A Light Insensitive Method for Contrast Enhancement of Insect Neurons Filled with a Cobalt-Lysine Complex

Modified protocols for cobalt-filling and silver intensification of neurons in the larval and adult stages of the moth, Manduca sexta, have led to improved neuronal visualization and minimal background staining. In particular, long distance projecting multisegmental in-terneurons. originating in the pterothoracic or terminal abdominal ganglion, were best visualized when a cobalt:lysine complex was used to fill hemi-connectives for several days at 4 C. Ganglia closest to the placement of tracer, which became flooded with cobalt:lysine during the filling period. were removed from the insect. This step eliminated the artifactual filling of neurons that may have taken up the tracer from such pooled regions. This led to a more accurate assessment of whether a multisegmental interneuron projected through the full length of nerve cord to the original site of tracer placement. The protocol for light insensitive silver intensification of cobalt-filled neurons was modified to include an important pH adjustment. NaOH was used to alter the pH of the protective colloid, sodium tungstate, to 10.4 or greater in solution. Especially in larvae. our techniques produced intensely stained cobalt-filled neurons within ganglia that remained transparent and relatively free of nonspecific silver deposition.     

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     

A Light Insensitive Method for Contrast Enhancement of Insect Neurons Filled with a Cobalt-Lysine Complex

Modified protocols for cobalt-filling and silver intensification of neurons in the larval and adult stages of the moth, Manduca sexta, have led to improved neuronal visualization and minimal background staining. In particular, long distance projecting multisegmental in-terneurons. originating in the pterothoracic or terminal abdominal ganglion, were best visualized when a cobalt:lysine complex was used to fill hemi-connectives for several days at 4 C. Ganglia closest to the placement of tracer, which became flooded with cobalt:lysine during the filling period. were removed from the insect. This step eliminated the artifactual filling of neurons that may have taken up the tracer from such pooled regions. This led to a more accurate assessment of whether a multisegmental interneuron projected through the full length of nerve cord to the original site of tracer placement. The protocol for light insensitive silver intensification of cobalt-filled neurons was modified to include an important pH adjustment. NaOH was used to alter the pH of the protective colloid, sodium tungstate, to 10.4 or greater in solution. Especially in larvae. our techniques produced intensely stained cobalt-filled neurons within ganglia that remained transparent and relatively free of nonspecific silver deposition.     

昆虫神经元染色的银增感法

NiCl2逆行染色是观察昆虫神经元结构的一个重要方法。这一方法不足之处是使一些神经末梢的分支不清晰,但可以通过神经元染色后的银增感方法来弥补。作者借对双斑蟋蟀Gryllus bimaculatusde Geet产卵瓣内的感觉神经元染色后的银增感介绍了这一方法,包括材料的解剖、染色、固定、增感、脱水、透明等。     

Distribution of subgroups of noradrenaline neurons in the coeliac ganglion of the guinea-pig

Summary The distributions within the coeliac ganglion of different chemically coded subgroups of noradrenaline neurons, and the relationships between these neurons and nerve fibres projecting to the ganglion from the intestine, have been assessed quantitatively by use of an immunohistochemical double-staining method. Noradrenaline (NA) neurons made up 99% of all cell bodies. Of these, 21% were also reactive for somatostatin (NA/SOM neurons), 53% were also reactive for NPY (NA/NPY neurons), and 26% were not reactive for either peptide. NA neurons without reactivity for any of the peptides whose localization was tested have been designated NA/-. A small percentage, about 1%, of neurons were reactive for both NPY and SOM. The three major types of NA neurons were arranged in clumps or ribbons throughout the ganglia, with a tendency for NA/SOM neurons to be medial and NA/NPY neurons to be lateral in the ganglia. A small group of neurons (<1%) encoded with dynorphin, NPY and vasoactive intestinal peptide (VIP) was encountered. VIP-immunoreactive nerve terminals, projecting to the ganglion from cell bodies in the intestine, ended around NA/SOM and NA/neurons but not around NA/NPY neurons. Thus, the VIP axons from the intestine end selectively around neurons that modify intestinal function (NA/SOM and NA/-neurons) but not around neurons, the terminals of which supply blood vessels (NA/NPY neurons).     

