Rapid activity-dependent sprouting of optic fibers into a local area denervated by application of beta-bungarotoxin in goldfish tectum

The retinotectal projection is known to be capable of extensive long-term expansion of connections, but it is not known how fast such changes can occur or what triggers sprouting of terminals. We studied sprouting of optic fibers into an area denervated by local microinjection of beta-bungarotoxin (β-BTX), a specific presynaptic neurotoxin with phospholipase A₂ activity that destroys nerve terminals at the neuromuscular junction. After injection of 0.1 pmol of β-BTX, the optic terminals fired spontaneously with decreasing amplitude and became silent within 1 to 2 h. Outside the injection zone, the retinotectal map was normal, so the silent zone was associated with a scotoma in the visual field. Horseradish peroxidase (HRP) staining of the entire optic nerve showed a denervated region at the injection site with beaded, degenerating fibers at its edge. Between 3 and 9 days later, optic units were recorded within the injection zone whose receptive fields lay just outside the scotoma in the visual field, indicating that intact surrounding terminals had sprouted into the area. These sprouts made functional connections, as indicated by field potential recordings and current source-density analysis. At this time, HRP staining also demonstrated retinal innervation within the injection zone. By 12 days, normal maps with no scotoma were recorded and HRP staining was normal at the injection site, indicating that the β-BTX-damaged fibers had regenerated to reclaim their tectal sites. The results show that the retinotectal projection of goldfish is very dynamic, since intact optic fibers can sprout into adjacent vacant postsynaptic territory within 2 to 3 days, much faster than previously reported. In a final experiment, we showed that this sprouting is activity-dependent, since it could be prevented by blocking retinal activity with intraocular tetrodotoxin (TTX) during the first 2 days postinjection, even though TTX block of activity does not block regeneration in this system. One possible mechanism for this rapidly triggered sprouting is that arachidonic acid liberated by β-BTX acts as a sprouting factor to attract surrounding healthy fibers into the denervated region but requires activity at the terminals to be effective. © 1996 John Wiley & Sons, Inc.     

Retinofugal and retinopetal projections in the teleost Channa micropeltes (Channiformes)

Summary Retinofugal and retinopetal projections were investigated in the teleost fish Channa micropeltes (Channiformes) by means of the cobaltous lysine and horseradish peroxidase (HRP) tracing techniques. Retinofugal fibers cross completely in the optic chiasma. A conspicious lamination is present in those parts of the optic tract that give rise to the marginal branches of the optic tract. This layering of optic fibers continues in the marginal branches to mesencephalic levels. Retinal projections to the preoptic and hypothalamic regions are sparse; they are more pronounced in the area of pretectal nuclei. The medial pretectal complex and the cortical pretectal nucleus are more fully differentiated than in other teleostean species. Further targets include the thalamus and the optic tectum. The course of major optic sub-tracts and smaller fascicles is described. Retinopetal neurons are located contralaterally in a rostral and a caudal part of the nucleus olfactoretinalis, and in a circumscribed nucleus thalamoretinalis. The present findings are compared with reports on other teleost species.     

Transneuronal uptake of horseradish peroxidase in the central nervous system of dipterous insects

Summary Horseradish peroxidase (HRP) applied to lesioned neurons in the retina and thoracic ganglia of the flies Musca, Calliphora and Drosophila labeled axon terminals, dendrites and perikarya of the severed neurons after anterograde or retrograde passage. In addition, HRP reaction product secondarily labeled intact neurons that are contiguous with injured nerve cells. In many cases labeling of optic lobe neurons remote from primarily filled ones was also seen (here called tertiary labeling). HRP labeling was extensive and both primarily and transneuronally filled neurons could be resolved in almost as much detail as Golgi-impregnated or cobalt-silver-labeled cells. Electron microscopy showed that in both primarily and secondarily filled neurons, reaction product was distributed diffusely in the cytoplasm.Transneuronal uptake of HRP was specific to certain types of neurons in the brain and thus displayed certain pathways. The pathways resolved by transneuronal labeling with HRP extend from the optic lobes to the thoracic ganglia and include visual neurons previously identified electrophysiologically and anatomically.Transneuronal HRP uptake, although believed to occur in vivo, could not be shown to be dependent on synaptic activity. Three other heme peptides tested were taken up by injured neurons, but showed no transneuronal labeling: lactoperoxidase, cytochrome c, and microperoxidase.     

