The decision by germ cells to differentiate and undergo either oogenesis or spermatogenesis takes place during embryonic development and Nanos plays an important role in this process. The present study was designed to investigate the expression patterns in rat of Nanos2-homologue protein in primordial germ cells (PGCs) over different embryonic developmental days as well as in spermatogonial stem cells (SSCs). Embryos from three different embryonic days (E8.5, E10.5, E11.5) and SSCs were isolated and used to detect Nanos2-homologue protein using immunocytochemistry, western blotting, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Interestingly, Nanos2 expression was detected in PGCs at day E11.5 onwards and up to colonization of PGCs in the genital ridge of fetal gonads. No Nanos2 expression was found in PGCs during early embryonic days (E8.5 and 10.5). Furthermore, immunohistochemical and immunofluorescence data revealed that Nanos2 expression was restricted within a subpopulation of undifferentiated spermatogonia (As, single type A SSCs and Apr, paired type A SSCs). The same results were confirmed by our western blot and RT-PCR data, as Nanos2 protein and transcripts were detected only in PGCs from day E11.5 and in undifferentiated spermatogonia (As and Apr). Furthermore, Nanos2-positive cells were also immunodetected and sorted using flow cytometry from the THY1-positive SSCs population, and this strengthened the idea that these cells are stem cells. Our findings suggested that stage-specific expression of Nanos2 occurred on different embryonic developmental days, while during the postnatal period Nanos2 expression is restricted to As and Apr SSCs.

Many problems in weed science may be solved by utilizing immunochemical techniques, although these techniques are currently underutilized by weed scientists. In this review, we describe some of the methods of greatest use to weed scientists. Antibodies may be raised against proteins, such as the targets of herbicide action or against the herbicide itself which can be linked to a larger molecule so that antibodies are elicited. Once specific antibodies are obtained, several techniques may be performed utilizing the binding phenomena of antibody-antigen interactions. Immunofluorescence and immunogold electron microscopy are utilized to obtain tissue and subcellular distributions of the protein or herbicide of interest. Quantitation of either protein or herbicide in a given sample may be performed by ELISA or “slot” and “dot” blotting. These protocols are less costly, more sensitive, and much less labor-intensive than most analytical methods. Molecular mass or charge alterations may be determined by electrophoresis and subsequent immunoblotting. With the increased exposure to immunological techniques by weed scientists and their potential utility, we predict that many more weed science problems will be addressed using these protocols.

Pheromone-binding proteins (PBPs) are believed to be involved in the recognition of semiochemicals. In the present study, western blot analysis, fluorescence-binding characteristics and immunolocalization of CmedPBP4 from the rice leaffolder, Cnaphalocrocis medinalis, were investigated. Western blot analysis revealed that CmedPBP4 showed obvious antenna-specific expression patterns in female and male antenna, and made a clearly different sex-biased expression. Immunocytochemical labeling revealed that CmedPBP4 showed specific expression in the trichoid sensilla. Competitive fluorescence binding assays indicated that CmedPBP4 could selectively recognize three sex pheromone components (Z13-18:Ac, Z11-16:Al and Z13-18:OH) and eleven rice plant volatiles, including cyclohexanol, nerolidol, cedrol, dodecanal, ionone, (−)-α-cedrene, (Z)-farnesene, β-myrcene, R-(+)-limonene, (−)-limonene, and (+)-3-carene. Meanwhile the CmedPBP4 detection of sex pheromones and host odorants was pH-dependent. Our results, for the first time, provide further evidence that trichoid sensilla might be play an important role in detecting sex pheromones and host plant volatiles in the C. medinalis moth. Our systematic studies provided further detailed evidence for the function of trichoid sensilla in insect semiochemical perception.