Brain phagocytes may empty tissue debris into capillaries

The possibility that brain phagocytes may empty remnants of degenerated neurons into capillaries has been studied in frogs. Degeneration of nerve fibers was brought about by transectioning the optic tract, the tectothalamic and tectoisthmic tracts, the postoptic commissure or the radial nerve. To help identification of phagocytozed degenerated neuronal elements, the transected fibers were filled either with horseradish peroxidase (HRP) or cobaltous-lysine complex. The survival times were 3, 4, 7, 27, 47 and 70 days after the application of the markers. The HRP-labeled structures were identified in 60 μm thick sections using diaminobenzidine as chromogen, while cobalt was precipitated in the form of cobaltous sulfide. Small pieces of these sections were further processed for electron microscopy. In each area of the brain and spinal cord investigated, microglial cells and astrocytic processes containing fragments of degenerated neuronal elements could be seen close to capillaries. In some cases a microglial or astrocytic process pierced the capillary basal lamina and seemingly delivered inclusion bodies into the cytoplasm of capillary endothelial cells and pericytes. In the inclusion bodies, which were usually large vesicles, fragments of HRP or cobalt-labeled or unlabeled membranes with a foamy appearance, or condensed myelin lamellae could be observed. These vesicles protruded the luminal membrane of the endothelial cell that was disrupted in some cases suggesting that the content of the inclusion body was discharged into the lumen of the capillary. These results give support to Penfield's hypothesis (1925) that glial cells may empty phagocytozed materials into capillaries.     

Metamorphosis of wing motor system in the silk moth,Bombyx mori L. (Lepidoptera: Bombycidae): Anatomy of the sensory and motor neurons that innervate larval mesothoracic dorsal musculature,stretch receptors,and epidermis

Anatomy of dorsal mesothoracic structures, such as muscles, sensory organs, and innervation, was studied in the silkworm, Bombyx mori L. (Lepidoptera : Bombycidae), and compared with the adult wing motor system. Musculature and nerve innervation were investigated by dissection and electron micrograph; and central projection of sensory fibers and morphology of somata and dendrites of motor neurons by cobalt back-filling, followed by silver intensification. There are 23 muscle bundles (DLM) and 2 stretch receptors (SR). The DLMs, SRs, and epidermis are innervated by a branch of the dorsal nerve trunk emerging from the mesothoracic ganglion (MSG). The branch bifurcates into a dorsal sensory branch of about 300 sensory fibers and a dorsal motor branch of 14 fibers. The sensory fibers project mainly to a longitudinal portion near the mid line in the ventral neuropil of MSG and the metathoracic ganglion. Several fibers extend into the prothoracic ganglion (PG) and a few into the subesophageal and 1st abdominal ganglia. At least 13 (probably 14) motor neurons send axons to DLMs: 9 (probably 10) in PG, and 4 in MSG. Their dendrites are located mostly on the dorsoipsilateral side of the neuropil, but several branches cross the mid line and give rise to many fine branches on the contralateral side. Comparison between the larval (present study) and adult motor system shows a significant similarity in the musculature, peripheral nerve pattern, and motor neurons with some peculiarities.     

Substance P dans les neurones sensitifs primaires innervant la pulpe dentaire,chez le cobaye

Primary sensory trigeminal neurons supplying the dental pulp of incisors in guinea pigs were labelled by retrograde axonal transport. Using an autometallographic intensification procedure, 48 h after injection of wheat germ agglutinin/colloidal gold in the pulp, gold particles were detected in the cytoplasm of the neurons as black granulations. A morphometric study showed a bimodal repartition of the labelled neurons of the ganglion. By submitting ganglion slices to an anti-substance P immunserum revealed by immunocytochemistry, it could be observed that, among the neurons supplying the dental pulp of incisors, the majority of the largest were substance P immunopositive while the smallest were substance P immunonegative. These observations suggest that there could be at least two different populations of nerve fibres supplying the guinea pig incisor dental pulp. Substance P negative neurons could express different neurotransmitters.     

Modified cobalt staining and silver intensification techniques for use with whole-mount gastropod ganglion preparations

While silver intensification of cobalt-filled cells is a common procedure for use with many arthropod preparations, it has not been routinely applied to gastropods. Several modifications in cobalt-staining techniques currently used with gastropods along with adaptations of silver intensification techniques used in insects are described. Cobalt was introduced into cells through axonal filling of cut nerve trunks or by either pressure injection or iontophoresis from intracellular, micropipette electrodes that had previously had their tips etched in dilute hydrofluoric acid. Etching produced consistent, sharp tips with large lumina. Further procedural modifications allowed complete, even intensification of neurons in large gastropod ganglia. These techniques have proved to be reliable and apparently broadly applicable, having been successfully used on three diverse gastropod species.     