Cellular localization of thiol-proteinase inhibitor in the epidermis of the newborn rat

Summary In cichlid, poecilid and centrarchid fishes luteinizing hormone releasing hormone (LHRH)-immunoreactive neurons are found in a cell group (nucleus olfactoretinalis) located at the transition between the ventral telencephalon and olfactory bulb. Processes of these neurons project to the contralateral retina, traveling along the border between the internal plexiform and internal nuclear layer, and probably terminating on amacrine or bipolar cells. Horseradish peroxidase (HRP) injected into the eye or optic nerve is transported retrogradely in the optic nerve to the contralateral nucleus olfactoretinalis where neuronal perikarya are labeled. Labeled processes leave this nucleus in a rostral direction and terminate in the olfactory bulb. The nucleus olfactoretinalis is present only in fishes, such as cichlids, poecilids and centrarchids, in which the olfactory bulbs border directly the telencephalic hemispheres. In cyprinid, silurid and notopterid fishes, in which the olfactory bulbs lie beneath the olfactory epithelium and are connected to the telencephalon via olfactory stalks, the nucleus olfactoretinalis or a comparable arrangement of LHRH-immunoreactive neurons is lacking. After retrograde transport of HRP in the optic nerve of these fishes no labeling of neurons in the telencephalon occurred. It is proposed that the nucleus olfactoretinalis anatomically and functionally interconnects and integrates parts of the olfactory and optic systems.     

Retinal projections in the catfish,Mystus vittatus (Bloch) as revealed by tracer studies with horseradish peroxidase

Summary The retinal efferents of the catfish, Mystus vittatus, were investigated with the use of the horseradish peroxidase (HRP) technique. Most retinal fibres extended contralateral to the eye that had received HRP label, while a few fascicles projected to the ipsilateral side without decussation in the optic chiasma. The contralateral fibres projected to the suprachiasmatic nucleus, the nucleus opticus dorsolateralis, the nucleus of the posterior commissure, the nucleus geniculatus lateralis, pretectal nuclear complex, and to two layers of the optic tectum, i.e., stratum fibrosum et griseum superficiale and stratum griseum centrale. The accessory optic tract arose from the inner area of the optic tract and extended ventromedially to the accessory optic nucleus. The ipsilateral fascicles projected to almost all the above mentioned nuclei, but these projections were comparatively sparse. The ipsilateral retinal projection was restricted to the rostral tectum.     

Dense HRP filling in pre-fixed brain tissue for light and electron microscopy

The use of neuroanatomical markers in tissues that have been pre-fixed has been virtually ignored, even though this approach could offer certain advantages over in vivo methods, in terms of convenience of application and choice of markers. We have found that HRP can be used on well-fixed brains of cats and goldfish to fill neurons, dendrites, axons, terminals, glial cells, and glial processes for high-resolution light microscopy and electron microscopy. Best results were obtained using brains that were perfusion-fixed with 2.5% depolymerized paraformaldehyde and 1.5% glutaraldehyde. Two methods of HRP application were used: optically guided injections of microliter quantities into various regions of cat brain, and optic nerve fills in goldfish by attaching an HRP-filled polyethylene tube for periods of 1 day to 2 weeks. HRP applied in these ways to pre-fixed tissue was found to fill neurons or glial cells with solid label in the anterograde and retrograde directions.     

