascular disease. METHODS: We investigated whether human umbilical cord blood (UCB)-derived MSC are able to differentiate in vitro along an endothelial lineage, by using flow cytometry, RT-PCR and immunofluorescence analyzes, as well as an Ab array method. RESULTS: When the cells were incubated for up to 3 weeks in the presence of VEGF, EGF and hydrocortisone, they began to express a variety of endothelial lineage surface markers, such as Flk-1, Flt-1, VE-Cadherin, vWF, VCAM-1, Tie-1 and Tie-2, and to secrete a specific set of cytokines. Differentiated cells were also found to be able to uptake low-density lipoprotein and form a tubular network structure. DISCUSSION: These observations have led us to conclude that UCB-derived MSC retain endothelial potential that is suitable for basic and clinical studies aimed at the development of vasculature-directed regenerative medicine.

Morphology of calretinin and tyrosine hydroxylase-immunoreactive neurons in the pig retina.

The morphology of calretinin- and tyrosine hydroxylase-immunoreactive (IR) neurons in adult pig retina was studied. These neurons were identified using antibody immunocytochemistry. Calretinin immunoreactivity was found in numerous cell bodies in the ganglion cell layer. Large ganglion cells, however, were not labeled. In the inner nuclear layer, the regular distribution of calretinin-IR neurons, the inner marginal location of their cell bodies in the inner nuclear layer, and the distinctive bilaminar morphologies of their dendritic arbors in the inner plexiform layer suggested that these calretinin-IR cells were AII amacrine cells. Calretinin immunoreactivity was observed in both A-and B-type horizontal cells. Neurons in the photoreceptor cell layer were not labeled by this antibody. The great majority of tyrosine hydroxylase-IR neurons were located at the innermost border of the inner nuclear layer (conventional amacrines). The processes were monostratified and ran laterally within layer 1 of the inner plexiform layer. Some of the tyrosine hydroxylase-IR neurons were located in the ganglion cell layer (displaced amacrines). The processes of displaced tyrosine hydroxylase-IR amacrine cells were also located within layer 1 of the inner plexiform layer. Some processes of a few neurons were located in the outer plexiform layer. A very low density of neurons had additional bands of tyrosine hydroxylase-IR processes in the middle and deep layers of the inner plexiform layer. The processes of tyrosine hydroxylase-IR neurons extended radially over a wide area and formed large, moderately branched dendritic fields. These processes occasionally had varicosities and formed "dendritic rings". These results indicate that calretinin- and tyrosine hydroxylase-IR neurons represent specific neuronal cell types in the pig retina.

Calcium-binding proteins calbindin D28K, calretinin, and parvalbumin immunoreactivity in the rabbit visual cortex.

The distribution and morphology of neurons containing three calcium-binding proteins, calbindin D28K, calretinin, and parvalbumin in the adult rabbit visual cortex were studied. The calcium-binding proteins were identified using antibody immunocytochemistry. Calbindin D28K-immunoreactive (IR) neurons were located throughout the cortical layers with the highest density in layer V. However, calbindin D28K-IR neurons were rarely encountered in layer I. Calretinin-IR neurons were mainly located in layers II and III. Considerably lower densities of calretinin-IR neurons were observed in the other layers. Parvalbumin-IR neurons were predominantly located in layers III, IV, V, and VI. In layers I and II, parvalbumin-IR neurons were only rarely seen. The majority of the calbindin D28K-IR neurons were stellate, round or oval cells with multipolar dendrites. The majority of calretinin-IR neurons were vertical fusiform cells with long processes traveling perpendicularly to the pial surface. The morphology of the majority of parvalbumin-IR neurons was similar to that of calbindin D28K: stellate, round or oval with multipolar dendrites. These results indicate that these three different calcium-binding proteins are contained in specific layers and cells in the rabbit visual cortex.

Coupling of the inositol 1,4,5-trisphosphate receptor and chromogranins A and B in secretory granules.

