Cryostat technique for central nervous system histofluorescence.

This paper offers a technique for obtaining monoamine histofluorescence in the CNS by means of formaldehyde perfusion followed by cryostat sectioning. No freeze-drying is involved. Cryostat sections are exposed to formaldehyde vapor to complete the fluorophore formation. The fluorescence thus obtained is bright, well localized, and does not require loading the animals with precursors. The anatomical distribution of the pathways is identical to that obtained with the classical technique. Furthermore, the fluorescence is reversible by sodium borohydride, and exhibits the expected changes in intensity with pharmacological manipulations. The sections can be exposed to a cold aqueous medium for as long as 15 min with minimal diffusion of fluorophore; this suggests potential for combining monoamine histofluorescence with other visualization techniques.     

Visualization of the Langerhans cells in the human vaginal epithelium by the L-dopa histofluorescence method

In this study we used the technique of L-DOPA histofluorescence for visualizing Langerhans cells in the human vaginal epithelium. Biopsy specimens were incubated with L-DOPA and sectioned by cryostat. The sections were exposed to formaldehyde vapour; during this passage, chemical conversion of L-DOPA into a strongly fluorescent compound occurred. Langerhans cells were clearly visualized in the vaginal epithelium; cell bodies and dendritic processes fluoresced sharply. The method is rapid and specific: it represents an useful tool for demonstrating Langerhans cells in the stratified squamous epithelium of vagina.     

Magnesium ions in catecholamine fluorescence histochemistry

Summary The effects of high concentrations of magnesium ions in the cryostat and Vibratome procedures for visualization of catecholamine fluorescence in the central nervous system have been investigated. In cryostat sections, obtained from specimens perfused with a formaldehyde and glyoxylic acid containing buffer, the addition of high concentrations of MgSO₄ to the perfusion solution enhances the fluorescence intensity and reduces the unspecific background fluorescence and the diffusion of the catecholamine fluorophore. This improves the visualization of all portions of the central catecholamine-containing neurons. Similar effects are obtained in the formaldehyde-Vibratome technique by the introduction of an immersion bath containing MgSO₄ after the sectioning procedure. The use of the magnesium perfusion or immersion steps furthermore increases the reproducibility of the Vibratome and cryostat techniques. The paper describes the improved Vibratome and cryostat techniques used in our laboratory.     

A simple, fast and reliable method for obtaining dopamine histofluorescence from mesencephalic cultures

A modified glyoxylic acid technique for obtaining dopamine histofluorescence from cultured mesencephalic cells is described. This method requires only two solutions: one contains glyoxylic acid, sucrose and monobasic potassium phosphate and is used at room temperature, the other is a Hepes buffered solution used at 37 C. Relatively high concentrations of a monoamine oxidase inhibitor and dopamine are added to the cultures to load dopaminergic neurons; the cell bodies and their processes take up and hold dopamine quickly and evenly. The cultures are dipped in a glyoxylic acid solution, dried in air, heated for 5 min and coverslipped with mineral oil. Since the cultures remain in their culture dishes, the entire procedure takes less than 2 hr. The green histofluorescence characteristic of dopamine is seen when the cultures are viewed by standard fluorescence microscopy. Various cell body types and sizes can be distinguished, as well as the complete extent of their processes and varicosities.     

A Simple, Fast and Reliable Method for Obtaining Dopamine Histofluorescence from Mesencephalic Cultures

A modified glyoxylic acid technique for obtaining dopamine histofluorescence from cultured mesencephalic cells is described. This method requires only two solutions: one contains glyoxylic acid, sucrose and monobasic potassium phosphate and is used at room temperature, the other is a Hepes buffered solution used at 37 C. Relatively high concentrations of a monoamine oxidase inhibitor and dopamine are added to the cultures to load dopaminergic neurons; the cell bodies and their processes take up and hold dopamine quickly and evenly. The cultures are dipped in a glyoxylic acid solution, dried in air, heated for 5 min and coverslipped with mineral oil. Since the cultures remain in their culture dishes, the entire procedure takes less than 2 hr. The green histofluorescence characteristic of dopamine is seen when the cultures are viewed by standard fluorescence microscopy. Various cell body types and sizes can be distinguished, as well as the complete extent of their processes and varicosities.     