Transport of pinocytic vesicles in the eye of a snail,Helix aspersa

The heads of small adult snails, Helix aspersa, were injected with horseradish peroxidase (HRP) for one to five hours before extirpating the eyes and preparing them cytochemically for electron microscopy. There was internalization of tracer by pinocytic vesicles (pinosomes) at the bases of types-I and -II sensory cells, ganglion cells and, in lesser amounts, by pigmented supportive cells. Vesicles and vacuoles filled with HRP were transported in two directions: lensward as far distad as the ends of the cells (retrograde) and brainward down the optic nerve (anterograde). We believe that the numerous reacted vacuoles in the cell somata are formed by fusion of vesicles, tubules and C-shaped organelles filled with tracer; we present evidence that they become secondary lysosomes. Sensory cell type II possesses more HRP-reacted vacuoles distally than the other retinal cells. Other vesicles are also described. There was no uptake of tracer by the distal ends of the retinal cells following injection HRP into the hemolymph. The swelling of the optic nerve, immediately behind the eye, contains more HRP-filled pinosomes and vacuoles than does the nerve below the dilatation. The significance of endocytosis and transport of pinosomes within the eye and down the optic nerve is discussed.     

Retinal projections in the caecilian Ichthyophis kohtaoensis (Amphibia,Gymnophiona)

Summary The retinal projections of the caecilian Ichthyophis kohtaoensis were investigated by anterograde transport of HRP. The optic tract forms two bundles in the diencephalon, a narrow medial bundle in the optic tectum, and a basal optic tract consisting of few fibres. Terminal fields are in the thalamus, pretectum, tectum, and as a circum-scribed basal optic neuropile in the tegmentum. Thalamic, pretectal and tectal projections are contralateral as well as ipsilateral. The reduced but existing visual projection corresponds to a reduced but existing visually guided behaviour.     

Superior sensitivity of conjugates of horseradish peroxidase with wheat germ agglutinin for studies of retrograde axonal transport.

We have compared the retrograde axonal transport of horseradish peroxidase (HRP), to the retrograde transport of HRP conjugated with wheat germ agglutinin (WGA). Morphometric studies have shown that WGA-HRP conjugates were 40 times more sensitive than free HRP, in the tracing of retrograde connections from the rat submandibular gland to the superior cervical ganglion. Also, WGA-HRP was more sensitive than free HRP in the tracing of retrograde connections from the rat tongue to the hypoglossal nucleus. Our findings with WGA-HRP are consistent with the observations by Schwab et al. who reported (-125I) WGA is a highly sensitive retrograde tracer (Brain Research 152:145, 1978 (22)).     

Establishment of neuronal connectivity during development of the Drosophila larval visual system

We used confocal microscopy in conjunction with specific antibodies and enhancer trap strains to investigate the development of specific neuronal connections in a simple model system, the larval visual system of Drosophila. We find that the establishment of axonal projections from the larval photoreceptor neurons to their central nervous system targets involves a series of discrete steps. During embryogenesis, the larval optic nerve contacts several different cell types, including optic lobe pioneer (OLP) neurons and a number of glial cells. We demonstrate that OLP neurons are present and project normally in glass (gl) mutant embryos in which the larval optic nerve fails to develop, suggesting that they do not depend on interactions with the larval optic nerve for differentiation and proper axonal projection. The OLPs fail to differentiate properly in disconnected (disco) mutant embryos, where appropriate connections between the larval optic nerve and its targets in the brain are not formed. The disco gene is expressed in the OLPs and may therefore act autonomously to direct the differentiation of these cells. Taken together, our results suggest that the OLPs act as an intermediate target required for the establishment of normal optic nerve projection and connectivity. © 1995 John Wiley & Sons, Inc.     

