Dendritic morphology and retinal distribution of tyrosine hydroxylase-like immunoreactive amacrine cells in Bufo marinus

Summary Tyrosine hydroxylase-like immunoreactive (TH-IR) amacrine cells (ACs) in the retina of metamorphosing and adult Bufo marinus were visualized, and their retinal distribution established, using immunohistochemistry on retinal wholemount and sectioned material. The somata of TH-IR ACs were located in the innermost part of the inner nuclear layer (INL). Their dendrites branched predominantly in the scieral sublamina of the inner plexiform layer (IPL), with sparse branching also in the vitreal sublamina. In the retinae of metamorphosing animals 592 ± 113 (mean ± S.D.) immunoreactive cells and in adult 5,670 ± 528 cells were found. Usually 1, 2 or 3 stem dendrites arose from the somata of TH-IR cells which branched 2 or 3 times. In the adult retinae the dendritic field sizes of immunoreactive cells were in the range of 0.059 ± 0.012 mm², which resulted in a considerable dendritic overlap across the retina. TH-IR cells were unevenly distributed over the retina, with 72 cells/mm² in the central temporal retina, 45–50 cells/mm² along the naso-temporal axis of the retina and 25 cells/mm² in the dorsal and ventral peripheral retina. The average density was 36 ± 6 cells/mm². A considerable number of TH-IR cells (range 52–133, n=4) were displaced into the ganglion cell layer (GCL) of the retina. The mean soma sizes of immunoreactive cells were significantly higher in the low density (95 ± 13 m²) than in the high cell density areas (86 ± 12 m²). There was also a slight but significant increase of the dendritic field sizes of these cells towards the low cell density areas of the retina. These observations show that the retinal distribution of TH-IR ACs parallels the non-uniform distribution of neurons of the INL demonstrated recently in Bufo marinus (Zhu et al. 1990). The class of TH-IR ACs appears to correspond to a subgroup of morphologically distinct dopaminergic ACs found in a number of other vertebrate species.On leave from Department of Anatomy, Zhanjiang Medical College, Guangdong, People's Republic of China     

The generation and changing retinal distribution of displaced amacrine cells in Bufo marinus from metamorphosis to adult.

Summary The generation and retinal distribution of displaced amacrine cells (DAs) were studied from metamorphosis to adult in the cane toad Bufo marinus. Displaced amacrine cells were identified by inducing chromatolytic changes in ganglion cells (GCs) following optic nerve section. Cells that did not chromatolyse in the ganglion cell layer (GCL) of the retina were regarded as DAs. The number of DAs increased considerably from an estimated 10000 at metamorphosis to 211000 in the adult toad, and was accompanied by a substantial decrease of average cell density. In contrast to the reported 6:1 cell density gradient of all cells of the GCL in adult toad (Nguyen and Straznicky 1989) only a shallow 1.6:1 density gradient of DAs from the visual streak to the dorsal and ventral retinal margins was detected. Consequently, the incidence of DAs increased from 15% of all cells of the GCL in the visual streak to 30% in the dorsal and ventral peripheral retina. These results indicate that the ratio of the newly generated DAs and GCs at the ciliary margin must be changing during development. More GCs are generated before and around metamorphosis then DAs, in contrast to the relative increase of the percentage of DAs generated after metamorphosis. The possible control of the numbers of DAs in the GCL is discussed.     

