Biomedical subjects

W D Eldred

Publications and source records attributed to W D Eldred.

At least 37 records · Page 2Linked to original sources

PubMed1991

Synaptic organization of two types of amacrine cells with CRF-like immunoreactivity in the turtle retina.

The ultrastructural features and synaptic contacts of two amacrine cell types with corticotropin-releasing factor-like immunoreactivity in the turtle retina were examined using electron immunocytochemistry. Type A cells were found only in the visual streak and had elongated dendritic arborizations that ran parallel to the visual streak. These cells arborized primarily in stratum 1 and near the border of strata 2 and 3 of the inner plexiform layer, with some processes extending into stratum 5. Type B cells were found only ventral to the visual streak and arborized primarily in a wide band in strata 4 and 5, with sparse dendritic arborizations in stratum 1. There was a diffuse cytoplasmic reaction product within each cell type; however, large labeled vesicles were rarely observed. Type A amacrine cells received many conventional synaptic contacts from amacrine cells in stratum 1 and at the border of strata 2 and 3, but only a small number of contacts in stratum 5. Bipolar synaptic contacts onto type A amacrine cells were observed in strata 1 and at the border of strata 2 and 3. The only positively identified synaptic outputs of type A cells were conventional synapses onto amacrine cells in strata 1 and at the border of 2 and 3. Type B amacrine cells received synaptic contacts from amacrine cells in strata 1 and 5, and bipolar cell synaptic input in stratum 5. They made conventional synapses onto amacrine cells in strata 1 and 5, and onto bipolar cells in stratum 5. We also found conventional synaptic contacts between unlabeled amacrine cells and type B amacrine cells outside of the primary layers of stratification. In addition, there were specialized junctions observed between type A cell profiles in stratum 1 and between type B cell profiles in stratum 5. The unique regional distributions of the type A and B cells, as well as their differences in synaptic connectivity, suggested that these amacrine cells play distinct physiological roles although they contain the same neuropeptide.

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PubMed1990

Synaptic analysis of amacrine cells in the turtle retina which contain tyrosine hydroxylase-like immunoreactivity.

This study examined amacrine cells in the retina of the turtle Pseudemys scripta elegans, which were labelled using an antiserum directed against tyrosine hydroxylase (an enzyme participating in catecholamine synthesis). These cells were investigated using both light and electron microscopy. Labelled somata were located in the inner nuclear layer near the border of the inner plexiform layer. The dendritic arborizations of these neurons were tristratified and arborized in strata 1 and 3 and near the border between strata 4 and 5. Serial tangential sections taken through the entire inner plexiform layer of a 1 mm-2 region in mid-peripheral retina were examined. All of the synapses associated with labelled profiles were counted and classified. The majority (84%) of the synapses involving labelled processes represented output, while the remaining 16% represented synaptic input. The synaptic output of the labelled processes was as follows: 87% onto unlabelled amacrine cells, 4% onto ganglion cells, 9% onto unidentified cell processes. None of the synaptic output from labelled processes was onto bipolar cells. The synaptic input to these labelled cells was from bipolar cells (29%) and from unlabelled amacrine cells (71%). A well labelled amacrine cell was serially sectioned and examined at the ultrastructural level to analyze its synaptic connectivity. Immunoreaction product was located diffusely throughout the cytoplasm and in large vesicles. The synaptic organization of the cell was directed primarily toward output. The labelled processes were postsynaptic and presynaptic to unlabelled amacrine cell processes in strata 1 and 3 and at the border between strata 4 and 5. Synaptic input from bipolar cells was seen exclusively near the border between strata 4 and 5. Labelled processes were presynaptic to ganglion cell processes in stratum 1 and at the border between strata 4 and 5, but not in stratum 3. Quantitative studies suggested that amacrine cell inputs and outputs were evenly distributed across the dendritic arborization, while bipolar cell inputs and outputs to ganglion cells were concentrated on the distal parts of the dendritic arborization. No labelled processes were seen in the outer plexiform layer, indicating that the cells with tyrosine hydroxylase-like immunoreactivity in the turtle retina were true amacrine cells and not interplexiform cells.