The proline-23-histidine line 3 (P23H-3) transgenic rat carries a human opsin gene mutation leading to progressive photoreceptor loss characteristic of human autosomal dominant retinitis pigmentosa. The aim of the present study was to evaluate neurochemical modifications in the P23H-3 retina as a function of development and degeneration. Specifically, we investigated the ion channel permeability of photoreceptors by tracking an organic cation, agmatine (1-amino-4-guanidobutane, AGB), which permeates through nonspecific cation channels. We also investigated the activity of ionotropic glutamate receptors in distinct populations of bipolar, amacrine, and ganglion cells using AGB tracking in combination with macromolecular markers. We found elevated cation channel permeation in photoreceptors as early as postnatal day 12 (P12) suggesting that AGB labeling is an early indicator of impending photoreceptor degeneration. However, bipolar, amacrine, or ganglion cells displayed normal responses secondary to ionotropic glutamate receptor activation even at P138 when about one half of the photoreceptor layer was lost and apoptosis and gliosis were observed. These results suggest that possible therapeutic windows as downstream neurons in inner retina appear to retain normal function with regard to AGB permeation when photoreceptors are significantly reduced but not lost.

Rod bipolar cells in Cebus apella monkey retina were identified by an antibody against the alpha isoform of protein kinase C (PKCα), which has been shown to selectively identify rod bipolars in two other primates and various mammals. Vertical sections were used to confirm the identity of these cells by their characteristic morphology of dendrites and axons. Their topographic distribution was assessed in horizontal sections; counts taken along the dorsal, ventral, nasal, and temporal quadrants. The density of rod bipolar cells increased from 500 to 2900 cells/mm2 at 1 mm from the fovea to reach a peak of 10,000–12,000 cells/mm2 at 4 mm, approximately 5 deg of eccentricity, and then gradually decreased toward retinal periphery to values of 5000 cells/mm2 or less. Rod to rod bipolar density ratio remained between 10 and 20 across most of the retinal extension. The number of rod bipolar cells per retina was 6,360,000 ± 387,433 (mean ± s.d., n = 6). The anti-PKCα antibody has shown to be a good marker of rod bipolar cells of Cebus, and the cell distribution is similar to that described for other primates. In spite of the difference in the central retina, the density variation of rod bipolar cells in the Cebus and Macaca as well as the convergence from rod to rod bipolar cells are generally similar, suggesting that both retinae stabilize similar sensitivity (as measured by rod density) and convergence.

Adult Corrigia vitta (Trematoda: Dicrocoelidea) inhabit the pancreatic duct of the fieldmouse, Apodemus sylvaticus, where, in numbers, they may occlude the duct lumen and prevent the flow of pancreatic secretions. Enzyme histochemical and immunocytochemical techniques, in conjunction with confocal scanning laser microscopy, have been used to examine the localization and distribution of cholinergic. serotoninergic (5-HT, serotonin) and peptidergic components of the nervous system of the adult worm. All three classes of neuronal mediator showed a common pattern of staining, occurring throughout the central and peripheral nervous systems. Of the four peptide immunoreactivities (IR) demonstrated (pancreatic polypeptide (PP), peptide YY (PYY), substance P (SP), FMRFamide), PP-IR was the most predominant, occurring not only within the central ganglia and longitudinal nerve cords, but also in subtegumental plexuses and in fibres associated with the egg-forming apparatus. PYY and FMRFamide IRs were evident throughout the central and peripheral nervous systems; FMRFamide immunostaining, in particular, highlighted innervation of the ootype and immunoreactive cell bodies around the Mehlis' gland. Both SP- and 5-HT-IRs were restricted to the cerebral ganglia, ventral nerve cords and associated cell bodies. The distribution pattems of these peptides and 5-HT within the nervous system of C. vitta suggest they are likely to function as neuronal mediators. PP, PYY and FMRFamide may also serve in regulating egg production.