Cloning and expression analysis of a muscarinic cholinergic receptor from the brain of ant,Polyrhachis vicina

Muscarinic acetylcholine receptors (mAchRs) are the predominant cholinergic receptors in the central and peripheral nervous systems of animals. They also have been found in various insect nervous systems. In this article, a full‐length cDNA of a pupative mAchR (PmAchR) was obtained from the brains of ant Polyrhachis vicina by homology cloning in combination with rapid amplification of cDNA ends. PmAchR encodes a 599‐amino acid protein that exhibits a high degree of homology with other mAchRs. Real‐time quantitative RT‐PCR analysis showed that PmAchR is differentially expressed in the brains of workers, males, and females. By in situ hybridization, it is revealed that PmAchR is widely expressed in different soma clusters of the brain, including the mushroom bodies, the antennal lobes, as well as the optic lobes (OL), and the most intensely staining is found in Kenyon cells. Nonetheless, there are more positive nerve fibers in the OL of males' brains than in females' and workers' brains. © 2011 Wiley Periodicals, Inc.     

The Binding of Bispecific Monoclonal Antibodies to the Solid Phase-Adsorbed Antigens

The ability of bispecific antibodies (Babs) formed by fusion of hybridomas and parent monoclonal antibodies (Mabs) to interact with the solid phase-adsorbed antigens was studied. Mabs specific to the three different antigens [horseradish peroxidase (HRP), human IgG (hIgG), and human myoglobin (Mb)] as well as Babs with the double specificity [antimyoglobin/antiperoxidase (anti-Mb/HRP) and anti-human IgG/antiperoxidase (anti-hIgG/HRP)] were used. It was shown by radioimmunological and immunoenzyme assays that parent Mabs bind to solid phase-adsorbed antigens considerably more effectively than Babs. The observed equilibrium binding constant (K _a) of antiperoxidase parental Mabs to immobilized HRP is 21 and 38 times higher than K _afor Babs binding sites (anti-Mb/HRP and anti-hIgG/HRP, respectively) to peroxidase. It was calculated that about 90–95% of all bound parental antiperoxidase Mabs were associated with immobilized HRP bivalently, and only about 5–10% were bound monovalently. On the contrary, parental Mabs against hIgG bind to the sorbed antigen essentially only monovalently. It was also shown that the avidity of anti-Mb/HRP Babs significantly increased when two antigens, Mb and HRP, were simultaneously adsorbed on the solid phase. These data imply that Babs bearing an enzyme-binding site (for example, binding to HRP) cannot be more effective than standard conjugates (e.g., enzyme-conjugated antibodies) in heterogeneous noncompetitive immunoassays.     

Feeding on poplar leaves by caterpillars potentiates foliar peroxidase action in their guts and increases plant resistance

Peroxidases (PODs) are believed to act as induced and constitutive defenses in plants against leaf-feeding insects. However, little work has examined the mode of action of PODs against insects. Putative mechanisms include the production of potentially antinutritive and/or toxic semiquinone free radicals and quinones (from the oxidation of phenolics), as well as increased leaf toughness. In this study, transgenic hybrid poplar saplings (Populus tremula × Populus alba) overexpressing horseradish peroxidase (HRP) were produced to examine the impact of elevated HRP levels on the performance and gut biochemistry of Lymantria dispar caterpillars. HRP-overexpressing poplars were more resistant to L. dispar than wild-type (WT) poplars when the level of a phenolic substrate of HRP (chlorogenic acid) was increased, but only when leaves had prior feeding damage. Damaged (induced) leaves produced increased amounts of hydrogen peroxide, which was used by HRP to increase the production of semiquinone radicals in the midguts of larvae. The decreased growth rates of larvae that fed on induced HRP-overexpressing poplars resulted from post-ingestive mechanisms, consistent with the action of HRP in their midguts. The toughness of HRP-overexpressing leaves was not significantly greater than that of WT leaves, whether or not they were induced. When leaves were coated with ellagitannins, induced HRP leaves also produced elevated levels of semiquinone radicals in the midgut. Decreased larval performance on induced HRP leaves in this case was due to post-ingestive mechanisms as well as decreased consumption. The results of this study provide the first demonstration that a POD is able to oxidize phenolics within an insect herbivore’s gut, and further clarifies the chemical conditions that must be present for PODs to function as antiherbivore defenses.     