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PubMed1989

Amacrine and ganglion cells with corticotropin-releasing-factor-like immunoreactivity in the turtle retina.

This study, which uses immunocytochemical methods at the light microscopical, level, examines the cell types in the turtle retina that contain corticotropin-releasing factor (CRF)-like immunoreactivity. Two anatomically distinct amacrine cell types are labeled when antiserum directed against ovine CRF is used to label the turtle retina. These cell types each have a different dendritic arborization pattern and regional distribution. Type A cells are found only in the visual streak and have elongated dendritic arborizations that run parallel to the visual streak. These cells arborize primarily in stratum 1 and near the border of strata 2 and 3, with some processes extending into stratum 5. Type B amacrine cells are found only ventral to the visual streak and arborize primarily in a wide band in strata 4 and 5 with sparse dendritic arborizations in stratum 1. No labeled amacrine cells of any type were found dorsal to the visual streak. The asymmetric dendritic arborizations of the type A amacrine cells and the different regional distributions of the A and B cell types suggest that these two amacrine cell types perform distinct physiological functions. In addition to these labeled amacrine cells, there are also some immunoreactive cell bodies in the ganglion cell layer. Rhodamine crystals were applied to the optic tectum to retrogradely label the ganglion cell bodies. Double label studies indicate that some of the rhodamine-labeled ganglion cells also contain CRF-like immunoreactivity. The localization of CRF-like immunoreactivity in two distinct amacrine cell types and in ganglion cells suggests that it may play multiple roles in visual processing in the turtle retina.

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PubMed1989

Immunocytochemical localization of glycine in the retina of the turtle (Pseudemys scripta).

We have localized glycine-like immunoreactivity to provide new anatomical detail about glycinergic neurons in the turtle retina. A rabbit antiserum directed against a glycine/albumin conjugate was used with standard fluorescent and avidin-biotin labeling techniques. Some processes in the outer plexiform layer and many processes in the inner plexiform layer, numerous somata in the inner nuclear layer, and isolated somata in the ganglion cell layer were immunoreactive. The vast majority of labeled neurons were amacrine cells. One class of amacrine cells had well-labeled somata near the inner nuclear/inner plexiform layer border, which gave rise to thick primary processes that entered the inner plexiform layer and arborized near the border of strata 1 and 2 and in stratum 3. A second class of glycinergic neurons, consisting of putative interplexiform cells, was unique in that it gave rise to dendritic arborizations in both the outer plexiform layer and the inner plexiform layer. Some of the immunoreactive neurons in the ganglion cell layer were apparently displaced amacrine cells, while others were probably true ganglion cells because they gave rise to labeled axons, and many labeled axons were visible in the ganglion cell axon layer. These results suggested that glycine played an extensive role in the turtle retina, and that it was involved in many diverse synaptic interactions in both the outer plexiform layer and the inner plexiform layer.

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PubMed1989

Localization of GABA- and GAD-like immunoreactivity in the turtle retina.