The secretory granules of neuroendocrine cells which contain large amounts of Ca(2+) and chromogranins have been demonstrated to release Ca(2+) in response to inositol 1,4,5-trisphosphate (IP(3)). Moreover, chromogranin A (CGA) has been shown to interact with several secretory granule membrane proteins, including the IP(3) receptor (IP(3)R). To determine whether the IP(3)Rs interact directly with chromogranins A and B (CGB), two major proteins of the secretory granules, we have used purified IP(3)R from bovine cerebellum in the interaction study with CGA and CGB, and have shown that chromogranins A and B directly interact with the IP(3)R at the intravesicular pH 5.5. Immunogold cytochemical study using the IP(3)R and CGA antibodies indicated that IP(3)R-labeled gold particles were localized in the periphery of the secretory granules, indicating the presence of the IP(3)Rs on the secretory granule membrane. To determine whether the IP(3)R and chromogranins A and B are physically linked in the cells, bovine type 1 IP(3)R (IP(3)R-1) and CGA or CGB are co-transfected into COS-7 cells and co-immunoprecipitation was carried out. Immunoprecipitation of the cell extracts demonstrated the presence of CGA-IP(3)R-1 and CGB-IP(3)R-1 complexes, respectively, indicating the complex formation between the IP(3)R and chromogranins A and B in native state.

Inositol 1,4,5-trisphosphate receptor/Ca2+ channel modulatory role of chromogranin A, a Ca2+ storage protein of secretory granules.

The secretory granules of neuroendocrine cells, which contain large amounts of Ca(2+) and chromogranins, have been demonstrated to release Ca(2+) in response to inositol 1,4,5-trisphosphate (IP(3)), indicating the IP(3)-sensitive intracellular Ca(2+) store role of secretory granules. In our previous study, chromogranin A (CGA) was shown to interact with several secretory granule membrane proteins, including the IP(3) receptor (IP(3)R), at the intravesicular pH 5.5 (Yoo, S. H. (1994) J. Biol. Chem. 269, 12001-12006). To examine the functional aspect of this coupling, we measured the IP(3)-mediated Ca(2+) release property of the IP(3)R reconstituted into liposomes in the presence and absence of CGA. Presence of CGA in the IP(3)R-reconstituted liposome significantly enhanced the IP(3)-mediated Ca(2+) release from the liposomes. Moreover, the number of IP(3) bound to the reconstituted IP(3)R increased. The fluorescence energy transfer and IP(3)R Trp fluorescence quenching studies indicated that the structure of reconstituted IP(3)R becomes more ordered and exposed in the presence of CGA, suggesting that the coupled CGA in the liposome caused structural changes of the IP(3)R, changing it to a structure that is better suited to IP(3) binding and subsequent Ca(2+) release. These results appear to underscore the physiological significance of IP(3)R-CGA coupling in the secretory granules.

Localization of calcium-binding protein parvalbumin-immunoreactive neurons in mouse and hamster visual cortex.

Calcium-binding proteins are thought to play important roles in regulating intracellular calcium in the central nervous system. In the present study, we investigated the distribution and morphology of neurons containing parvalbumin in the visual cortex of mouse and hamster. The calcium-binding proteins were localized using immunocytochemistry. Parvalbumin-immunoreactive neurons were located in all layers except layer I. The highest density of parvalbumin immunoreactivity was found in layer V of both mouse and hamster. The labeled neurons varied in morphology. The majority of the parvalbumin-immunoreactive neurons both in mouse and hamster visual cortex was stellate and round, or oval with multipolar dendrites. These results indicate that the calcium-binding protein parvalbumin is contained in specific layers and in selective cell types of the mouse and hamster visual cortex. The distribution of parvalbumin in the mouse visual cortex is very similar to that of hamster.

Metabotropic glutamate receptor mGluR2/3 immunoreactivity in the mouse superior colliculus: co-localization with calbindin D28K.

