Function of Catecholamine-containing Neurones in Mammalian Central Nervous System

SEVERAL chemical substances are involved in synaptic transmission in the mammalian central nervous system^1–3. The Falck-Hillarp technique⁴ has demonstrated noradrenaline, dopamine and 5-hydroxytryptamine within nerve cell bodies and terminals^5,6 and the belief that these amines act as neurohumours is strengthened by observations that nerve fibre activation leads to their release from the terminals^7,8. Histo-chemical evidence suggests that discrete systems of neurones can be identified by their content of particular amines and it seems possible that such neurohumorally homogeneous systems have a functional as well as a chemical identity. Before the anatomical distribution of amine-containing neurones had been described, Brodie and Shore⁹ proposed that noradrenaline functions as the central neurohumour of the sympathetic and 5-hydroxytryptamine of the parasympathetic system. This suggestion has not been supported by anatomical evidence; the amine-containing neurones form systems of small diameter fibres of very diffuse terminal distribution, which do not correspond to recognized ascending or descending pathways^5,6, although amine-containing neurones in invertebrates have been identified as sensory systems¹⁰.     

Fluorescence and electron microscopy of amine-storing enterochromaffin-like cells in tracheal epithelium of mouse

Summary The tracheo-bronchial mucosa of the mouse has been found to contain an extensive system of argyrophilic epithelial cells. In the trachea the cells morphologically resemble enterochromaffin cells. Normally, these enterochromaffin-like cells contain no fluorogenic amine, as revealed by the Falck-Hillarp formaldehyde technique. On the other hand the cells have the capacity to take up and decarboxylate 3,4-dihydroxyphenylalanine (DOPA) or 5-hydroxytryptophan (5-HTP); the amine formed is stored in the cytoplasm in a reserpine-sensitive store. This capacity to produce and store amines under experimental conditions may reflect the presence in the tracheal enterochromaffin-like cells of an amine which can not be demonstrated with available fluorescence histochemical techniques. In the electron microscope the tracheal enterochromaffin-like cells were identified by a positive argyrophil reaction and by their capacity to accumulate radioactivity after administration of ³H-DOPA or ³H-5-HTP as revealed by autoradiography. The radioactive labelling was associated with cytoplasmic electron-dense granules (800–1000 Å), suggesting that the amine formed was stored in these granules. Accordingly, the granules stained argentaffin after DOPA-pre-treatment of the animal. It is suggested that, like similar cells in the gastric mucosa, these argyrophilic enterochromaffin-like cells constitute an endocrine system in which amines are of cytophysiological importance.     

Morphologic and cytochemical characteristics of amine-containing globule leukocytes in rat tracheal epithelium

Amine-containing cells in the tracheal epithelium are typically of the small-granule type (diameter approximately 100 nm). However, in the rat, another amine-containing cell type has been identified that possesses the amine-handling features of the APUD-series of cells (amine precursor uptake and decarboxylation) but not the ultrastructural characteristics. It has been postulated that these cells may be related to cutaneous melanocytes. In this study, fluorescent cells were present in the laryngeal and tracheal epithelial lining of adult Sprague-Dawley rats following freeze-drying and exposure to formaldehyde vapor (FIF or formaldehyde-induced fluorescence). Microspectrofluorimetry revealed an emission maximum at 493 nm. The excitation maximum could not be calculated but appeared to be around or below 350 nm (to record spectra below requires the use of quartz optics). Yellow fluorescence also emanated from serotonin-containing mast cells (excitation and emission maxima: 401/515 nm). Tracheal segments processed according to the aqueous formaldehyde ( AFIF ) technique, for the demonstration of 5- hydroxytryptophan (5-HTP) or serotonin (5-HT), failed to identify fluorescent cells in the epithelial lining even though connective-tissue mast cells were evident. Subsequent treatment of AFIF -fixed sections with formaldehyde and HCl vapors ( AFIF -HCl) resulted in the formation of a fluorogenic compound within numerous cells in the tracheal lining (455/537 nm). This spectral shift and increase in intensity of fluorescence following acidification are characteristic for standards and/or cells that contain tryptamine, tryptophan, or peptides with NH2-terminal tryptophan and are markedly different from microspectrofluorimetric data reported for the phenylethylamines or serotonin. It is therefore postulated that these cells contain a closely related beta-(3-indolyl) ethylamine-like compound, serotonin excluded. The morphology of the fluorescent cells was similar when prepared according to the FIF or AFIF -HCl techniques. Conjunctive staining, the examination of a single section first by fluorescence microscopy and subsequently by other histochemical and cytochemical methods, demonstrated that the fluorescent granules were also methylene blue, alcian blue, periodic-acid Schiff, and ferric- fericyanide positive. Subsequent correlative electron microscopic examination of Epon-embedded AFIF -HCl-treated tracheal sections demonstrated that these amine-containing cells were globule leukocytes.     