Application of the aluminum-formaldehyde (ALFA) histofluorescence method for demonstration of peripheral stores of catecholamines and indolamines in freeze-dried paraffin-embedded tissue,cryostat sections and whole-mounts

Summary This paper describes new procedures for highly sensitive visualization of monoamine stores in peripheral tissues, taking advantage of the recently introduced aluminum-catalysed formaldehyde (ALFA) reaction. The tissues are exposed to an aluminum sulphate solution (with or without formaldehyde fixation) in a perfusion and/or immersion step, followed by formaldehyde vapour treatment. Procedures are described for freeze-dried, paraffin embedded tissue, cryostat sections and whole mount preparations. For all these tissue preparations the ALFA method gives a highly sensitive and precise demonstration of catecholamine-containing neurons and 5-HT-containing cells in a variety of peripheral tissues. For freeze-dried tissue and cryostat sections the ALFA method represents an improvement in comparison with other available methods. This is particularly noticeable for the very delicate adrenergic nerves in such organs as the thyroid, ovary, pancreas and the gastrointestinal tract.     

Catecholamine neurons in Aplysia: improved light-microscopic resolution and ultrastructural study using paraformaldehyde and glutaraldehyde (FaGlu) cytochemistry

We have modified the formaldehyde-glutaraldehyde (FaGlu) histofluorescence method of Furness, Costa, and Blessing (1977a) and Furness, Costa, and Wilson (1977b) to examine wholemounts and sections of both juvenile and adult ganglia as well as peripheral tissues of Aplysia californica. FaGlu fluorescence is the result of a reaction between formaldehyde and tissue catecholamines to produce water-insoluble (fixed) fluorophores. In serially sectioned cerebral ganglia, 70-80 positive neurons were observed (many in clusters of 10-20 cells), many more than were found using the glyoxylic acid technique. Catecholamine-containing varicosities were densely packed in localized portions of the neuropil of all central ganglia. Exclusive localization in the neuropil of presumed dopamine release sites is similar to that previously found for the neuropeptide SCP but differs from the widespread ramification of varicose neurites containing 5-HT, FMRFamide, and ELH. The FaGlu technique also enabled us to study the ultrastructure of catecholamine-containing neurons. In contrast to the larger vesicles found in serotonergic and histaminergic neurons, these dopaminergic neurons contain 70 nm dense-cored vesicles.     

Vacuumless freezing-drying: Its application in catecholamine histochemistry

Summary Details of a simple technique to prepare tissues for the catecholamine reaction are described. Fresh cryostat sections are dried above phosphorous pentoxyde at –25⁰C for 15 hours. Ice evaporates from the sections under normal atmospheric pressure at this temperature. Frozen-dried cryostat sections are treated with formaldehyde gas like other frozendried specimens and observed under the fluorescence microscope immediately. The patterns obtained are at least comparable with those obtained by the original Falck procedure.     

Specific effect of 5,6-dihydroxytryptamine on the monoamine fluorophore of the frog's gustatory cells

Summary A specific formaldehyde-induced yellow fluorescence, suggesting the presence of serotonin-like monoamine has been demonstrated in the gustatory cells of the frog.The fungiform papillae of frogs were examined fluorescence-histochemically after intraperitoneal injection of 5,6-dihydroxytryptamine. The results indicated that the fluorophore of gustatory cells was affected selectively by the drug injection: the yellow fluorescence was transiently enhanced 3 hours after the drug injection, thereafter being reduced rapidly. The effect of 5,6-dihydroxytryptamine was long-lasting with the reduction of the yellow fluorophore persisting at least for the experimental duration of 14 days. A single injection of 6-hydroxydopamine induced a complete depletion of noradrenaline fluorescence from adrenergic nerve terminals, while the fluorescence of gustatory cells was not affected by a high dose of the drug.The present results with pharmacologic treatments further support the view that the gustatory cell of the frog contains a serotonin-like monoamine.     

Glutathione localization by a novel o-phthalaldehyde histofluorescence method

Summary Glutathione in tissues forms an intense fluorophore with a solution ofo-phthalaldehyde at room temperature. We have studied the loss of glutathione from tissue sections and find that it is not measurable from thick sections. The fluorescence spectra of the induced fluorophore between glutathione ando-phthalaldehyde are identical in model and tissue sections, while depletion of hepatic glutathione by diethyl maleate produces a comparable fall in fluorescence measured biochemically or histochemically. This simple method is specific as interfering substances, such as spermine and spermidine, produce very weak fluorescence under the conditions employed.     