We have studied the distribution of mGluR2/3 in the mouse superior colliculus (SC) with antibody immunocytochemistry and the effect of enucleation on this distribution. We also compared this labeling to that for calbindin D28K. Anti-mGluR2/3-immunoreactive (IR) cells formed distinctive laminar patterns within the lower optic and upper intermediate gray layers. By contrast, anti-calbindin D28K-IR cells formed obvious laminar patterns in three layers: one within the zonal and upper superficial gray layers, a second within the optic and intermediate gray layers, and the third within the deep gray layer. The distribution of mGluR2/3-IR cells thus matches the second layer of calbindin D28K cells. Two-color immunofluorescence revealed that more than half (52.5%) of mGluR2/3-IR cells were also labeled with antibody to calbindin D28K. The majority of mGluR2/3-IR cells were small to medium-sized round/oval or stellate cells. Immunoreactivity for mGluR2/3 was clearly reduced in the contralateral SC following unilateral enucleation. The present results show that mGluR2/3 has a unique cellular sublaminar organization in SC that includes some calbindin D28K-IR cells. The effects of enucleation suggest that the retinal projection may control the expression of mGluR2/3 in some cells in the mouse SC.

Immunocytochemical localization of calretinin containing neurons in retina from rabbit, cat, and dog.

Calcium homeostasis is critical for many neuronal functions, yet the distribution of calcium-binding protein is not always conserved among species, even between closely related species. We decided therefore to study the distribution of one of these calcium-binding proteins calretinin, in retina from rabbit, cat, and dog. Calretinin was localized using antibody immunocytochemistry. Calretinin immunoreactivity was found in numerous cell bodies in the ganglion cell layer in all three animals. These cells had small to medium-sized somas. Large ganglion cells, however, were not labeled using antiserum against calretinin. In the inner nuclear layer, calretinin immunoreactivity was found in many neurons in all three species. The regular distribution of neurons, the inner marginal location of their cell bodies in the inner nuclear layer, and the distinctive bilaminar morphologies of their dendritic arbors in the inner plexiform layer suggested that these calretinin-positive cells were AII amacrine cells. Calretinin immunoreactivity was observed in both A- and B-type horizontal cells in cat and dog retina. However, horizontal cells in the rabbit retina were not labeled by this antibody. Neurons in the photoreceptor cell layer were not labeled by this antibody. The present study suggests that calretinin immunoreactivity is present in several populations in the retina. In particular, calretinin labels AII amacrine cells and a subpopulation of ganglion cells in all three animals. Horizontal cells, however, were not labeled in rabbit.

The major cell populations of the mouse retina.

We report a quantitative analysis of the major populations of cells present in the retina of the C57 mouse. Rod and cone photoreceptors were counted using differential interference contrast microscopy in retinal whole mounts. Horizontal, bipolar, amacrine, and Müller cells were identified in serial section electron micrographs assembled into serial montages. Ganglion cells and displaced amacrine cells were counted by subtracting the number of axons in the optic nerve, learned from electron microscopy, from the total neurons of the ganglion cell layer. The results provide a base of reference for future work on genetically altered animals and put into perspective certain recent studies. Comparable data are now available for the retinas of the rabbit and the monkey. With the exception of the monkey fovea, the inner nuclear layers of the three species contain populations of cells that are, overall, quite similar. This contradicts the previous belief that the retinas of lower mammals are "amacrine-dominated", and therefore more complex, than those of higher mammals.

Calretinin and calbindin D28K immunoreactivity in the superficial layers of the rabbit superior colliculus.