Aminergic neurons in the anterior nervous system of the rat acanthocephalan moniliformis dubius

The cerebral ganglion and nerve tracts of Moniliformis dubius show intense, specific, green fluorescence that is also associated with the lateral and apical sensory bulbs. Radioenzymatic assays showed that high levels of dopamine were present but only small amounts of the catecholamines norepinephrine and epinephrine were identified. Incubations of the proboscis sac in dilute solutions of dopamine increased fluorescence while incubations in reserpine resulted in loss of fluorescence. Nonfluorogenic amine octopamine was also detected radioenzymatically. Neutral red vitally stained a number of cells in the cerebral ganglion and the nerve tracts extending from the ganglion. Electron microscopy showed that many neurons contained electron-dense vesicles. The close association of the fluorescing, amine-containing nerve tracts with the sensory bulbs suggests that they may play a functional role in sensory reception and transmission in M. dubius. This is the first report on the presence of biogenic amines in the Acanthocephala.     

Fluorometric quantitation of amino sugars in the picomole range.

A fluorometric method has been developed for the convenient and quantitative assay of amino sugars over the concentration range of 10 nm to 6 mm. Linear results are obtained for reaction mixtures containing 6 pmol to 60 nmol hexosamine. The procedure involves the condensation of amino sugars with the fluorogenic reagent o-phthalaldehyde, at alkaline pH in the presence of 2-mercaptoethanol. Relative fluorescence intensities are then determined using excitation and emission wavelengths of 340 and 455 nm, respectively. The presence of 2-mercaptoethanol in reaction mixtures not only enhanced sensitivity of the assay, but also defined the excitation/emission spectra. Under the conditions described, amino acids were also found to react with o-phthalaldehyde, yielding fluorescence intensities similar to those of amino sugars. These results suggest the applicability of fluorescence techniques in automated amino sugar analyses, as well as the potential interference of other compounds containing primary amines.     

Studies on the reaction of fluorescamine with primary amines

Fluorescamine is a useful reagent for the fluorometric assay of primary amines. The extent of the reaction between fluorescamine and primary amines, as well as the fluorescence intensities of the resulting fluorophors depend on pH, solvent composition and reagent concentration. Optimum values for these variables further depend on the amine under study. The influence of these parameters on the fluorogenic reaction of representative amines, and on their fluorophoric derivatives has been investigated, and the results are reported here.     

Evidence for anterograde transport of secretory granules in processes of gastric paracrine (somatostatin) cells

Certain disseminated endocrine-like cells have previously been found to give off long cytoplasmic processes which end with small bulbous expansions on the membranes of other cell types. It is believed that the process-carrying cells control the functions of the receiving cells by local and directed (paracrine) secretion of messenger molecules (peptides, biogenic monoamines) through their processes. Following injections of amine precursors paracrine cells take up and convert these to the corresponding amines, which can be cytochemically visualized by the Falck-Hillarp formaldehyde-induced fluorescence technique. As the amines are stored in the cytoplasmic (secretory) granules of the cells, they form useful markers for studies of granule turnover and transport. By injecting, at different time intervals, two different precursors (L-5-hydroxytryptophan and L-3,4-dihydroxyphenylalanine), resulting in amines giving different fluorescence colours in the Falck-Hillarp procedure, we have been able to separately label old and new secretory granule fractions in different fluorescence colours. Examination of such double-labelled paracrine cells (mostly gastric somatostatin cells) indicates that their secretory granules are transported in a proximo-distal direction in the paracrine cell processes (" paraxons "). This finding strongly supports the concept that paracrine cells control the functions of the cells they contact by way of directed, process-mediated delivery of their secretory products.     