gamma-aminobutyric acid (GABA) has been reported to be an important neurotransmitter in the retinas of many species. This immunocytochemical study detailed the localization of antigens resembling GABA and glutamic acid decarboxylase (GAD, an enzyme involved in the synthesis of GABA), in retinal neurons in the turtle, Pseudemys scripta elegans. GABA-like immunoreactivity was present within somata in the inner and outer regions of the inner nuclear layer, within somata in the ganglion cell layer, and in processes in the outer plexiform layer, inner plexiform layer, and ganglion cell axon layer. GAD-like immunoreactivity was found in somata in the inner and outer regions of the inner nuclear layer and in processes in the inner and outer plexiform layers. Cell counts indicated more somata with GABA-like than GAD-like immunoreactivity in the inner nuclear layer. Double-label studies showed that every somata in the inner nuclear layer which had GAD-like immunoreactivity also had GABA-like immunoreactivity, but that many somata had only GABA-like immunoreactivity. The stratification of immunoreactivity within the inner plexiform layer was analyzed using a scanning densitometer. We described the strate within the inner plexiform layer such that S0 represented the inner nuclear layer/inner plexiform layer border and S100 represented the inner plexiform layer/ganglion cell layer border. Analysis of GAD-like labeling yielded seven distinct strata with peak densities at positions S8, S19, S28, S42, S59, S75, and S93. GABA-like labeling provided five distinct strata with peak densities at positions S17, S28, S67, S84, and S95. The strata with peaks of GABA-like immunoreactivity at S17 and S28 were in statistically identical locations to corresponding strata with GAD-like immunoreactivity. The strata with GABA-like immunoreactivity at S67, S84, and S95 did not have statistically identical peaks of correlated GAD-like immunoreactivity, although there were corresponding strata with GAD-like immunoreactivity nearby. Antiserum directed against GABA failed to produce labeled strata at positions corresponding to the strata with GAD-like immunoreactivity at S8 and S42. In summary, our results indicated that the antisera we used, which were directed against GABA and GAD, produced significantly different labeling in the inner nuclear layer, inner plexiform layer, and the ganglion cell body and axon layers of the turtle retina. Until the physiological significance of these differences is resolved, studies employing these markers to investigate the function of GABA in the turtle retina should be interpreted with caution.

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PubMed1988

Synaptic analysis of amacrine cells with neuropeptide Y-like immunoreactivity in turtle retina.

Neuropeptide Y-like immunoreactivity has been localized previously within three classes of amacrine cells in the turtle retina. We have used the avidin-biotin with horseradish peroxidase technique to label these neurons for examination at the ultrastructural level to answer the following questions. Where are the synaptic contacts of these neurons made? What types of neurons are involved pre- and postsynaptically? What is the intracellular distribution of the immunoreactivity? Processes with neuropeptide Y-like immunoreactivity were located primarily within three regions of the inner plexiform layer: stratum 1, stratum 3, and at the border between strata 4 and 5. In all three regions the processes with neuropeptide Y-like immunoreactivity received synaptic contacts from both unlabeled amacrine and bipolar cells, but the majority of the synaptic input in all three regions was from unlabeled amacrine cells. Processes with neuropeptide Y-like immunoreactivity were presynaptic to unlabeled amacrine cells in all three regions, but also formed contacts onto unlabeled bipolar cells in the region between strata 4 and 5. The immunoreactivity within these cells gave rise to a diffuse reaction product that was distributed throughout the cytoplasm and within large vesicles. This localization of neuropeptide Y-like immunoreactivity within large vesicles suggests that this peptide may play a neuromodulatory role. Such a role would be consistent with previous studies of neuropeptides in the turtle retina.

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PubMed1988

Neuropeptide Y-immunoreactive amacrine cells in the retina of the turtle Pseudemys scripta elegans.

Antiserum directed against neuropeptide Y selectively labeled certain amacrine cells in the turtle retina. The cell types, sizes, dendritic stratification, regional distribution, and degrees of immunolabeling were examined. The results indicated that three morphologically distinct cell types were labeled: types A, B, and C. Computer rotation of digitized data from camera lucida drawings was used to study dendritic stratification. The type A somata were large (11.5 micron in diameter), well-stained, and located in the third tier of the inner nuclear layer. Type A somata gave rise to well-stained processes which arborized within the inner plexiform layer in strata 1 and 3 and at the border between strata 4 and 5. Processes in stratum 1 were sparse and delicate with small boutons. Processes in stratum 3 were numerous and often coarse, with many large and small boutons. At the border between strata 4 and 5 the processes were frequently numerous but slender, with many small boutons. Occasional immunolabeled processes were found in the ganglion cell layer. The somata of the type B cells were smaller (9.0 micron in diameter) and gave rise to single labeled processes which descended into the inner plexiform layer and divided quickly into two secondary processes. These secondary processes gave rise to lightly labeled dendritic fields which arborized primarily in strata 2 and 4. The type C cells were usually observed at the periphery of the retina and had large somata (12.0 micron in diameter) with simple, but very elongated, dendritic arborizations in strata 1, 4, and 5. Observations also showed that type A and B cells were often found in close proximity to each other and suggested that dendrites of these cells made contact with each other. The labeled neurons were distributed relatively evenly throughout the retina except for the visual streak where they were sparse. This study indicates that neuropeptide Y-like immunoreactivity is found in more than one anatomically distinct class of amacrine cells in the turtle retina.