New fluorophore-forming reactions for histochemical visualization of N-acetylated and tertiary indolamines using glyoxylic acid,aluminum-formaldehyde and trifluoroacetic acid anhydride as reagents

Summary N-acetylated and tertiary indolamines, some of which are possible neurotransmitter candidates in the CNS, cannot be visualized with the standard Falck-Hillarp histofluorescence method and very little is known about their cellular localization. The present investigation demonstrates that glyoxylic acid (GA), formaldehyde (FA) in combination with aluminum ions (the ALFA method) and trifluoroacetic acid anhydride (TFAA) are capable of forming fluorescent compounds from N-acetylated (e.g. melatonin and N-acetyl-5-hydroxytryptamine) and tertiary (e.g. bufotenin) indolamines in histochemical protein models. With GA and FA-aluminum more vigorous reaction conditions were required for demonstration of these compounds compared to those needed for optimal visualization of primary catecholand indolamines (prolonged reaction time and higher concentration of GA and FA and aluminum ions). The fluorophore formation from N-acetylated and tertiary indolamines, which represents a new reaction principle in amine fluorescence histochemistry, is proposed to proceed as follows. In the first step, the indole reacts in 2-position with the reagent. The intermediate formed is dehydrated in the second step, yielding a strongly fluorescent 2-methylene derivative, which either per se or as the corresponding autoxidized dimer constitutes the main fluorophore. TFAA and related anhydrides represent new and potent reagents for histochemical visualization of N-acetylated indolamines such as melatonin. In contrast to the GA and ALFA reactions the optimal formation of fluorphores with TFAA required only mild reaction conditions (2–10 min at 0–20° C). The main fluorophore formed from melatonin has been identified and the reaction with TFAA is proposed to proceed as follows. An unstable intermediate, the isoimidinium carboxylate, is formed in the first step and this compound is then cyclized to form the fluorophore, 6-methoxy-1-methyl-3,4-dihydro--carboline. The GA and ALFA methods are already widely used for visualization of catecholamine systems. The fluorescence microscopical and microspectrofluorometric analysis did not, however, veveal any specific structures containing N-acetylated or tertiary indolamines in the rat CNS. The TFAA reaction was highly specific for N-acetylated indolamines when applied to protein models. However, in tissue a disturbing background fluorescence appeared, which under all reaction conditions tested, developed concomitantly with the specific fluorescence from melatonin. The problem with this background reaction has to be solved before the TFAA reaction can be applied for demonstration of N-acetylated indolamines in tissue.     

Diaminobenzidine Induces Fluorescence in Nervous Tissue and Provides Intrinsic Counterstaining of Sections Prepared for Peroxidase Histochemistry

3,3'-Diaminobenzidine (DAB) is widely used as a chromogen for visualization of horseradish peroxidase activity in neuroanatomical tracing experiments and in immunohistochemistry. The product of the enzymatically catalyzed oxidation of DAB by hydrogen peroxide is brown and nonfluorescent. In frozen sections of formaldehyde fixed rat and mouse brain that had been exposed to DAB either alone or with hydrogen peroxide, we observed strong greenish fluorescence in myelinated nerve fibers and in the somata of some neurons. This fluorescence was not associated with brown coloration and was not due to endogenous peroxidase activity. Extractions, blocking reactions, and other histochemical tests indicate that the fluorescence resulted from the combination of DAB with aldehyde groups that were formed by oxidation of unsaturated linkages in lipids. DAB induced fluorescence provides a simple and useful demonstration of background anatomy in sections that also contain specifically localized deposits of peroxidase activity.     

Comparative anatomy of brain monoaminergic neurons in New World and Old World monkeys

A comparison of the distribution of brain monoamine neurons in several New World and Old World monkeys was undertaken using the Falck-Hillarp formaldehyde histofluorescence technique. The overall organization of the monoamine neurons was very similar in all species, although subtle variations were found. Catecholamine (noradrenaline and dopamine) and indoleamine (serotonin) cell bodies corresponding to groups A1–A7, A8–A10, and B1–B9, respectively were found throughout the brainstem. A few catecholamine (dopamine) cells equivalent to groups All and A12 in the diencephalon were also observed. Noradrenaline neurons, rather than those of the dopamine and serotonin systems, tended to be less numerous in the New World monkeys. Ascending catecholamine and indoleamine fiber bundles were observed in most monkeys. It is interesting that fibers corresponding to the “ventral noradrenaline bundle” appeared to be much finer in the common marmoset and tamarin than in other species. In addition, a substantial catecholamine (noradrenaline) innervation of the diencephalon was noted in all the Old World monkeys, while a much lower overall terminal density was apparent in the New World forms.     