The changing distribution of neurons in the inner nuclear layer from metamorphosis to adult: a morphometric analysis of the anuran retina

Summary The generation and changing distribution of neurons of the inner nuclear layer (INL) in the retina of two anuran species, Bufo marinus and Xenopus laevis, were studied from metamorphosis to adult. Morphometric studies were undertaken at six developmental stages in Bufo and four in Xenopus. The number and thickness of neurons in the INL were established in 29 predetermined retinal locations from serial sections of the eyes cut vertically or horizontally. The total number of neurons in the INL increased from metamorphosis to adult from 826000 ± 185 to 18760000 ± 562 (mean ± SD) in Bufo and from 308000 ± 25 to 877000 ± 31 in Xenopus. Over the same period the surface area of the INL increased about 50-fold from 2 mm² to 96 mm² in Bufo and 5-fold from 2.5 mm² to 13 mm² in Xenopus. In Bufo the difference between the highest cell number (centraltemporal retina) and the lowest cell number in a sample area (dorsal and ventral peripheral retina) was 2.11 at metamorphosis. This ratio increased to 3.41 in the adult. Both the cell number and cell density per sample area in the INL was found to be higher along the nasotemporal meridian of the eye overlying the visual streak of the ganglion cell layer (GCL) of the retina. The retinal distribution of neurons in the INL did not change significantly during postmetamorphic growth in Xenopus. At metamorphosis a 1.71 difference was found between the highest neuron number (retinal ciliary margin) and lowest neuron number (retinal centre) decreasing to 1.51 in the adult. Retinae were labelled with ³H-thymidine in 15 mm Bufos and examined 2, 6, 12 and 18 weeks later. Higher rates of cell addition to the nasal and temporal poles of the INL were found compared with that at the dorsal and ventral poles. The retinal radial growth at the ciliary margin of the dorsal, ventral, nasal and temporal poles between the time of isotope injection and 18 weeks survival was found to be uneven; more radial elongation occurred at the nasal, dorsal and ventral poles and less at the temporal pole. These observations suggest that (a) the neuron distribution of the INL in adult animals approximates that of the GCL and (b) the visual streak-like area of the INL in Bufo develops by a sustained differential cell addition at the temporal and nasal poles of the retina.On leave from the Department of Anatomy, Zhanjiang Medical College, Guangdong,People's Republic of China     

The development and the topographic organization of the retinal ganglion cell layer in Bufo marinus

Summary The number and distribution of neurons in the retinal ganglion cell layer were studied from the metamorphic climax to adulthood in the toad Bufo marinus. Retinal wholemounts stained with cresyl violet showed that total neuron numbers increased from 55,000 at metamorphic climax to about 950,000 in adult animals. During the same time the entire retinal area increased 46-fold from an average 3.4 mm² to 157 mm². The morphological character of the neurons and their density across the retina changed during development. In metamorphosing animals, the neurons of the ganglion cell layer had a uniform appearance and their density increased slightly from the centre to the dorsal ciliary margin. After metamorphosis a high neuron density area, the visual streak, evolved in the retinal centre, resulting in the formation of a 6 to 1 density gradient from the visual streak out to the dorsal and ventral retinal poles in adult animals. Optic fibre numbers in juvenile and adult optic nerves were estimated to be 330,000 and 745,000, respectively, corresponding to similar ganglion cell numbers. One optic nerve was sectioned in a few animals and 4 weeks later the number of intact neurons — assumed to be displaced amacrine cells (DA) — was estimated. They amounted to 80,000 in juvenile and 189,000 in adult animals or about 20% of the total neuron population of the retinal ganglion cell layer, the remaining 80% being GC. A 1.7 to 1 density gradient of DA from the visual streak out to the dorsal and ventral retinal periphery was established. These results show that the visual streak evolves after metamorphosis from an originally uniform neuron distribution of the retinal ganglion cell layer. The possible mechanisms of the formation of the visual streak are discussed.     