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PubMed1988

Ganglion cells in the turtle retina contain the neuropeptide LANT-6.

This study investigated the presence of the neurotensin-related hexapeptide, LANT-6, in retinal ganglion cells and their central projections in the turtle Pseudemys scripta elegans. Immunocytochemical techniques demonstrated that many of the cells in the ganglion cell layer of the turtle retina could be labeled with an antiserum specific for LANT-6. Radioimmunoassay and chromatographic analysis confirmed the presence of LANT-6-related peptides in retina, as well as brain. Several molecular forms of LANT-6 were observed, some larger than LANT-6. Characterization of the cells labeled in the ganglion cell layer in terms of their cell body size and their dendritic arborization patterns revealed that at least 6 specific LANT-6-positive cell types were present in the ganglion cell layer. Morphologically, the LANT-6-positive cells strongly resembled turtle ganglion cells, as previously described. In addition, two other lines of evidence supported this interpretation. First, double-label studies were performed in which retinal ganglion cells projecting to the tectum were retrogradely labeled by HRP injected into the tectum (using a cobalt chloride color-modified DAB reaction product) and immunocytochemically labeled with DAB using the antiserum against LANT-6. These double-label studies revealed that many of the LANT-6-positive cells in the ganglion cell layer in the portion of the retina labeled retrogradely by the HRP injection did project to the tectum. Within the retrogradely labeled portion of the retina, LANT-6-positive cells that were not labeled retrogradely, as well as neurons labeled retrogradely that did not contain LANT-6 were also observed. Second, the central projections of LANT-6-positive cells of the ganglion cell layer were examined by studying the effects of monocular enucleation on the distribution of LANT-6-positive fibers in the central projection targets of the turtle retina. Two to 8 weeks after enucleation, a substantial reduction in LANT-6-positive fibers was observed in all retinal target areas contralateral to the enucleated eye. Radioimmunoassay and chromatographic studies confirmed the presence of LANT-6-related peptides in the turtle brain and corroborated the reduction of LANT-6 observed in the contralateral tectum following monocular enucleation. Previous studies have demonstrated that LANT-6-related material is present in cells of the ganglion cell layer in a variety of vertebrates. The present results indicate that LANT-6 is in ganglion cells and that it may play a role in neurotransmission between retinal ganglion cells and their central target areas.

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PubMed1987

Immunocytochemical localization of LANT-6-like immunoreactivity within neurons in the inner nuclear and ganglion cell layers in vertebrate retinas.