Physiological methods to study biofilm disinfection

This report reviews the development of a rapidin situ approach to study the physiological responses of bacteria within biofilms to disinfectants. One method utilized direct viable counts (DVC) to assess the disinfection efficacy when thin biofilms were exposed to chlorine or monochloramine. Results obtained using the DVC method were one log higher than plate count (PC) estimates of the surviving population after disinfection. Other methods incorporated the use of fluorogenic stains, a cryotomy technique to yield thin (5-m) sections of biofilm communities and examination by fluorescence microscopy. The fluorogenic stains used in this approach included 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which indicates cellular electron transport activity and Rhodamine 123, which responds specifically to proton motive force. The use of these stains allowed the microscopic discrimination of physiologically active bacteria as well as heterogeneities of active cells within thicker biofilms. The results of experiments using these techniques with pure culture and binary population biofilms on stainless steel coupons indicated biocidal activity of chlorine-based disinfectants occurred initially at the bulk-fluid interface of the communities and progressed toward the substratum. This approach provided a unique opportunity to describe the spatial response of bacteria within biofilms to antimicrobial agents and address mechanisms explaining their comparative resistance to disinfection in a way that has not been possible using traditional approaches. Results obtained using this alternative approach were also consistently higher than PC data following disinfection. These observations suggest that traditional methods involving biofilm removal and bacterial enumeration by colony formation overestimate biocide efficacy. Hence the alternative approach described here more accurately indicates the ability of bacteria surviving disinfection to recover and grow as well as demonstrate spatial heterogeneities in cellular physiological activities within biofilms.     

A fluorescence-quenched chitopentaose for the study of endo-chitinases and chitobiosidases.

A new fluorogenic substrate displaying intramolecular fluorescence energy transfer (FRET) has been synthetized from NI,NII,NIII, NIV-tetra-acetyl-chitopentaose. Two molecules, a fluorophore (5-(2-aminoethyl) amino-1-naphtalene-sulfonic acid; EDANS) and a quenching group (dimethylaminophenylazophenyl; DAB) were chemically introduced on to the chitopentaose, one at each end. Among eight enzymes tested, only endo-chitinase and chitobiosidase activities could be specifically assayed by monitoring the variation of fluorescence after enzymatic hydrolysis of this substrate. Chitobiases and N-acetyl-beta-glucosaminidases are not active on the compound, the presence of a bulky chromogenic group at the 2 position of the nonreducing end of the subtrate preventing the binding and thus hydrolysis by these two exo-enzymes. The observation that chitobiosidases are able to hydrolyse a chitooligosaccharide functionalized on both extremities demonstrates the possibility of an endo-action for this class of chitinases, which are generally classified as exo-enzymes. This fluorogenic chitooligosaccharide should prove to be very useful for the detection and the convenient assay of chitinolytic activities at nanomolar concentrations.     

Immunohistochemical evidence for the presence of melatonin in the pineal gland,the retina and the Harderian gland

Summary The presence of melatonin is demonstrated in the pineal gland, the retina and the Harderian gland in some mammalian and non-mammalian vertebrates, using a specific fluorescence labelled antibody technique. Four different potent antibodies against melatonin have been used and compared. In the pineal gland of hamsters, mice, rats and snakes, specific fluorescence, mostly restricted to the cytoplasm of the cells, is detected in pinealocytes. Fluorescence is also detected in the pineal organ of fishes, tortoises and lizards, but it has not been possible, from cryostat sections of fresh tissue, to assert which kind of cell is reacting (photoreceptor cells or interstitial ependymal cells). In the retina, fluorescence is almost exclusively restricted to the outer nuclear layer. In the Harderian gland of mammals and reptiles, fluorescence is localized in the secretory cells of the alveoli and mostly restricted to the cytoplasm surrounding the nucleus. These results are discussed in relation to the concept of melatonin synthesis at extrapineal sites independent of pineal production.Parts of this work have been presented in the Xth Conference of Comparative Endocrinologists, Sorrento, May 20–25, 1979 (Vivien-Roels and Dubois 1980) and the VIth International Congress of Endocrinology, Melbourne, February 10–16, 1980 (Vivien-Roels et al. 1980)The author wishes to thank Professor Lutz Vollrath who has accepted her in his laboratory for a short period, Doctor George M. Bubenik for his suggestions and critical remarks, Dr. L.J. Grota for producing the melatonin diazobenzoic acid-BSA and Dr. Castro for preparing one of the melatonin derivates     

DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet

Using a bisubstituted caspase-3 target sequence: aspartate-glutamate-valine-aspartate, (z-DEVD)2 peptide derivative of the fluorophore, cresyl violet, we have obtained a cell permeant, fluorogenic, caspase substrate capable of detecting the site-specific presence of functionally active, caspase-3 and caspase-7 up-regulation within intact apoptotic cells. Addition of this substrate to induced and noninduced cell culture populations allows for the rapid site-specific detection of caspase up-regulation without the requirement for a wash step. We demonstrate here the use of (z-DEVD)2-cresyl violet substrate for the detection of apoptosis induction in Jurkat, THP-1, and MCF-7 cells using fluorescence microscopy and 96-well fluorescence plate reader analysis. Intracellular up-regulated DEVDase activity, which was clearly visible by fluorescence microscopy and 96-well fluorescence plate reader measurements, showed greater than 6-fold increases in fluorescence output in induced versus noninduced Jurkat cell samples. A simple fluorogenic substrate conversion method is demonstrated here for detecting apoptosis induction within intact living cells.     

A reduction-triggered fluorescence probe for sensing nucleic acids

We have developed a reduction-triggered fluorescence probe with a new fluorogenic compound derivatized from Rhodamine for sensing oligonucleotides. The chemistry to activate the compound involves the reaction between the azide group of rhodamine derivatives and the reducing reagents, with the fluorescence signal appearing after reduction of the azide group. The signal/background ratio of this fluorogenic compound reached 2100-fold enhancement in fluorescence intensity. Dithio-1,4-threitol or triphenylphosphine as reducing reagents were successfully utilized for this chemistry to be introduced into the DNA probe. The genetic detection requires that two strands of DNA bind onto target oligonucleotides, one probe carrying a reducible fluorogenic compound while the other carries the reducing reagents. The reaction proceeds automatically without any enzymes or reagents under biological conditions to produce a fluorescence signal within 10-20 min in the presence of target DNA or RNA. In addition, the probe was very stable under biological conditions, even such extreme conditions as pH 5 solution, pH 10 solution, or high temperature (90 degrees C) with no undesirable background signal. The probes were successfully applied to the detection of oligonucleotides at the single nucleotide level in solution and endogenous RNA in bacterial cells.     

Fluram induces species-dependent C and G bands in mammalian chromosomes, revealing heterogeneous distribution of chromosomal proteins

Fluram (Fluorescamine; 4-phenylspiro(furan-2(3H),1'-phthalan)-3,3'-dione) is a fluorogenic reagent, which permits the detection of primary amines by forming highly fluorescent pyrrolinone derivatives. This reagent has been used on methanol-acetic acid fixed metaphase chromosomes of mouse and man and proved to be very effective in differentiating chromosome regions in both genomes. Mouse centromeric heterochromatin is highly reactive, showing intense fluorescence in all centromeric regions, whereas human chromosomes show no fluorescence in such regions. In addition, a G-like banding pattern is also obtained in both types of chromosomes. The differential reactivity of each chromosome region showed by this method demonstrates a heterogeneous distribution of chromosome proteins, resulting in a chromosome banding pattern, which is in this case species dependent.     

Sensing of carbon dioxide by a decrease in photoinduced electron transfer quenching.