Lateral ocellar nerve projections in the dragonfly brain

Summary The central projections of the lateral ocellar neurons of the dragonfly were examined using whole nerve cobalt iontophoresis, supplemented by sectioning of the nerve and brain for inspection in the light and electron microscopes. At E.M. level the presence of cobalt in filled axon profiles and cell bodies was confirmed by analysis of X-ray energy spectra in the microscope.The pathways, cell body sites and terminal arborizations of four large (7–25 m diameter) lateral ocellar neurons are described. Two of these fibers arborize in the ipsilateral posterior neuropil of the protocerebrum and two cross the brain and arborize in the contralateral posterior neuropil. Within each half of the posterior neuropil, two spatially separated regions of ocellar input have been identified. These regions receive median ocellar input plus input from either the ipsi- or contralateral ocellus, but not both. The arborizations of the contralateral fibers are more extensive than those of the ipsilateral fibers.One of the contralateral neurons crosses the brain in the region of the protocerebral bridge giving off a collateral in that region before descending to the posterior neuropil. This collateral arborizes almost immediately in a region receiving input from arborizations of a number of small ocellar neurons (those less than 5 m in diameter) from the ipsilateral ocellar nerve, together with small neurons from the median ocellar nerve, forming a region in each half of the brain which receives input from all three ocelli. The small lateral ocellar neurons associated with these arborizations have cell bodies adjacent to the lateral ocellar tracts.This work was supported in part by National Institute of Health Grants 2 RO1 EY-00777 and 1 KO4 EY-00040     

Cobalt mapping of the nervous system: how to avoid artifacts

Infusion of cobalt ions into cut axons is an established method for tracing neuron projections in the central nervous system. Artifacts, where unintended neurons are stained, however, have been reported, leading to difficulties in interpretation. Experiments in the locust Schistocerca gregaria Forsk?l show that such artifacts can be induced through damage to axons caused by cutting peripheral nerves and by using high cobalt chloride concentrations (0.4M and above). Mixtures of cobalt and nickel chlorides and nickel chloride alone were introduced into different branches of the same nerve and developed with rubeanic acid to give precipitates of different colors in the two sets of axons. Preparations were examined with the light microscope, where mixing of ions would appear as intermediate colors, and by x-ray probe microanalysis. No evidence for leakage of metal ions from the filled axons or for ion uptake by other axons could be detected, provided that low concentrations of cobalt and nickel chlorides were used and nerve cutting was reduced to a minimum by making preparations in vivo. If extreme conditions are avoided when making the preparation, the risk of producing artifacts is minimized, thus enabling the cobalt method to be used with greater confidence for describing neuronal projections.     

Metal cofactors of lysine-2,3-aminomutase.

Lysine-2,3-aminomutase from Clostridium SB4 contains iron and sulfide in equimolar amounts, as well as cobalt, zinc, and copper. The iron and sulfide apparently constitute an Fe-S cluster that is required as a cofactor of the enzyme. Although no B12 derivative can be detected, enzyme-bound cobalt is a cofactor; however, the zinc and copper bound to the enzyme do not appear to play a role in its catalytic activity. These conclusions are supported by the following facts reported in this paper. Purification of the enzyme under anaerobic conditions increases the iron and sulfide content. Lysine-2,3-aminomutase purified from cells grown in media supplemented with added CoCl2 contains higher levels of cobalt and correspondingly lower levels of zinc and copper relative to enzyme from cells grown in media not supplemented with cobalt. The specific activity of the purified enzyme increases with increasing iron and sulfide content, and it also increases with increasing cobalt and with decreasing zinc and copper content. The zinc and copper appear to occupy cobalt sites under conditions of insufficient cobalt in the growth medium, and they do not support the activity of the enzyme. The best preparations of lysine-2,3-aminomutase obtained to date exhibit a specific activity of approximately 23 units/mg of protein and contain about 12 g atoms of iron and of sulfide per mol of hexameric enzyme. These preparations also contain 3.5 g atoms of cobalt per mol, but even the best preparations contain small amounts of zinc and copper. The sum of cobalt, zinc, and copper in all preparations analyzed to date corresponds to 5.22 +/- 0.75 g atoms per mol of enzyme. An EPR spectrum of the enzyme as isolated reveals a signal corresponding to high spin Co(II) at temperatures below 20 K. The signal appears as a partially resolved 59Co octet centered at an apparent g value of 7. The 59Co hyperfine splitting (approximately 35 G) is prominent at 4.2 K. These findings show that lysine-2,3-aminomutase requires Fe-S clusters and cobalt as cofactors, in addition to the known requirement for pyridoxal 5'-phosphate and S-adenosylmethionine.     