The localization and distribution of cholinergic, serotoninergic (5-HT, serotonin) and peptidergic components of the nervous system of adult Cephalochlamys namaquensis (Cestoda: Pseudophyllidea) have been determined using enzyme histochemical and immunocytochemical techniques interfaced with light and confocal scanning laser microscopy. All three classes of neuroactive substance showed a similar pattern of staining, occurring extensively throughout the central and peripheral nervous systems of the parasite. There were some minor regional differences in staining, suggesting specific roles for certain classes of neurone, and nerve cell bodies were most evident following immunostaining for serotonin. The general overlap in the distribution of staining may be indicative of some co-localization of neurotransmitter and/or neuromodulatory substances.

Immunocytochemical techniques were used to localize strychnine-sensitive glycine receptors in cat retina. Light microscopy showed staining in processes ramifying throughout the inner plexiform layer and in cell bodies of both amacrine and ganglion cells. At the electron-microscopic level, receptor immunoreactivity was seen to be clustered at sites postsynaptic to amacrine cells. In contrast, bipolar cells were neither presynaptic nor postsynaptic elements at sites of glycine receptor staining. Double-label studies verified the presence of glycine immunoreactivity in amacrine terminals presynaptic to glycine receptors. These findings support a role for glycine as an inhibitory neurotransmitter in amacrine cells.

This paper describes changes in the appearance and distribution of microglia in postnatal cat retina as demonstrated by a new antibody, H386F. This fractionated IgM antibody was created via an intrasplenic immunization of a single BALB/C mouse with about 2–3 × 105 large, whole cells isolated from 46 minced cat retinae. To confirm that the labeled cells are microglia, the staining properties of H386F were compared with those of four commercially available antibodies, OX-33, OX-41, OX-42, and ED-1, that have been used by others to distinguish between microglia and other cells in rat brain. These experiments show that H386F is the only antibody of the five to label only microglia in both the cat retina and hippocampus.

A fundamental anatomical feature of retinal neurons is that they form planar mosaics. Each mosaic can be described by its density, pattern, and regularity (non-randomness). As part of ongoing studies to quantitatively describe the anatomy of regenerated retina in the goldfish, we determined the planimetric density and regularity of the mosaic of dopaminergic interplexiform cells in patches of regenerated retina and compared this to the mosaic generated de novo. In addition, we selectively ablated dopaminergic neurons with the neurotoxin 6–hydroxydopamine (6–OHDA) before inducing local regeneration and determined whether or not the absence of the extant dopaminergic neurons modulated the planimetric density or number of regenerated ones. The results showed that dopaminergic neurons are regenerated at higher planimetric densities and in less orderly arrays than normal. Furthermore, there was no statistical difference in the density or number of regenerated cells in normal retinas and retinas treated with 6–OHDA.

Microspectrophotometry and immunocytochemistry with several antivisual pigment antibodies were used to study visual cells of the Siberian sturgeon, Acipenser baeri Brandt. The retina contained rods and three morphological types of cones: large cones with oil drops, small cones with oil drops, and cone-like cells without oil drops. Rods and cone-like drop-free cells were found to possess porphyropsin-549, while the large oil drop-bearing cones contained red-sensitive (P613), green-sensitive (P542), and blue-sensitive (P462) visual pigments. The immunocytochemical staining pattern with three antibodies to visual pigment proteins also revealed one visual pigment in rods and three visual pigments in cones. Rods were labeled with all three antibodies, while the majority of large cones (type I), presumably the red-sensitive ones, were negative with the polyclonal serum AO against bovine opsin. A less-frequently occurring large cone type (type II) was stained by all three antibodies including mAb COS-1 specific to middle-to-long-wave visual pigments in birds and mammals, and is thought to be green-sensitive. An even less-frequent large cone type (type III, probably the blue-sensitive one) did not bind COS-1. The small cones with oil droplets showed immunoreactivities similar to either type II or type III cones. The oil drop-free small photoreceptor exhibited a staining pattern identical with that of rods. These results indicate that the immunocytochemical approach can be used to reveal photoreceptor-specific neural connections in the sturgeon retina.