Small lesions in the primary visual cortex of rats cause a specific reorganization of associational connections

Summary Neuroplastic changes in associational connections were investigated 3 weeks after the intrinsic organization of the visual cortex of rats had been partially damaged by small cylindrical lesions (type I). These lesions caused the degeneration of short intracortical connections and associational connections that form patches in the primary and secondary visual areas. The resulting terminal degeneration disappeared within 20 days p.o. after which only some fiber degeneration was evident in the infragranular layers.Patches of terminal degeneration reappeared in the vicinity of the stab wounds, when the associational connections between the retrosplenial and the primary visual cortex had been secondarily interrupted by elongated lesions (type II), which penetrated the paramedian cortex and subcortical white matter. When type-II lesions were made in the intact cortex, patches of degeneration were absent, although in both cases some terminal degeneration was diffusely distributed in the primary visual cortex.Horseradish peroxidase (HRP) was applied to sites similar to those where type-I lesions were applied. In the intact cortex, HRP caused a granular labeling of numerous neurons in various positions including the retrosplenial cortex and patches of the postero-median visual cortex. HRP was also applied to type-I lesions that had been made 3 weeks earlier. In these cases, apparently HRP labeled the same subpopulations of neurons as it did in the intact cortex. However, a fraction of the labeled neurons showed a Golgilike staining (e.g., 27% of the labeled neurons in the retrosplenial cortex) only when HRP was applied to stab wounds.These results suggest that the breakdown of corticocortical connections in foci of the primary visual cortex causes a focal augmentation of specific associational connections, which are weak and diffusely distributed in the intact adult cortex of rats. Re-innervation originates from subpopulations of associative neurons in the retrosplenial and postero-median visual cortex. Preliminary experiments indicate that the failure of neonatal treatment with 6-OHDA to suppress this lesion-induced plasticity is not dependent on an intact noradrenergic innervation.     

Morphology of the larval and adult brains of the monarch butterfly, Danaus plexippus plexippus, L

Cell population and neuropile morphology of larval and adult brains of the monarch butterfly, Danaus plexippus plexippus, L., are compared. The larval brain is in continuous transition, the processes of adult brain development being underway from the earliest larval stages. It is characterized by a less diverse population of cells and more homogenous fiber areas than those of the adult. Neuroblasts, which divide to form the neurones of the adult brain, occur either in discrete proliferation centers or scattered among the larval ganglion cells. The larval brain contains, in addition to small homogeneous antennal centers and a distinct larval optic center, rapidly developing adult optic centers, corpora pedunculata, and protocerebral bridge. The larval brain lacks a central body. Major differences between larval and adult brains are clearly related to the increased dependence of the adult upon sensory input from the eyes and antennae.     

Cholinergic and GABAergic neuronal elements in the pineal organ of lampreys, and tract-tracing observations of differential connections of pinealofugal neurons

The putative cholinergic and GABAergic elements of the pineal organ of lampreys were investigated with immunocytochemistry to choline acetyltransferase (ChAT) and γ-aminobutyric acid (GABA), and by acetylcholinesterase (AChE) histochemistry. For comparison we also carried out immunocytochemistry to serotonin (5-HT) and a tract-tracing investigation of the two types of projecting cells, i.e., ganglion cells and long-axon photoreceptors. Most photoreceptors were ChAT-immunoreactive (ChAT-ir) and AChE-positive, while ganglion cells and the pineal tract were ChAT-negative and AChE-negative or only faintly positive. These results strongly suggest the presence of a cholinergic system of photoreceptors in the lamprey pineal organ. GABA-ir fibers that appear to originate from faintly to moderately stained ganglion cells were observed in the pineal stalk. Immunocytochemistry to 5-HT indicated the presence of two types of 5-HT-ir cells, bipolar cells and ganglion-like cells. The connections of the ganglion cells and long-axon photoreceptors were also studied by application of DiI to the pineal stalk in fixed brains or of biotinylated dextran amine (BDA) to one of the main targets of pinealofugal fibers (optic tectum or mesencephalic tegmentum) in isolated brains in vitro. Some long-axon photoreceptors and ganglion cells were labeled from the optic tectum. However, BDA application to the tegmentum exclusively labeled ganglion cells in the pineal organ. These results indicate that the two morphological types of afferent pineal neuron have different projections. No labeled cells were observed in the parapineal organ in BDA experiments, indicating that this organ and the pineal organ are involved in different neural circuits.     