Calretinin and calbindin D28K were localized in the superficial layers of rabbit superior colliculus (SC). Calretinin and calbindin D28K-immunoreactive (-IR) neurons were concentrated in the upper superficial gray layer. Calretinin-IR fibers were found in the optic layer. The majority of calretinin-IR cells were small- to medium-sized vertical fusiform neurons and neurons with round or stellate-shaped somas with small varicose dendrites. The morphology of calbindin D28 K-IR neurons was different from that of calretinin neurons. Anti-calbindin D28K-IR neurons usually had fusiform cell bodies and a thick primary dendrite with small branches forming a dendritic bouquet. Two-color immunofluorescence revealed that no cells expressed both proteins. Following unilateral enucleation a marked reduction of calretinin-IR fibers in the contralateral side to the enucleation was found. Enucleation appeared to have no effect on the cell bodies labeled with either protein. The results suggest the anti-calretinin immunoreactivity in the superficial layer of rabbit SC contrasts starkly with that of other animals.

Glutamate containing neurons in the cat superior colliculus revealed by immunocytochemistry.

Glutamate is the probable neurotransmitter of both retinal and cortical afferents to the cat superior colliculus (SC). The present study shows that glutamate is also contained in many postsynaptic neurons in SC. The distribution, morphology, and ultrastructure of neurons in SC were examined using glutamate antibody immunocytochemistry. Labeled cells were widely distributed throughout, but a specific laminar pattern was evident. Relatively few cells were found in the zonal and upper superficial gray layers (SGL). A dense band of intensely labeled neurons was found within the deep superficial gray and upper optic layers. Many cells were also labeled in the deeper layers. Labeled cells had varied sizes and morphologies. Soma diameters ranged from 9-67 microns, with a mean of 22 microns. Cells with stellate, vertical fusiform, and multipolar morphologies were labeled. Cells in the deep subdivision all had morphologies and sizes typical of projection neurons. To determine if labeled cells in the dense band were also projection neurons, WGA-HRP was injected into the lateral posterior nucleus and these sections were double-labeled with the glutamate antibody. Over one-half of cells in the dense band that were labeled by HRP were also obviously labeled by antibody. At the electron-microscope level, both medium- and large-sized neurons were also labeled by glutamate antibodies. These cells had different but characteristic morphologies.

Glutamate-like immunoreactivity in the cat superior colliculus and visual cortex: further evidence that glutamate is the neurotransmitter of the corticocollicular pathway.

Biochemical studies provide evidence that the pathway from visual cortex to the superior colliculus (SC) utilizes glutamate as a neurotransmitter. In the present study, we have used immunocytochemistry, visual cortex lesions, and retrograde tracing to show directly by anatomical methods that glutamate or a closely related analog is contained in corticocollicular neurons and terminals. A monoclonal antibody directed against gamma-L-glutamyl-L-glutamate (gamma glu glu) was used to localize glutamate-like immunoreactivity in both the superior colliculus (SC) and visual cortex (VC). Unilateral lesions of areas 17-18 were made in four cats to determine if gamma glu glu labeling was reduced in SC by this lesion. WGA-HRP was injected into the SC of 10 additional cats in order to determine if corticocollicular neurons were also labeled by the gamma glu glu antibody. A distinctive dense band of gamma glu glu immunoreactivity was found within the deep superficial gray and upper optic layers of SC where many corticotectal axons are known to terminate. Both fibers and cells were labeled within the band. Immunoreactivity was also found in cells and fibers throughout the deep layers of SC. Measures of total immunoreactivity (i.e. optical density) in the dense band were made in sections from the SC both ipsilateral to and contralateral to the lesions of areas 17-18. A consistent reduction in optical density was found in both the neuropil and in cells within the dense band of the SC ipsilateral to the lesion. A large percentage of all corticocollicular neurons that were retrogradely labeled by WGA-HRP also contained gamma glu glu. These results provide further evidence that the corticocollicular pathway in mammals is glutamatergic. The results also suggest that visual cortex ablation alters synthesis or storage of glutamate within postsynaptic SC neurons, presumably as a result of partial deafferentation.

Immunocytochemical localization of calcium-binding protein calretinin containing neurons in cat visual cortex.