Distance-dependent interactions between the rod and the cone systems in goldfish retina

Summary We recorded from ganglion cell axons in the optic tracts of self-respiring goldfish, and examined the interaction of rod-effective and cone-effective stimuli within their receptive fields. The summation of influences due to the rod system and the long wavelength sensitive cone system was analyzed by the methods of response-summation and sensitivity-summation. Different spatial relationships between the rod- and cone-effective stimuli allowed examination of distance-dependent effects.Both the response-summation and sensitivity-summation analyses showed a difference in non-linearity between a configuration in which the rod-and cone-effective stimuli were spatially overlapped and a configuration in which they were not. This difference in both analyses demonstrates a distance dependent interaction between the rod and cone systems. Both analyses also showed a difference in non-linearity between a configuration in which the rod- and cone-effective stimuli were nearby (but not overlapped) and one in which they were more distant. This demonstrates that the interaction is not limited to receptors that are immediate neighbors. An estimate of the strength of interaction in each case showed that the differences among the three configurations were relatively slight, indicating a broad spread of the effect. The interaction was found to be relatively powerful; assuming a specific simple model for the interaction mechanism, we found that each system exerts an effect upon the other which accounts for about 1/3 of its signal.Supported by grant no. 5 R01 EY 01951 from the National Eye Institute of NIHSupported by Research Fellowship no. 5 F32 NS05438 from NINCDS of NIH     

Morphological classification and retinal distribution of large ganglion cells in the retina ofBufo marinus

Summary The retrograde transport of horseradish peroxidase (HRP) and cobaltic-lysine complex (CLC) was used to morphologically characterize large ganglion cells (GCs) and to determine their distribution in retinal wholemounts and in sectioned material in the retina ofBufo marinus. Large GCs, amounting to about 0.5% of total GC population, were defined to be those with very large dendritic field sizes varying between 0.1 mm² to 0.6 mm² and cell soma sizes of between 100 m² to 400 m². These cells were subdivided into 3 major groups, Types I, II and III, on the basis of their dendritic field sizes, arborization patterns and the strata of dendritic branching within the inner plexiform layer (IPL). The majority of large neurons (about 90%) were classified as Type I GCs with symmetrical dendritic arbor. These cells had either bistratified branching in the scierai and vitreal sublaminae of the IPL (65% of Type I Cells) or unistratified branching in the scleral (26%) or in the vitreal (9%) sublamina. Their dendritic field sizes increased linearly from the retinal centre from 0.13 mm±0.02 mm² (mean and S.D.) to 0.58±0.11 mm² in the retinal periphery. Type II GCs (about 9% of the large GC population) were characterized by an asymmetrical dendritic arborization directed towards the ciliary margin with unistratified branching in the scierai sublamina of the IPL. The mean dendritic field sizes of these cells were 0.26±0.09 mm². Type III GCs, the least frequent (about 1%) category of large GCs had sparsely branching, elongated dendritic branching aligned approximately parallel with the nasotemporal axis of the retina. The unistratified dendritic branches of these neurons were located in the vitreal sublamina of the IPL with a mean dendritic field size of 0.42±0.11 mm². The dendritic field sizes of Types II and III GCs did not increase with retinal eccentricity. Type I GCs were distributed unevenly across the retina, the density being greatest in the visual streak, along the nasotemporal meridian of the retina. The dendritic field sizes of these cells increased towards the retinal periphery, resulting in a constant dendritic field coverage factor across the retina. Each retinal point was covered by the dendritic fields of 4–5 adjacent GCs. In contrast, Types II and III GCs had only discontinuous dendritic coverage. The identification of morphological types of large GCs with previously described functional classes of GCs in the anuran retina is discussed.On leave from the Department of Anatomy, University Medical School, Pécs, Hungary     

Peroxidation of lipids and degeneration of photoreceptors in the retina of rats with avitaminosis E

The formation of peroxidation products of lipids (hydroperoxides, dialkyl peroxides, intermolecular cross linkages) is sharply intensified in the retina of rats with alimentary avitaminosis Ein vivo and degeneration of the photoreceptors (mainly the layer of outer segments of the rods) develops. The experimental results are discussed on the basis of views regarding the antioxidant mechanism of action of -tocopherolin vivo.Laboratory of Physical chemistry of Biological Membranes, Departament of Biochemistry, Biological Faculty, M. V. Lomonosov Moscow State University. Institute of Chemical Physics, Academy of Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 4, pp. 411–413, April, 1977.     