LANT-6 is a hexapeptide (H-Lys-Asn-Pro-Tyr-Ile-Leu-OH) isolated from chicken small intestine, which resembles the COOH-terminal half of neurotensin, except for the amino acid substitutions Lys/Arg and Asn/Arg. The present report concerns the immunocytochemical staining of vertebrate retinas using an antiserum directed against LANT-6. In the retinas from goldfish, bird and turtle, cells in both the inner nuclear and ganglion cell layers were labeled, but in the frog no cells were labeled specifically and in the rat only cells in the ganglion cell layer were labeled. Labeled cell bodies in the inner nuclear layer gave rise to processes which were seen primarily within the following laminas of the inner plexiform layer (IPL): in the goldfish, lamina 3; chicken, laminae 1, 3 and 4; and turtle, laminae 3, 4 and 5. The cell bodies of the labeled neurons in the ganglion cell layer gave rise to dendrites which entered the IPL and axons which descended to the optic fiber layer. The cells with LANT-6-like immunoreactivity were distributed in both the central and peripheral parts of the retina in all the species examined except frog. Measured by radioimmunoassays, the levels of LANT-6-like-immunoreactivity in extracts of turtle, chicken, and goldfish retinas were 5-30 times those for neurotensin-like immunoreactivity, however no LANT-6-like immunoreactivity was detected in frog. Multiple chromatographic analyses indicated that while the LANT-6-like immunoreactivity in chicken retina was indistinguishable from synthetic LANT-6, LANT-6-like immunoreactivity in turtle and goldfish retinas was primarily associated with large molecular forms. Treatment of turtle LANT-6-like immunoreactivity with pepsin, an enzyme known to mimic processing for neurotensin precursors, yielded 3 major peptides, one of which co-chromatographed with synthetic LANT-6. The present immunocytochemical localization of LLI within cells in the inner nuclear and ganglion cell layers, coupled with the biochemical characterization of LANT-6 in the vertebrate retinas and brains, suggests that neuropeptides such as LANT-6 may play a role in visual processing both within the retina and within the visual pathways to the brain.

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PubMed1987

A simple device for the computer quantification of depth measurements in thick light microscope sections.

The use of computerized techniques to characterize quantitatively the anatomy of individual neurons has been increasing. One difficulty has been the quantification of the z-axis or depth measurements within thick light microscopic sections. In the present report we describe a simple device which employs an incremental optical encoder to transduce the movements of the focusing knob of the microscope so that depth information can be recorded directly by a computer. A resolution of 0.13 micron over a range of approximately 8.5 cm is achieved. The mechanical interface to the microscope is simple and applicable to a wide variety of microscopes. Interfacing circuits which allow the optical encoder to be used with an IBM-PC compatible computer are presented and described. The accuracy of the depth measurements is limited only by the mechanical tolerances of the focusing mechanism and by the optics of the microscope.

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PubMed1987

Neurocircuitry of two types of neurotensin-containing amacrine cells in the turtle retina.

The ultrastructural features and synaptic contacts of two types of neurotensin-containing amacrine cells in turtle retina were examined by electron immunocytochemistry, and the retinal peptides were characterized using radioimmunoassay and high-pressure liquid chromatography. The two types of cell were distinguished on the basis of their sizes, dendritic arborizations, synaptic connections and cytoplasmic staining characteristics. Type A cells had lightly labeled cytoplasm and large vertically elongated cell bodies which gave rise to a single primary process which in turn branched and ramified as smooth tapering processes in stratum 3 of the inner plexiform layer. Type A cells received approximately equal synaptic input from amacrine and bipolar cells. Type A amacrines had much more overall synaptic input than synaptic output, and they made conventional synaptic contacts onto bipolar, amacrine, and ganglion cells. Type B cells had a much darker-staining cytoplasm and a smaller cell body which gave rise to numerous delicate beaded dendrites which arborized in strata 3, 4 and 5 of the inner plexiform layer. Type B cells received primarily amacrine and some bipolar cell input. Type B cells had equal amounts of synaptic input and output and they made conventional synaptic contacts onto amacrine, bipolar, and ganglion cells. Whereas there were numerous large vesicles (120 nm diameter) that stained for neurotensin in both types of cells, conventional synaptic vesicles (60 nm diameter) were not labeled. In several cases these large labeled vesicles appeared to fuse with the cell membrane in non-synaptic regions and release their contents into extracellular space, which suggested a non-synaptic release of the neurotensin from type A neurons. Immunochemical and chromatographic studies demonstrated that the neurotensin-related material in retina was indistinguishable from neurotensin found in brain. These results are consistent with a neuroactive role for the neurotensin present within the large vesicles. The differences in the synaptic contacts and dendritic arborizations of the two amacrine cell types suggest they play distinctive functions in visual processing.