The distribution and morphology of neurons containing calretinin in area 17 of the cat visual cortex were studied. The calcium-binding protein calretinin was localized by antibody immunocytochemistry. Most of the calretinin-labeled neurons were located in layers I, II, and III. There were few calretinin-labeled cells in the other layers. The labeled neurons varied in morphology. The majority of the labeled neurons had small round or oval somas with long processes traveling perpendicular to the pial surface. Many small multipolar neurons were also labeled by this antibody. These results indicate that the calcium-binding protein calretinin is contained both in specific layers and selective cell types in the cat primary visual cortex.

A population of wide-field bipolar cells in the rabbit's retina.

We have stained an unusual population of retinal bipolar cells. When the low molecular weight tracer biocytin was injected into the vitreous body of rabbits, it subsequently accumulated in the somata and processes of a population of wide-field bipolar cells. The cells have 2-4 primary dendrites. Their dendritic arbors span a field 50 to 200 microns in diameter. The axonal arbors are sparse and often highly asymmetric. The longest dimension of the axonal arbor ranges from 100 to 300 microns. The cells are moderately evenly spaced. They make up less than 1% of the total population of bipolar cells in the rabbit retina. With the whole population stained, regularities in the spatial arrangement of nearby cells can be recognized. Their dendrites often run to a common point, where they have the appearance of making contact with each other. A similar arrangement is seen for the cells' axonal arbors, so that the whole population is spatially linked in both the outer retina and the inner. The exact nature of the points of conjunction cannot be learned from light microscopy. One possibility is that the processes run together because they contact a common target. If so, the target structures (one in the outer retina and one in the inner) must be sparse. An alternative is that the points of conjunction represent synapses or gap junctions among wide-field bipolars of this type.

Choline acetyltransferase-immunoreactive patches overlap specific efferent cell groups in the cat superior colliculus.

Fibers containing acetylcholine (ACh) form distinct patches in the dorsal intermediate gray layer (IGL) of the cat superior colliculus (SC). Although these patches are known to overlap several afferent projections to SC, it is not known whether they are associated with specific postsynaptic cell groups. We have examined the relationship of these ACh fiber patches to specific efferent cell groups by combining retrograde transport of horseradish peroxidase (HRP) with choline acetyltransferase (ChAT) immunocytochemistry. Successful HRP injections were made into the predorsal bundle (PB), the tecto-pontine-bulbar pathway (TPB) and the cuneiform region (CFR), the inferior olive (IO), the dorsolateral pontine gray nucleus (PGD), and the pedunculopontine tegmental nucleus (PPTN). The distribution of HRP-labeled neurons which project to these targets was mapped by a computer-based microscope plotter. Distinct clusters of HRP-labeled neurons in the IGL were seen after three injections into the mesencephalic reticular formation that involved the caudal TPB and cuneiform region (CFR), and after one injection into the medial accessory nucleus of IO. As many as seven clusters of labeled neurons were found in some sections through the caudal one-half of SC after the TPB/CFR injections. Each cluster consisted of 3-20 cells, all of which were small to medium in size. In sections also tested for ChAT, the cell clusters in the TPB/CFR cases were found to overlap precisely the ACh patches in the IGL. In addition, SC neurons projecting to the IO formed clusters above the ChAT patches and in the intermediate white layer (IWL) of SC. None of the other HRP injections produced any obvious cell clusters in the deep layers of SC. These results are the first to show that specific cell groups, distinguished by size and projection site, form clusters that match the patch-like innervation of cholinergic afferents to SC. This modular organization may correspond to saccade-related cells that have also been reported to be organized into clusters in the cat SC.

Selective accumulation of diamidino yellow and chromomycin A3 by retinal glial cells.