Simultaneous demonstration of adrenergic and non-adrenergic nerve fibres

Summary A technique for simultaneous demonstration of adrenergic and non-adrenergic nerve fibres is described, using methylene blue staining and fluorescence microscopy after formaldehyde treatment. The procedure is applicable to whole mounts as well as to microtome sections.     

Development of a novel FRET immunosensor technique

We report on a novel technique to develop an optical immunosensor based on fluorescence resonance energy transfer (FRET). IgG antibodies were labeled with acceptor fluorophores while one of three carrier molecules (protein A, protein G, or F(ab')2 fragment) was labeled with donor fluorophores. The carrier molecule was incubated with the antibody to allow specific binding to the Fc portion. The labeled antibody-protein complex was then exposed to specific and nonspecific antigens, and experiments were designed to determine the 'in solution' response. The paper reports the results of three different donor-acceptor FRET pairs, fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate, Texas Red/Cy5, and Alexa Fluor 546/Alexa Fluor 594. The effects of the fluorophore to protein conjugation ratio (F/P ratio) and acceptor to donor fluorophore ratios between the antibody and protein (A/D ratio) were examined. In the presence of specific antigens, the antibodies underwent a conformational change, resulting in an energy transfer from the donor to the acceptor fluorophore as measured by a change in fluorescence. The non-specific antigens elicited little or no changes. The Alexa Fluor FRET pair demonstrated the largest change in fluorescence, resulting in a 35% change. The F/P and A/D ratio will affect the efficiency of energy transfer, but there exists a suitable range of A/D and F/P ratios for the FRET pairs. The feasibility of the FRET immunosensor technique was established; however, it will be necessary to immobilize the complexes onto optical substrates so that consistent trends can be obtained that would allow calibration plots.     

Descending monoaminergic pathways in the primate spinal cord.

The distribution of monoamine axons and terminals within the spinal cord of a primate (Macaca mulatta) was studied with the Falck-Hillarp histofluorescence technique for the demonstration of biogenic amines. Catecholamine and indoleamine varicosities appeared qualitatively similar to those previously reported for the rat although the indoleamine terminals were difficult to visualize and were not studied in great detail. Catecholamine fibers innervate the substantia gelatinosa, marginal layer, intermediolateral cell column, ventral horn and the region surrounding the central canal. The location of monoamine axons, as revealed by spinal cord ligation, corresponds to that in the rat and cat with the exception of the dorsolateral region of white matter where fluorescent axons are not visible in the primate.     

Formaldehyde-Induced Fluorescence as a Means for Differentiating Epinephrine Cells from Norepinephrine Cells in Adrenal Medulla

The Falck and Hi Harp technique for the cellular localization of catecholamines by formaldehyde-induced fluorescence was applied to rat, mouse, and Syrian hamster adrenal. Some medullary cells revealed an unexpected orange-brown fluorescence. In guinea pig or rabbit adrenals, which store predominantly epinephrine, orange-brown fluorescence was not readily observed. It was found in Syrian hamsters only at the medullary periphery, where norepinephrine-producing cells are known to occur. Orange-brown fluorescence was depleted by administration of reserpine and intensified by nialamide plus DOPA. The same cell clusters which stained specifically for norepinephrine with ferric ferricyanide were found in adjacent sections to exhibit orange-brown fluorescence. Only by reducing the temperature of the formaldehyde reaction could sections of hamster adrenal showing only yellow-green fluorescence be obtained. These data suggest that the orange-brown fluorescence might result from' polymerization, oxidation, or both, of the isoquinoline produced by the norepinephrine-formaldehyde reaction under conditions slightly more vigorous than optimal and in the presence of high concentrations of norepinephrine     