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PubMed1985

Ultrastructure and synaptic contacts of enkephalinergic amacrine cells in the retina of turtle (Pseudemys scripta).

Bistratified amacrine cells of the turtle retina containing enkephalin-like immunoreactivity were examined with the electron microscope with the aid of peroxidase immunocytochemical techniques. Our goal was to determine the nature and the location of the synaptic contacts of these cells and the intracellular localization of the immunoreactivity. There was a diffuse reaction product throughout the cytoplasm which coated the surfaces of all the organelles and a dense reaction product which filled the core of some large cytoplasmic vesicles (130 nm in dia.). These labeled amacrine cells received conventional synaptic contacts from other unlabeled amacrine cells and ribbon synaptic contacts from unlabeled bipolar cells, in both the proximal and distal inner plexiform layer. These enkephalin-positive amacrine cells made conventional synaptic contacts containing unlabeled synaptic vesicles (60 nm in dia.), with ganglion cells in the proximal inner plexiform layer and with bipolar cells in the distal inner plexiform layer. These results suggest that enkephalin-like material coexists with another neurotransmitter within these neurons and that these amacrine cells are able to integrate information from both amacrine cells and bipolar cells and provide synaptic input to bipolar cells, ganglion cells, and possibly other amacrine cells.

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PubMed1985

The co-occurrence of a substance P-like peptide and cholecystokinin-8 in a fiber system of turtle cortex.

Single-label and double-label immunohistochemical techniques were used to demonstrate the coexistence of substance P-like immunoreactivity (SPLI) and cholecystokinin-8-like immunoreactivity (CCK-8-LI) in an extensive fiber system within the telencephalic cortex of turtle. All SPLI-containing fibers and terminals of this system contain CCK-8-LI and vice versa. The fibers of this system course from more medial cortical regions to more lateral ones, originating either from neurons in the more medial cortices or from extracortical neurons, the axons of which ascend the medial wall of the cortex. The precise location of the neurons that give rise to this cortical projection system is uncertain, but a hypothalamic location seems most likely at present. The fibers and terminals of this system are found throughout the entire mediolateral and rostrocaudal extent of the telencephalic cortex of turtle and are largely confined to the cell body layer of the cortex. Fewer SPLI/CCK-8-LI-containing fibers are found in pyriform (olfactory) cortex than in the other cortices. Ultrastructural studies indicate that SPLI/CCK-8-LI-containing terminals make asymmetric synapses on cell bodies or their proximal dendrites. Both SPLI and CCK-8-LI are found in large dense core vesicles in these labeled terminals. Labeled terminals also contained numerous small, round, unlabeled vesicles clustered near synaptic release sites and a number of unlabeled large dense core vesicles. Quantification of the percentage of the large dense core vesicles that were labeled in SP-labeled terminals, in CCK-8-labeled terminals, and in terminals labeled for both SP and CCK-8 provided suggestive evidence that SPLI and CCK-8-LI must be contained within the same large dense core vesicles. Radioimmunoassay indicated that the SP/CCK-8-containing system of turtle cortex contains 0.93 +/- 0.090 pg of SP/microgram of cortical tissue protein and 0.31 +/- 0.11 pg of CCK-8/micrograms of cortical tissue protein. The CCK-8-like material in turtle cortex coelutes with CCK-8-sulfate, using gradient elution high pressure liquid chromatography (HPLC). The SP-like material, although immunologically highly similar to undecapeptide SP (Reiner, A., J. E. Krause, K. T. Keyser, W. D. Eldred, and J. F. McKelvy (1984) J. Comp. Neurol. 226: 50-75), does not coelute with undecapeptide SP using gradient elution HPLC.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1984

The distribution of substance P in turtle nervous system: a radioimmunoassay and immunohistochemical study.