Metamorphosis of Wing Motor System in the Silk Moth, Bombyx mori: Origin of Wing Motor Neurons

The origin of the peripheral nerve and motor neurons that innervate the adult mesothoracic dorsal longitudinal muscles (DLMs) was examined in the silk moth, Bombyx mori . The anatomical features of the peripheral nerve and motor neurons were investigated by dissection, electron microscopy, and cobalt back-fill staining at different pupal stages. These studies showed that the peripheral nerve (IIN1c) that innervates the adult DLMs originates from a branch (db branch) of the larval mesothoracic dorsal nerve that innervates the larval DLMs. During metamorphosis the larval nerve shortens or lengthens locally without change in its basic branching pattern, and the db branch moves towards the mesothoracic ganglion to become the IIN1c. All the adult DLM motor neurons are from larval ones. Nine of the 14 larval DLM motor neurons survive during metamorphosis to become adult DLM motor neurons, and 5 disappear in early pupal stages.     

Multisegmental cobalt filling of the dorsal giant fibers in the nervous system of the earthworm,Lumbricus terrestris

Summary The anatomical organization of the two dorsal giant fiber systems of the earthworm Lumbricus terrestris is demonstrated in whole mounts and serial-section reconstructions based on backfillings of the ventral nerve cord with cobalt chloride. Both the medial and lateral fiber systems can be labeled selectively over more than ten body segments. They show a characteristic segmental pattern of collaterals with some modification in tail segments and of dorsal plasma protrusions in the unpaired medial giant fiber presumably representing openings in the myelin sheath. We found no multisegmental cobalt transport in other large neurons of the nerve cord. Cobalt passes through the segmentai septa between consecutive axonal elements of the metameric giant fibers and presumably also through commissural contacts between specific collaterals of the lateral giant fibers. Since these sites of contact are known to represent electrical synapses, cobalt coupling may, in L. terrestris, correlate with functional electrotonic coupling.Abbreviations CL collateral of lateral giant fiber - CM collateral of medial giant fiber - GIN giant interneuron - LGF lateral giant fiber - MGF medial giant fiber - SN segmental nerve     

Transient expression of the calcitonin receptor by enteric neurons of the embryonic and early post-natal mouse

Calcitonin receptor-immunoreactivity (CTR-ir) was found in enteric neurons of the mouse gastrointestinal tract from embryonic day 13.5 (E13.5) to post-natal day 28 (P28). CTR-ir occurred in cell bodies in ganglia of the myenteric plexus extending from the esophagus to the colon and in nerve cells of the submucosal ganglia of the small and large intestines. CTR-ir was also found in vagal nerve trunks and mesenteric nerves. Counts in the ileal myenteric plexus revealed CTR-ir in 80% of neurons. CTR-ir was clearly evident in the cell bodies of enteric neurons by E15.5. The immunoreactivity reached maximum intensity between P1.5 and P12 but was weaker at P18 and barely detectable at P28. The receptor was detected in nerve processes in the intestine for only a brief period around E17.5, when it was present in one to two axonal processes per villus in the small intestine. In late gestation and soon after birth, CTR-ir was also evident in the mucosal epithelium. The perinatal expression of CTR within the ENS suggests that the calcitonin/CTR system may have a role in the maturation of enteric neurons. Signals may reach enteric neurons in milk, which contains high levels of calcitonin.     

Neuronal distribution and subcellular localization of HCNP-like immunoreactivity in rat small intestine

A novel peptide, hippocampal cholinergic neurostimulating peptide (HCNP), originally purified from young rat hippocampus, affects the development of specific cholinergic neurons of the central nervous system in vitro. In this study, HCNP-like-immunoreactive nerve processes and nerve cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the circular muscle layer and around the submucosal blood vessels. In the submucosal and myenteric plexuses, some HCNP-like-immunopositive nerve cell bodies and nerve fibers were present. The reaction product was deposited on the membranes of various subcellular organelles, including the rough endoplasmic reticulum, Golgi saccules, ovoid electron-lucent synaptic vesicles in axon terminals associated with submucosal and myenteric plexuses, and the outer membranes of a few mitochondria. The synaptic vesicles of HCNP-like-positive terminals were 60–85 nm in diameter. The present data provide direct immunocytochemical evidence that HCNP-like-positive nerve cell bodies and nerve fibers are present in the submucosal and myenteric plexuses of the rat small intestine. An immunohistochemical light microscopic study using mirror-image sections revealed that in both the submucosal and myenteric ganglia, almost all choline acetyltransferase (ChAT)-immunoreactive neurons were also immunoreactive for HCNP. These observations suggest (i) that HCNP proper and/or HCNP precursor protein is a membrane-associated protein with a widespread subcellular distribution, (ii) that HCNP precursor protein may be biosynthesized within neurons localized in the rat enteric nervous system, and (iii) that HCNP proper and/or HCNP precursor protein are probably stored in axon terminals.     