Peroxidase-anti-peroxidase immunocytochemistry, applied on serial semithin epoxy resin sections, was used to examine the localization of endogenous GABA in horizontal cells in the retina of a marine teleost, the dragonet (Callionymus lyra L.). The immunostaining shows that not only the external H1 cone horizontal cells label with antibodies against GABA, but also the H2 and H3 cone horizontal cells in the inner nuclear layer. The distribution of the H1 cells corresponds to that of the single cones. They are square-patterned and in the dorsal retina their density equals 20,000 cells/mm2. The estimated density of the immunostained H2 and the H3 cells in the dorsal retina is 9500 and 1300 cells/mm2, respectively. The H2 and H3 cells are not geometrically arranged, but nearest-neighbor analysis shows that these horizontal cell types do have a very regular disposition. We suggest that GABA is the likely neurotransmitter substance used by all cone horizontal cell types in teleost retina.

γ-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 strata 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.

Neonatal subcutaneous administration of the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) to hamsters results in a marked depletion of serotonin (5-HT) in cortex and an increase in the concentration of this amine in the superior colliculus (SC). To determine whether this increase was associated with an alteration in the synaptic organization of 5-HT-containing axons in the superficial gray layer of the SC, immunocytochemistry was combined with electron microscopy. In normal adult hamsters, only 4.0% of 500 5-HT-immunoreactive profiles make synaptic contacts in the superficial gray layer of the hamster's SC. In 5,7-DHT-treated animals, examination of 400 individual profiles indicated that 25.5% of 5-HT-positive profiles made synaptic contacts (P < 0.05). Given the recently demonstrated effect of 5-HT on retinotectal transmission in this species, the present results suggest that the functional organization of the SC may also be markedly altered in animals that sustain neonatal 5,7-DHT administration.

Light-microscopic immunocytochemistry was used to study serotonin (5HT)-containing retinal cells in the lamprey (Lampetra fluviatilis). Observations of sections and flatmounted retinas enabled us to distinguish four principal types of 5HT-immunoreactive neurons, on the basis of the localization of their somata and the arrangement of their processes in the inner plexiform layer, (IPL). Type 1 cell bodies (9 μm mean diameter) were numerous and were found in the innermost row of the inner nuclear layer (INL). They sent their processes into a dense plexus in sublamina a of the IPL. Type 2 cell bodies (12 μm mean diameter) were observed near the inner limiting membrane, their processes forming a plexus in sublamina b of the IPL. Most of the type 3 cells were bistratified, their cell bodies (similar in dimension to type 1) were located in the INL and their dendrites projected to both plexuses. Type 4 cell bodies (15 μm mean diameter) were observed in the middle of the IPL and could be compared with the interstitial described elsewhere. Their processes probably ended in the 5HT plexus of sublamina b but because of their sinuous course in the IPL, we could not affirm this fact. Most of 5HT immunoreactive cells were thought to be amacrine cells, but the presence of some thin processes emerging either from the soma or the primary dendrite, principally in type 1 and 2 cells, raises the possibility that some ganglion cells could be 5HT immunoreactive. The organization of the 5HT processes into two plexuses located in sublaminae a and b of the IPL resemble the functional ON and OFF pathway seen in the other vertebrates.

Newborn rhesus monkeys were raised under monocular (lid suture, aphakia, aphakia corrected optically with contact lenses, and occlusion with opaque occluder lenses) and under binocular visual deprivation conditions (aphakia combined with occlusion or optical undercorrection of the fellow eye). Routine immunohistochemical methods with an antibody to the calcium-binding protein parvalbumin (PV) were used to examine the distribution of PV+ neurons and PV+ processes in the LGN of these monkeys. Under all rearing conditions, we found no obvious difference in PV density in neurons in any lamina, although in all monocularly deprived and in two of the three binocularly deprived monkeys neurons connected to the deprived eye were of reduced size. Furthermore, PV-immunoreactive processes in the neuropil of deprived laminae were as numerous and of the same morphologies as those in nondeprived laminae or as in normal controls. Thus, in the LGN of rhesus monkeys, the calcium-binding protein parvalbumin is resistant to monocular as well as binocular visual deprivation during the postnatal maturation process of the visual system.