We described a new approach to sensing of carbon dioxide based on photoinduced electron transfer (PET) quenching. Fluorophores like naphthalene and anthracene are known to be quenched by unprotonated amines by the PET mechanism. We examined the fluorescence spectral properties of two amine-containing fluorophores, 1-naphthylmetylamine (NMA) and 9-ethanolaminomethylanthracene (EAA). When dissolved in an organic solvent, both fluorophores displayed increased intensity when equilibrated with gaseous carbon dioxide. In the case of NMA, we found that the mean lifetime increased with increasing partial pressures of CO(2). The intensity and lifetime changes of NMA are completely reversible when CO(2) is removed by purging with argon. Our results are consistent with decreased quenching by the covalently linked amino groups when CO(2) is dissolved in the solution. At present, we are not certain whether the increased intensity is due to protonation of the amino groups or to carbamate formation. In either event, these results suggest that CO(2) can be detected directly using amine-containing fluorophores without the use of bicarbonate and a pH-sensitive fluorophore.     

Rapid assay to detect possible natural substrates of proteases in living cells

Proteolysis is a regulatory step in many physiological processes, but which proteases in what cellular sites are involved in activation or degradation of which peptides is not well known. We developed a rapid assay consisting of living cells and fluorogenic protease substrates to determine which bioactive peptides are possible natural substrates of a specific protease with the multifunctional or moonlighting protein CD26/dipeptidyl peptidase IV (DPPIV) as a model. CD26/DPPIV catalyzes cleavage of peptides from the amino terminus of peptides with proline at the penultimate position. Many biologically active peptides, such as beta-casomorphin1-5, contain proline in the penultimate position. We incubated living Jurkat cells, which are T cells that lack CD26/DPPIV, and CD26/DPPIV-transfected Jurkat cells in the presence of the fluorogenic substrate [Ala-Pro]2-cresyl violet (Magic Red) and beta-casomorphin1-5. Fluorescent cresyl violet was generated by CD26/DPPIV-transfected Jurkat cells but not by wild-type Jurkat cells with a Km of 3.7 microM. beta-Casomorphin1-5 appeared to be a possible natural substrate of CD26/DPPIV, because it inhibited production of fluorescence competitively (Ki = 60 microM). The assay using living cells and a fluorogenic protease substrate is an efficient system to determine whether specific peptides are possible natural substrates of a particular protease.     

Structural and microspectrofluorometric studies on glial cells from the periventricular and arcuate nuclei of the rat hypothalamus

Summary Fluorescence spectroscopy and electron microscopic techniques have been employed to investigate a class of glial cells that is characterized by the presence in their cytoplasm of large fluorescing inclusions that stain with paraldehyde-fuchsin or chrome-hematoxyline-alum. In the periventricular nucleus the cells have been identified as a population of astrocytes whose inclusions emit an orange-red fluorescence. In the arcuate nucleus there are, in addition to an overwhelming majority of such astrocytes, also some microglial cells with similar characteristics. The ability of the latter to emit any kind of fluorescence has not yet been established. The fluorescence maximum of these astrocytic inclusions was found to be at 640 nm when excited at 405 nm. The data obtained suggest that the fluorescence observed is due to the presence of porphyrins in the astrocytic inclusions. In the majority of our electron microscopic pictures the inclusions lack a bounding membrane. By contrast, neuronal lipofuscin has an outer membrane. In cryostat sections, the lipofuscin emits a yellow fluorescence when excited at 400–410 Onm.     

The formaldehyde-fluorescamine method

Summary Formaldehyde reacts with primary amino groups to derivatives which are unable to react with the fluorogenic primary amino group probe, fluorescamine. Paradoxically, however, certain specific cell systems continue to display strong fluorescamine-induced fluorescence after formaldehyde pretreatment. Among such formaldehyde-fluorescamine (FF) positive cell systems are certain peptide- and protein-secreting cells as well as all hitherto investigated types of cancer cells. We have now optimized the cytochemical FF method by using microfluorometry in combination with systematically varied reaction conditions. In addition, the quantitative data indicate that in FF positive cells, formaldehyde pretreatment causes a paradoxical increase in the fluorescence yield with fluorescamine. This has tentatively been ascribed to quenching phenomena, associated with closely spaced primary amino groups. Work with alternative fluorogenic amino group probes (MDPF and OPT) show that these display the same spectrum of tissue selectivity as fluorescamine, but that the latter remains the reagent of choice for the cytochemical FF reaction.     