We applied the fluorescent DNA stains diamidino yellow (DY) and chromomycin A3 to rat and rabbit retinas in vivo and in vitro. They accumulated in the nuclei of a subpopulation of cells of the inner nuclear layer. The number and distribution of the fluorochrome-accumulating cells were similar to those of the Müller glia, and double-labeling experiments showed that the cells accumulating DY or chromomycin A3 contained oriented filaments of vimentin. The fluorochromes also accumulated in the sparse astrocytes and oligodendrocytes located among the myelinated fibers of the rabbit central retina. Specific accumulation in retinal glia occurred only when the fluorochromes were applied to living retinas. If the plasma membranes were disrupted by fixation or exposure to detergent, most retinal cells were stained. This indicates that the locus of specificity is the entry of the molecules into the cells. When applied to living retinas, other DNA stains selectively accumulate in subclasses of retinal neurons. Why DNA-binding molecules should selectively cross the membranes of either retinal neurons or retinal glia remains an unsolved problem.

Novel zinc finger motif in the basal transcriptional machinery: three-dimensional NMR studies of the nucleic acid binding domain of transcriptional elongation factor TFIIS.

Transcriptional elongation provides a key control point in the regulation of eukaryotic gene expression. Here we describe homonuclear and 15N-heteronuclear 3D NMR studies of the nucleic acid binding domain of human transcriptional elongation factor TFIIS. This domain contains a Cys4 Zn(2+)-binding site with no homology to previously characterized Cys4, Cys6, or Cys2-His2 Zn fingers. Complete 1H and 15N NMR resonance assignment of a 50-residue TFIIS peptide-Zn2+ complex is obtained. Its solution structure, as determined by distance geometry/simulated annealing (DG/SA) calculations, exhibits a novel three-stranded antiparallel beta-sheet (designated the Zn ribbon). Analogous sequence motifs occur in a wide class of proteins involved in RNA or DNA transactions, including human basal transcriptional initiation factor TFIIE. A three-dimensional model of the TFIIE Cys4 domain is obtained by DG-based homology modeling. The role of the TFIIS Zn ribbon in the control of eukaryotic transcriptional elongation is discussed.

Organization and synaptic connections of cholinergic fibers in the cat superior colliculus.

The cat superior colliculus (SC) receives a dense cholinergic input from three brainstem nuclei, the pedunculopontine tegmental nucleus, the lateral dorsal tegmental nucleus, and the parabigeminal nucleus (PBG). The tegmental inputs project densely to the intermediate gray layer (IGL) and sparsely to the superficial layers. The PBG input probably projects only to the superficial layers. In the present study, the morphology of choline acetyltransferase (ChAT)-immunoreactive axons and synaptic endings in the superficial and deep layers of the SC was examined by light and electron microscopy to determine whether these cholinergic afferents form different types of synapses in the superficial and deep layers. Two types of fibers were found within the zonal (ZL) and upper superficial gray layers (SGL): small diameter fibers with few varicosities and larger diameter fibers with numerous varicosities. Quantitative analysis demonstrated a bimodal distribution of axon diameters, with one peak at approximately 0.3-0.5 micron and the other at 0.9-1.0 micron. On the other hand, ChAT-immunoreactive fibers in the IGL were almost all small and formed discrete patches within the IGL. Two types of ChAT-immunoreactive synaptic profiles were observed within the ZL and upper SGL using the electron microscope. The first type consisted of small terminals containing predominantly round synaptic vesicles and forming asymmetric synaptic contacts, mostly on dendrites. The second type was comprised of varicose profiles that also contained round synaptic vesicles. Their synaptic contacts were always symmetric in profile. ChAT-immunoreactive terminals in the IGL patches contained round or pleomorphic synaptic vesicles, and the postsynaptic densities varied from symmetric to asymmetric, including intermediate forms. However, no large varicose profiles were observed. This study suggests that cholinergic fibers include at least two different synaptic morphologies: small terminals with asymmetric thickenings and large varicose profiles with symmetric terminals. The large varicose profile in the superficial layers is absent in the IGL. This result suggests that the cholinergic inputs that innervate the superficial layers and the patches in the IGL of the cat SC differ in their synaptic organization and possibly also in their physiological actions.