Effects of Trifolium pratense and Cimicifuga racemosa on the endometrium of wistar rats

Objective

The aim of this study was to evaluate the effects of Trifolium pratense and Cimicifuga racemosa upon the endometrium of castrated female Wistar rats, comparing these results with a placebo and estradiol valerate.

Methods

Thirty-two adult castrated female Wistar rats were divided into four groups (eight rats per group) that receiving either tap water, estradiol valerate, isoflavones from T. pratense or deoxyactein from C. racemosa daily. The doses used were equivalent to normal doses used in humans. The results were analyzed by endometrial histology and the expression of α-estrogen receptor and protein Ki67. Both α-receptor and Ki67 expressions were determined by immunohistochemical and morphometric analysis.

Results

Endometrium histology stayed atrophic with both herbal extracts, but T. pratense supplementation increased the expression of α-estrogen receptors when compared to the placebo group, without protein Ki67 expression enhancement. Both herbal extracts presented a lower Ki67 expression when compared to the placebo group.

Conclusion

T. pratense presented α-estrogen receptor stimulation in the endometrium without increasing cell proliferation. Both herbal extracts reduced endometrial proliferation in comparison to the placebo group.     

Computer analysis of photochemical changes in the human retina

The first stage of the visual process involves the absorption of quanta by light-sensitive pigments in the retinal receptors. The photochemical consequences of this event can be measured in vivo by the technique of fundus reflectometry, and rapidly analyzed by a computer. This paper describes the principles of fundus reflectometry and considers the programming requirements for on-line data acquisition and processing of analog signals in a small laboratory computer.     

The morphology and laminar distribution of cortico-pulvinar neurons in the Rhesus monkey

Summary Using autoradiography and the horseradish peroxidase method, the morphology and laminar distribution of cortico-pulvinar neurons and the reciprocity of connections between pulvinar and cortex were examined in five Rhesus monkeys which had received medial, lateral and inferior pulvinar nucleus injections of both tritiated amino acids and horseradish peroxidase.Cortico-pulvinar neurons were identified in one heterotypical cortical area (area 17) and in many homotypical areas in frontal (areas 45, 46, 11, 12), parietal (5, 7), occipital (18, 19) and temporal (20, 21, 22) lobes. The cortico-pulvinar neurons were pyramidal in shape and ranged in size from small to large. In heterotypical cortex they were found in layers V and VI whereas in area 17 they were found only in layer Vb. Reciprocal connections between pulvinar and cortex were a feature of homotypical but not heterotypical cortex.     

Multiple types of Ca2+ channels in visceral smooth muscle cells

Single-channel currents were recorded from two classes of Ca²⁺ channels in visceral smooth muscle cells isolated from the stomach of the toad, Bufo marinus: a class of small-conductance channels (approximately 11 pS) and a class of large-conductance channels (approximately 26 pS). Small-conductance channels were present in a majority of patches and gave rise to a slowly inactivating current (t_1/2 250 ms at 0 mV). Openings of large-conductance channels could be unequivocally resolved only in the presence of the dihydropyridine Ca²⁺ agonist Bay K 8644. Two subtypes of the large-conductance channels were found — those with a very slow rate of decay (> 500 ms) and those with a faster one (< 100ms). Large-conductance channels resemble L-type Ca²⁺ channels of other preparations. Small-conductance channels do not fit unambiguously into the other existing categories (i.e., N or T). Correspondence between single-channel and macroscopic Ca²⁺ currents is discussed.     