The distribution of a substance P-like material in turtle brain, spinal cord, dorsal root ganglion, and retina was determined using radioimmunoassay (RIA) and immunohistochemistry. High levels of a substance P-like material were found in turtle neural tissue, particularly in basal telencephalon, hypothalamus, and tegmentum. In many regions, the concentration of a substance P-like material in turtle nervous tissue was found to be similar, in a region-to-region comparison, to that previously reported for birds and mammals, particularly for the more "phylogenetically conservative" parts of the nervous system (such as the basal ganglia, tegmentum, and hypothalamus). The slopes of substance P RIA dose-response curves for tissue extracts from nearly all regions of the turtle nervous system examined were parallel to a standard dose-response curve for synthetic substance P (SP). The immunohistochemical results, with anti-substance P antisera from guinea pig or rabbit, or with a monoclonal antibody, were consistent with the RIA data. Regions showing high concentration of an SP-like material by RIA were observed to contain numerous neurons and/or fibers containing an SP-like material. The immunohistochemical results provide evidence for the presence in turtle of numerous SP-containing pathways, several of which (e.g., an SP-containing strionigral pathway, an SP-containing striopallidal pathway and an SP-containing dorsal root ganglia-spinal dorsal horn pathway), have been described in birds and mammals. The present results thus suggest that the neuropeptide SP has had a largely stable evolutionary history as a transmitter or modulatory agent during amniote brain evolution.

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PubMed1983

Characterization and quantification of peptidergic amacrine cells in the turtle retina: enkephalin, neurotensin, and glucagon.

Immunocytochemical methods were used for selective labeling and characterization of amacrine cells of the turtle (Pseudemys scripta elegans) retina which contained neuropeptide-like immunoreactivity (leu-enkephalin, glucagon, and neurotensin). Processes of amacrine cells arborized in specific strata of the inner plexiform layer (IPL). Different strata were defined in relation to the boundaries of the IPL. Zero represented the strata nearest the inner nuclear layer and 100 represented the strata nearest the ganglion cells. Antisera directed against leu-enkephalin labeled approximately 7,300 amacrine cells in a single turtle retina which were concentrated in the region of the visual streak and decreased in density toward the periphery. In retinal regions outside the visual streak the labeled neurons were similar in size and shape with dendritic arbors which lacked a particular orientation. In contrast, in the visual streak, there were particular neurons which were labeled with enkephalin antiserum which had elongated dendritic arbors that ran parallel to the streak. Both types of amacrine cells with enkephalinergic immunoreactivity sent their dendrites into the 0-20 region and the 65-100 region of the IPL. Antisera directed against glucagon labeled approximately 2,500 amacrine cells in a single turtle retina. These cells were concentrated in areas near the visual streak. Amacrine cells labeled with glucagon antiserum had dendritic arbors which were asymmetrically skewed toward one end of the cell and ramified in the 0-20 strata with sparse projections at the 40 and 80 strata of the IPL. Antisera directed against neurotensin labeled 12,800 amacrine cells in a single turtle retina. These cells were concentrated in the region of the visual streak. Two distinct amacrine cell types were labeled selectively. One type had a large, vertically oriented cell body (10 X 14 micron) which gave rise to a single 2-micron-thick process that branched and ramified within the 45-70 strata. The other amacrine cell type with neurotensin-like immunoreactivity had a smaller cell body (8 micron) that sent numerous thin dendrites into the same 45-70 strata. The present results indicated that various neuropeptides were present in amacrine cells of the turtle retina and that a specific neuropeptide could be found in more than one anatomical type of amacrine cell. Each anatomical type of amacrine cell had a unique dendritic arborization which ramified within particular strata within the IPL.

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PubMed1983

Comparison of fixation and penetration enhancement techniques for use in ultrastructural immunocytochemistry.