Improved fixation and cobalt-glucose oxidase-diaminobenzidine intensification for immunohistochemical demonstration of corticotropin-releasing factor in rat brain

An optimal fixation method and intensification procedure may be required in brain immunohistochemistry to obtain intense and widespread staining for a specific antigen, in cases where ordinary fixation and conventional immunohistochemistry result in only partial demonstration of the antigen. In the present study of localization of corticotropin-releasing factor immunoreactivity (CRFI) in rat brain, the importance of such intensification is shown. We describe a fixation procedure in which perfusion of rat brain with Bouin's solution is followed by a PBS wash and a further perfusion with either Zamboni's fluid or 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), for subsequent investigation of the detailed localization of CRFI in cerebral cortex and subcortical structures. The cobalt-glucose oxidase-diaminobenzidine (Co-GOD) intensification method has been modified to increase the sensitivity of immunostaining by reducing the concentration of glucose oxidase, which is added to the final incubation solution as a generator of hydrogen peroxide. The use of cobalt acetate instead of cobalt chloride appears to slightly suppress background staining in the Co-GOD method. Combination of the two modified procedures was applied to visualize intense and widespread CRFI in a variety of rat brain regions, including median eminence, cerebral cortex, and central amygdaloid nucleus.     

Biological sulfane sulfur

A voltammetric method for determining cyanide-reactive sulfane sulfur in biological materials is described. Samples are incubated with a sulfurtransferase, a thiolic cofactor, and cyanide. Thiocyanate formed and/or residual cyanide may then be determined electrochemically with either a silver rotating disk electrode or a dropping mercury electrode in differential pulse mode to provide estimates of sulfane sulfur content. The thiocyanate-based procedure is preferable, particularly when samples contain either serum albumin or inorganic sulfide.     

The stomatogastric nervous system of the house cricket Acheta domesticus L. II. Iontophoretic study of neuron anatomy

The anatomy of neurons of the stomatogastric nervous system of Ascheta domesticus was studied using heavy metal iontophoresis through cut nerve ends followed by silver intensification. Nineteen categories of neuron are described and compared with neurons known from the stomatogastric nervous system of other insects. Possible functions for the neurons are suggested. Motor neuron candidates are suggested for all parts of the gut served by the stomatogastric nervous system, and axons of sensory neurons of the anterior pharynx are located. There are four neuron types that cannot readily be assigned motor, sensory, or interneuron functions: large dorsal cells of the frontal ganglion; the two neurons of the nervus connectivus, and two categories of neurons in the median neurosecretory cell group of the pars intercerebralis, the axons of which are contained in the stomatogastric nerves.     

Evaluation of Inferior Alveolar Nerve Regeneration by Bifocal Distraction Osteogenesis with Retrograde Transportation of Horseradish Peroxidase in Dogs

Background

Bifocal distraction osteogenesis has been shown to be a reliable method for reconstructing segmental mandibular defects. However, there are few reports regarding the occurrence of inferior alveolar nerve regeneration during the process of distraction. Previously, we reported inferior alveolar nerve regeneration after distraction, and evaluated the regenerated nerve using histological and electrophysiological methods. In the present study, we investigated axons regenerated by bifocal distraction osteogenesis using retrograde transportation of horseradish peroxidase in the mandibles of dogs to determine their type and function.

Methods and Findings

Using a bifocal distraction osteogenesis method, we produced a 10-mm mandibular defect, including a nerve defect, in 11 dogs and distracted using a transport disk at a rate of 1 mm/day. The regenerated inferior alveolar nerve was evaluated by retrograde transportation of HRP in all dogs at 3 and 6 months after the first operation. At 3 and 6 months, HRP-labeled neurons were observed in the trigeminal ganglion. The number of HRP-labeled neurons in each section increased, while the cell body diameter of HRP-labeled neurons was reduced over time.

Conclusions

We found that the inferior alveolar nerve after bifocal distraction osteogenesis successfully recovered until peripheral tissue began to function. Although our research is still at the stage of animal experiments, it is considered that it will be possible to apply this method in the future to humans who have the mandibular defects.