The present study was performed as part of a systematic examination of glycine's coexistence with other classical transmitters and neuropeptides in neuronal populations of the larval tiger salamander retina. Substance P immunocytochemistry was combined with either glycine immunocytochemistry or autoradiography of glycine high-affinity uptake to examine whether tiger salamander substance P-amacrine cells express these glycine markers. Double-label analyses revealed two populations of substance P-amacrine cells that express glycine immunoreactivity and glycine high-affinity uptake. The large majority of double-labeled cells were situated in the innermost cell row of the inner nuclear layer, while a smaller number were located in the inner nuclear layer in the second cell row distal to the inner plexiform layer. Double-label immunocytochemistry revealed that these double-labeled cells accounted for 91.7% of substance P-immunoreactive amacrine cells. A slightly lower percentage (90.1%) of substance P-amacrine cells were found to exhibit a glycine high-affinity uptake mechanism. Substance P-amacrine cells that did not co-label for markers of glycine activity were situated in the innermost cell row of the inner nuclear layer. Substance P-immunoreactive displaced amacrine cells were not observed to co-label for either glycine immunoreactivity or glycine high-affinity uptake.

The present study reveals that the large majority of substance P-amacrine cells in the larval tiger salamander retina co-express markers of glycine activity. This finding suggests a functional diversity in the population of tiger salamander substance P-amacrine cells relative to their coexisting relationship with a major inhibitory neurotransmitter.

Two physiologically distinct, HRP-marked turtle retinal ganglion cells were examined for their morphology, GABAergic, glycinergic, and bipolar cell synaptic inputs, using electron-microscopic autoradiography and postembedding immunocytochemistry. One cell was a color-opponent, transient ON/OFF ganglion cell. Its center response to red was a sustained hyperpolarization, and its center response to green was a depolarization with increased spiking at onset. The HRP-injected cell most resembled G6, from previous Golgi-impregnation studies (Kolb, 1982; Kolb et al., 1988). It was a narrow-field bistratified cell, whose two broad dendritic strata peaked at approximately levels L20–25 (sublamina a) and L60 (sublamina b) of the inner plexiform layer. Bipolar cell synapses onto G6 were found evenly distributed between its distal and proximal dendritic strata, spanning L20–75. These inputs probably originated from several different bipolar cells, reflecting the complexity of the center response. GABAergic inputs were found onto both the distal and proximal strata, from near L20–L85. Only a few glycinergic inputs, confined to dendrites at L50–70, were observed.

A second ganglion cell type that we physiologically characterized and HRP-injected had sustained ON-center, sustained OFF-surround responses. Two examples were studied; both were bistratified in sublamina b, near L60–70 and L85–100, with branches up to near L40. They resembled G10, from previous Golgi-impregnation studies (Kolb, 1982; Kolb et al., 1988). One cell was partially reconstructed to look at the distributions of GABAergic and glycinergic amacrine cell, and bipolar cell inputs. Although synapses from bipolar cells were equally divided between the two major dendritic strata of G10, the inputs to the distal stratum were close to the soma, and the inputs to the more proximal stratum were on the peripheral dendrites. This arrangement may reflect input from two distinct types of ON-bipolar cell. GABAergic and glycinergic inputs to G10 costratified to both strata and to the distal branches; but where glycinergic inputs were found distributed throughout the arbor, GABAergic inputs appeared to be confined to peripheral dendrites. We hypothesize on the neural elements involved and the circuitry that may underlie the physiologically recorded receptive fields of these two very different ganglion cell types in the turtle retina.