Mathematical modeling of a single-cell enzyme assay

A quantitative assay of beta-galactosidase activity in single cells of Saccharomyces cerevisiae has been developed using a fluorogenic substrate and flow cytometry [reported in Wittrup & Bailey, Cytometry, 9,394 (1988)]. The beta-galactosidase activity is expressed in yeast from the Escherichia coli lacZ gene under the control of the yeast GAL10 promoter, and is used as a marker for multicopy plasmid content. A nonfluorescent fluorogenic substrate is enzymatically cleaved by intracellular beta-galactosidase to form a fluorescent product. The accumulation of fluorescent product in single cells was found to depend on bulk substrate concentration and single-cell enzyme activity in a fashion that could not be described by a Michaelis-Menten kinetic rate form. It has been demonstrated that diffusion limitation rather than enzyme activity can determine the level of single-cell fluorescence under certain assay conditions, and a mathematical model has; been formulated which accounts for substrate and product diffusion. Guided by the mathematical model, the assay conditions were modified to allow measurement of single-cell enzyme activity rather than diffusion rates.     

Evidence for anterograde transport of secretory granules in processes of gastric paracrine (somatostatin) cells

Summary Certain disseminated endocrine-like cells have reviously been found to give off long cytoplasmic processes which end with small bulbous expansions on the membranes of other cell types. It is believed that the process-carrying cells control the functions of the receiving cells by local and directed (paracrine) secretion of messenger molecules (peptides, biogenic monoamines) through their processes. Following injections of amine precursors paracrine cells take up and convert these to the corresponding amines, which can be cytochemically visualized by the Falck-Hillarp formaldehyde-induced fluorescence technique. As the amines are stored in the cytoplasmic (secretory) granules of the cells, they form useful markers for studies of granule turnover and transport. By injecting, at different time intervals, two different precursors (L-5-hydroxytryptophan and L-3,4-dihydroxyphenylalanine), resulting in amines giving different fluorescence colours in the Falck-Hillarp procedure, we have been able to separately label old and new secretory granule fractions in different fluorescence colours. Examination of such double-labelled paracrine cells (mostly gastric somatostatin cells) indicates that their secretory granules are transported in a proximo-distal direction in the paracrine cell processes (paraxons). This finding strongly supports the concept that paracrine cells control the functions of the cells they contact by way of directed, process-mediated delivery of their secretory products.This work was reported in part at the 29th Congress of the International Union of Physiological Sciences, Sydney, Australia, August 28–September 3, 1983     

Analysis by fluorescence microscopy of the development of compartment-specific gene expression during sporulation of Bacillus subtilis.

The use of a fluorogenic substrate, 5-octanoylaminofluorescein-di-beta-D-galactopyranoside, for beta-galactosidase has made it possible to visualize enzyme activity in individual cells of sporulating populations of Bacillus subtilis by fluorescence microscopy. lacZ fusions to different sporulation-associated genes have been used to investigate the cell compartmentalization of gene expression during sporulation. A strain with a lacZ fusion to sspA, a gene which is transcribed by E-sigma G at a late stage of sporulation, displayed predominantly compartment-specific fluorescence. Expression of the early-expressed spoIIA locus, which includes the structural gene for sigma F, was seen not to be compartmentalized. Populations of strains with lacZ fusions to gpr and dacF, genes which are transcribed by E-sigma F at intermediate stages of sporulation, included some organisms showing uncompartmentalized fluorescence and others showing compartment-specific fluorescence; the proportion showing compartment-specific fluorescence increased in samples taken later in sporulation. Several possible explanations of the results obtained with gpr and dacF are considered. A plausible interpretation is that sigma F activity is initially not compartmentalized and becomes compartmentalized as sporulation progresses. The progression to compartmentalization does not require the activities of the sporulation-specific factor sigma E or sigma G but may require some product of sigma F activity.