The origin of central pinealopetal nerve fibers in the Mongolian gerbil as demonstrated by the retrograde transport of horseradish peroxidase

The location of perikarya and nerve fibers projecting via the habenular and posterior commissures from the brain into the pineal organ of the Mongolian gerbil was investigated by the use of the retrograde horseradish peroxidase (HRP)-tracing method. After microiontophoretic or hydraulic injection of the tracer into the superficial pineal gland via a glass micropipette, and after survival periods of 6 to 48 h, the animals were transcardially perfused and the brains processed for the histochemical demonstration of the enzyme. In the pineal stalk 15 to 20 nerve fibers, including 4 to 7 myelinated elements, were traced back to the brain. HRP-labeled perikarya were located in the medial and lateral habenular nuclei as well as in the nucleus of the posterior commissure. Few fibers projected rostrally to perikarya in the paraventricular nucleus of the hypothalamus. A striking and persistent finding was the labeling of fibers that, in the habenular area, bent laterad and continued ventral to the optic tract. These fibers originated from perikarya located in the dorsal nucleus of the lateral geniculate body. These results strongly suggest a central innervation of the pineal organ in the Mongolian gerbil originating from hypothalamic and limbic areas of the brain as well as from the optic system.     

Visualization of the retino-hypothalamic projection in the rat by cobalt precipitation

Summary The technique of cobalt sulfide precipitation combined with Timm's sulfide-silver method for intensification of heavy metals was used to delineate the retino-hypothalamic projection of the rat. Freshly isolated rat brains were dissected and a solution of cobaltous chloride was applied to one of the cut optic nerves. Sixteen hours later, after cobalt ions had passed into the brain along the entire length of the optic fibers, the preparation was treated with ammonium sulfide to precipitate the cobalt as cobalt sulfide. In thick light microscopic sections, cobalt-filled axons were visualized as black fibers against a light gold background. Such fibers were observed to leave the posterior medial portion of the optic chiasm and, after arching dorsally, to project into the posterior fifth of the suprachiasmatic nucleus (SCN), as well as into the rostral part of the arcuate nucleus. Neither bifurcation of these axons nor looping of the axons back to the chiasm was seen. Most fibers projected to the SCN contralateral to the filled nerve, but the projection represented less than 0.1 % of the total number of fibers in one optic nerve. These observations are considered to be graphic evidence of a retino-hypothalamic projection. The interpretation of the cobalt method is discussed, as are the functions of the connections that have been observed.This work was supported by the Nuffield FoundationWe are grateful to Mr. Clifford Jeal of the Department of Pathology for excellent advice on photomicrography     

The ultrastructure of somatic neuromuscular junctions in the tick Amblyomma variegatum (Fabr.)

Summary Injections of large doses of horseradish peroxidase (HRP) into the telencephalon of the squirrel fish (Holocentrus rufus) revealed the first anatomical evidence for a visual thalamo-telencephalic projection in a teleost. The central optic nucleus of the thalamus appears to be the only visual thalamic nucleus projecting to the telencephalon in this species. Since the central optic nucleus has a large tectal input but not a direct one from the retina, it is suggested that a retino-geniculo-telencephalic pathway does not exist in this species. Acknowledgements. The author is grateful to Drs. J. Maldonado, Dietrich Meyer and Henning Scheich for encouragement and support in this endeavor. The study was supported by: National Institutes of Health grant EY-02014 and EY-03264, a NIH grant to Dr. José del Castillo and the German Science Foundation (SFB 45)