GABA-immunoreactive photoreceptors in the retina of an anuran, Pelobates fuscus

We have recently started to unravel the retinal neurochemistry of an anuran species, the spadefoot toad (Pelobates fuscus), because of its unique lifestyle. The immunolabelling experiments included tests to localize the major inhibitory transmitter, gamma-aminobutyric acid (GABA) to subsets of retinal neurons, using commercially available antibodies. Apart from the regular GABA-immunoreactive pattern observed formerly in other anurans, certain structures in the photoreceptor layer were also regularly labeled for GABA. The soma diameter of the labeled cells is 5-6 microm and the outer segment seems to be unlabeled. In resin-embedded preparations GABA-positive photoreceptor cells were identified as cones based on their sparse distribution and short outer segments. If these cells release GABA as a transmitter, it may act on the second order cells, from which certain horizontal and bipolar cells have functional GABA receptors. Alternatively, GABA may influence the cones themselves through autoreceptors.     

Temporo-nasal asymmetry in the accretion of retinal ganglion cells in late larval and postmetamorphic Xenopus

Summary The spatial pattern of cell production and retinal growth were studied in Xenopus between stage 60 and two months after metamorphosis using ³H-proline and ³H-thymidine autoradiography. The position and the number of the ganglion cells labelled with ³H-thymidine were determined. The area of the unlabelled retina due to growth since ³H-proline administration at stage 60 was measured. Both retinal area measurements and counts of labelled ganglion cells showed 30–40% higher values in the temporal than in the nasal retinal half. The greater cell production and area accretion were even more pronounced between the temporal and the nasal retinal quadrants. The results on the temporoventral crescentic retinal growth rule out the possibility that from midlarval stages onwards the retinal and the tectal growth patterns are matched.     

Co-localization of serotonin and GABA in neurons of the Xenopus laevis retina

Serotonin-synthesizing neurons in the retina of Xenopus laevis have been identified using anti-phenylalanine hydroxylase (PH) antibody which recognizes tryptophan 5-hydroxylase, the rate-limiting enzyme for serotonin synthesis. Double-labelling experiments, using anti-PH antibody and anti-serotonin antibody/5,7-dihydroxytryptamine (5,7-DHT) uptake, have shown that some serotonin-like immunoreactive/5,7-DHT-labelled neurons exhibit PH-like immunoreactivity (PH-LI) (serotonin-synthesizing neurons), but the others do not (serotonin-accumulating neurons). In the present study, triple-labelling experiments were performed using 5,7-DHT uptake and antibodies raised against GABA and PH, to determine the possible co-localization of -aminobutyric acid (GABA) in serotonin-synthesizing and/or -accumulating neurons in the Xenopus retina. All 5,7-DHT-labelled bipolar cells lacked PH-LI; all of them were immunoreactive to GABA. In contrast, all 5,7-DHT-labelled large amacrine cells exhibited PH-LI, but none of them expressed GABA-LI. Small amacrine cells labelled with 5,7-DHT but not PH-LI exhibited GABA-LI, whilst the small amacrine cells with PH-LI lacked GABA-LI. These observations indicate that GABA is co-localized in serotonin-accumulating amacrine and bipolar cells, whereas serotonin-synthesizing large and small amacrine cells do not contain GABA-LI.     

The frequency of fim genes among Salmonella serovars

Salmonella serovars were examined for the presence of fim gene sequences using specific DNA probes. All strains, regardless of their ability to express surface-associated fimbriae, retain a considerable amount of DNA homologous to the gene probes used. The phenotypically non-fimbriate FIRN and non-FIRN strains of S. typhimurium retain detectable amounts of fim gene sequences and, therefore, may not be genotypically non-fimbriate. The MS adhesin can be expressed by type 2 fimbriate bacteria when they are transformed with discrete regions of the fim gene cluster. However, this conversion to a hemagglutinating phenotype is not associated with a small region of DNA. Therefore, the inability of type 2 fimbria-producing strains of Salmonella to mediate hemagglutination does not appear to be due to a small deletion in a single fim gene.     