Electron microscopic immunohistochemistry, although generally providing good localization, has often failed to produce satisfying ultrastructural preservation. Techniques that result in well-preserved tissue ultrastructure often hinder penetration of immunological reagents or render antigens non-immunoreactive. These are particularly serious limitations in studies of central nervous system and retina. We have evaluated several fixatives, including picric acid, high pH paraformaldehyde, and glutaraldehyde with subsequent sodium borohydride treatment, and penetration enhancement techniques, including buffered-ethanolic treatment and freeze--thaw, for their applicability in the retina. Our best fixation was achieved with 1 hr in 4% paraformaldehyde and 0.2% glutaraldehyde (pH 7.4) followed by overnight fixation in 4% paraformaldehyde (pH 10.4). Treatment with sodium borohydride after glutaraldehyde fixation restores much of the immunoreactivity that would otherwise be undetectable. The penetration of immunological reagents can be greatly increased by using either a buffered-ethanolic series or by freezing the tissue after careful cryoprotection. Using these methods we have been able to achieve specific immunological staining throughout the full thickness of retinal slices, up to 500 microns across, while preserving good ultrastructure. The methods should prove useful in the immunocytochemical localization of many different antigens in a variety of tissues.

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PubMed1982

Localization of substance P-like and enkephalin-like immunoreactivity within preganglionic terminals of the avian ciliary ganglion: light and electron microscopy.

The avian ciliary ganglion receives its only recognized input from the nucleus of Edinger-Westphal. This is known to be a cholinergic input. In the present study, using fluorescein isothiocyanate and peroxidase-antiperoxidase immunohistochemical methods, substance P-like and enkephalin-like immunoreactivity has been found within preganglionic terminals of the avian ciliary ganglion. The ciliary ganglion is known to consist of two distinct cell populations: small choroid cells that project to the smooth muscle coat of the choroid and large ciliary neurons that send axons to both the iris and the ciliary body. Preganglionic terminals on choroid cells consist of small boutonal endings, whereas ciliary neurons receive a calyx-like cap ending around the hilus of the cell. Substance P-like and enkephalin-like immunoreactivity was localized to preganglionic axons and to both boutonal and calyx-like terminations upon cells of the ciliary ganglion. Electron microscopic studies of both substance P-like and enkephalin-like immunoreactive terminals revealed small clear core vesicles (approximately 58 nm in diameter) and two sizes of dense core vesicles (approximately 85 and approximately 119 nm in diameter). Immunoreactive staining was observed only in the smaller dense core vesicles. The unlabeled clear core vesicles were clustered at synaptic release sites, while the immunoreactive and larger unlabeled dense core vesicles usually were not near these synaptic specializations. These observations strongly imply that neuropeptides co-occur with acetylcholine in preganglionic axons of the ciliary ganglion.

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PubMed1981

Multiple classes of photoreceptors and neurons in the frontal organ of Rana pipiens.

An attempt has been made to characterize the photoreceptors and neurons of the frontal organ of Rana pipiens and the synaptic contacts of these cells, in order to provide morphological correlates for previous physiological findings. Neurons, glial cells, and photoreceptors with two different appearances can be seen in normal anatomical preparations. The photoreceptors make ribbon synaptic contacts onto both neurons and other photoreceptors. Retrograde transport of either cobalt or horseradish peroxidase through the frontal nerve results in labeling of neurons which are often located near the exit point of the frontal nerve from the frontal organ. Some of the dendritic processes of these neurons ramify among the neurons themselves, while others ramify among the photoreceptors. In addition, the frontal organ contains two photoreceptor types which synaptically contact both neurons and other photoreceptors. In some frontal organs labeled with cobalt (but never in peroxidase-labeled preparations) certain photoreceptors were also labeled. The labeled photoreceptors were probably the result of transcellular cobalt movement from labeled neurons, and not the result of some photoreceptors having axons in the frontal nerve. These results are discussed in conjunction with physiological evidence that chromatic interactions occur within single photoreceptors, each of which contains a single visual pigment with two physiologically active states.

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