Simultaneous monoamine histofluorescence and neuropeptide immunocytochemistry

Summary A method was developed that allows the analysis of neuropeptides and monoamines in a single tissue section by the application of the unlabeled antibody method for peptide staining to tissue sections freeze-dried for formaldehyde-induced monoamine histofluorescence. The hypothalamic magnocellular system of male albino rats served as a model for this study; neurons were stained with anti-neurophysin sera, which mark the vasopressin- and oxytocin-associated proteins. Neurophysin-containing perikarya appeared to be surrounded by catecholamine-containing varicosities. This phenomenon was seen to varying degrees within the supraoptic and paraventricular nuclei. The juxtaposition of varicosities and peptidergic neurons suggests an afferent fiber-target neuron relationship that might favor a functional interaction between monoamines and neuropeptides.Supported by USPHS Grant and postdoctoral NS15816 (JRS) fellowship AG01575 (THM)     

New aspects on factors determining the sensitivity of the formaldehyde and glyoxylic acid fluorescence histochemical methods for monoamines

Summary The fluorophore and fluorescence yield from tryptamine and 3-methoxytyramine in histochemical protein models have been compared in the standard formaldehyde reaction, the acid-catalyzed formaldehyde reaction, the formaldehyde-ozone reaction, and the aluminum-formaldehyde reaction. In the standard formaldehyde reaction both the fluorophore and fluorescence yields are low. However, the other reactions give a dramatic increase in fluorescence intensity (18–20 times) from tryptamine and 3-methoxytyramine whereas only minor changes (up to 100% increase) in fluorophore yield are observed. It is concluded that the relative fluorescence intensity of each fluorophore molecule formed in the three modifications of the formaldehyde reaction is much higher than that of the molecules formed in the standard formaldehyde reaction. It has previously been demonstrated that the fluorophores formed from dopamine in the gaseous formaldehyde and glyoxylic acid reactions have a much higher (10 times) relative fluorescence intensity than the synthetic fluorophores. The present experiments show that if the histochemical models are dissolved in buffer after the reaction and new models are made from this solution, the fluorescence intensity of the fluorophores formed in the reaction is drastically reduced and becomes comparable to that of the synthetic ones. The results of this and our previous studies indicate that hitherto unknown fluorescence enhancing mechanisms play a major role for the fluorescence yield, i.e. the sensitivity, in the various formaldehyde and glyoxylic acid methods. One possible explanation to the high relative fluorescence intensity of the fluorophores formed in the histochemical reactions could be an energy transfer between, e.g. the non-fluorescent intermediary reaction products (the tetrahydro derivatives) and the fluorophores (the dihydroisoquinolines and dihydro--carbolines). Such an energy transfer is probably attenuated in the dissolved models, where the distances between and orientations of the various molecules have been changed.Abbreviations DA dopamine - FA formaldehyde - GA glyoxylic acid - 3-MT 3-methoxytyramine - 4-MT 3-hydroxy-4-methoxyphenylethylamine - T tryptamine - DHC dihydro--carbolines - THC tetrahydro--carboline - 2-Carb.Me-DHIQ 2-carboxymethyl-3,4-dihydroisoquinolinium compound - THIQ-1-COOH tetrahydroisoquinoline-1 carboxylic acid     

Histochemical demonstration of monoaminergic and cholinesterase-positive nerve fibres in regenerating rat submandibular gland autografts

Summary A cholinesterase localization method and a monoamine histofluorescence technique were used to locate nerve fibres in regenerating rat submandibular gland autografts. Experimental rats had a portion of one submandibular gland excised and cut into small fragments which were autografted immediately into the middle one-thrid of the tongue. Control rats had a portion of one submandibular gland removed and discarded, and their tongues were sham-operated. Seven to ten weeks later, the rats were killed and the tongues were removed, frozen and sectioned in a cryostat. A light microscopical study of the tongue sections subjected to the cholinesterase technique showed that the submandibular gland autografts contained many nerve fibres that exhibited cholinesterase activity. These cholinesterase-positive nerve fibres were distributed throughout the autografts. The fibres were associated with the numerous duct-like structures and the less numerous acini. In addition, ultraviolet illumination of tongue sections after treatment with a glyoxylic acid mixture revealed histofluorescent monoaminergic nerves within the autografts. These fibres were less prominent than the cholinesterase-positive fibres and appeared to run primarily along blood vessels within the autografts. The results suggest that autonomic nerves are present within regenerating submandibular gland autografts.