Morphology and retinal distribution of tyrosine hydroxylase-like immunoreactive amacrine cells in the retina of developingXenopus laevis

Summary The development of neurons immunoreactive to tyrosine hydroxylase (TH-IR) in the retina ofXenopus laevis was investigated from stage 53 tadpoles to adult, by using an antibody against tyrosine hydroxylase. At all developmental stages, most of the immunoreactive somata were located in the inner nuclear layer, and a few in the ganglion cell layer. Immunoreactive processes arborised in the scleral and vitreal sublaminae of the inner plexiform layer, indicating that these cells were bistratified amacrine cells. However, occasionally a few immunoreactive processes were observed projecting to the outer plexiform layer, suggesting the presence of THIR interplexiform cells. The number of immunoreactive amacrine cells in the inner nuclear layer per retina increased from 204 at stage 53 tadpole to 735 in adult, while the number of immunoreactive amacrine cells in the ganglion cell layer did not change significantly over the same period. Retinal area increased from 1.95 mm² at stage 53 to 23.40 mm² in the adult, and correspondingly cell density in the inner nuclear layer decreased from 104/mm² to 31/mm². At all stages there was an increasing density towards the ciliary margin, but this gradient decreased with age. The soma size of immunoreactive amacrine cells increased with age, and was consistently larger in the central than in the peripheral retina. Dendritic field size was estimated to increase 13-fold, from stage 53 to adult. This study shows that tyrosine hydroxylase-like immunoreactive amacrine cells are generated continuously throughout life, that after metamorphosis the retina grows more by stretching than by cell generation at the ciliary margin, and that the increase of dendritic field size is proportional to the increase in retinal surface area.On leave from Department of Anatomy, Zhanjiang Medical College, Guangdong, People's Republic of China     

Synergistic interaction between the Potyvirus, Turnip mosaic virus and the Crinivirus, Lettuce infectious yellows virus in plants and protoplasts

Lettuce infectious yellows virus (LIYV), the type member of the genus Crinivirus in the family Closteroviridae, is specifically transmitted by the sweet potato whitefly (Bemisia tabaci) in a semipersistent manner. LIYV infections result in a low virus titer in plants and protoplasts, impeding reverse genetic efforts to analyze LIYV gene/protein functions. We found that synergistic interactions occurred in mixed infections of LIYV and Turnip mosaic virus (TuMV) in Nicotiana benthamiana plants, and these resulted in enhanced accumulation of LIYV. Furthermore, we examined the ability of transgenic plants and protoplasts expressing only the TuMV P1/HC-Pro sequence to enhance the accumulation of LIYV. LIYV RNA and protein titers increased by as much as 8-fold in these plants and protoplasts relative to control plants. LIYV infections remained phloem-limited in P1/HC-Pro transgenic plants, suggesting that enhanced accumulation of LIYV in these plants was due primarily to increased replication efficiency, not to greater spread.     

Stretch-activated ion channels in smooth muscle: a mechanism for the initiation of stretch-induced contraction

As in many smooth muscle tissue preparations, single smooth muscle cells freshly dissociated from the stomach of the toadBufo marinus contract when stretched. Stretch-activated channels have been identified in these cells using patch-clamp techniques. In both cell-attached and excised inside-out patches, the probability of the channel being open (P _o) increases when the membrane is stretched by applying negative pressure to the extracellular surface through the patch pipette. The increase inP _o is mainly due to a decrease in closed time durations, but an increase in open time duration is also seen. The open-channel current-voltage relationship shows inward rectification and is not appreciably altered when K⁺ is substituted for Na⁺ as the charge-carrying cation in Ca²⁺-free (2 mM EGTA) pipette solutions bathing the extracellular surface of the patch. The inclusion of physiological concentrations of Ca²⁺ (1.8 mM) in pipette solutions (containing high concentrations of Na⁺ and low K⁺) significantly decreases the slope conductance as well as the unitary amplitude. The channel also conducts Ca²⁺, since inward currents were observed using pipette solutions in which Ca²⁺ ions were the only inorganic cations. When simulating normal physiological conditions, we find that substantial ionic current is conducted into the cell when the channel is open. These characteristics coupled with the high density of the stretch-activated channels point to a key role for them in the initiation of stretch-induced contraction.