Formaldehyde-induced Fluorescence Research Articles

Quantitative Analysis of Formaldehyde-induced Fluorescence in Paraffin-embedded Specimens of Malignant Melanomas and Other Melanocytic Lesions.

Inter-observer agreement is problematic in the histopathological diagnosis of melanoma and melanocytic naevi, even among expert pathologists. Formaldehyde-induced fluorescence (FIF) has been used for histochemical demonstration of catecholamines, 5-hydroxytryptamine and their immediate precursors. FIF can detect melanogenic activity and may be useful in differentiating malignant melanoma from other melanocytic lesions. The fluorescence of various types of melanocytic lesions has been previously studied quantitatively in formalin-fixed and paraffin-embedded sections. This study compared 2 sets of excitation and emission bands: 450-490 nm excitation/510-560 nm absorption filters (filter unit A) and 480 nm excitation/510< nm absorption filters (filter unit B). Higher FIF was observed with filter unit A than with filter unit B. FIF intensity of central regions was found to be higher than that of the peripheral regions. Mean FIF was significantly higher in malignant melanomas than in naevi. Fluorescence imaging with filter unit A gave better diagnostic performance. In conclusion, quantitative measurement of FIF is a useful marker of malignant potential.

Gamma‐aminobutyric acid B Receptor Immunoreactivity in the Mouse Adrenal Medulla

The present study examined gamma-aminobutyric acid B (GABAB ) receptor, GABA, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS) immunoreactivities in the mouse adrenal medulla. GABAB receptor immunoreactivity was seen in numerous chromaffin cells and in a few ganglion cells of the adrenal medulla. By using a formaldehyde-induced fluorescence (FIF) method, GABAB receptor immunoreactivity was observed in numerous adrenaline (A) cells, but not in noradrenaline (NA) cells showing blue-white fluorescence. This suggests that GABAB receptors may be present in the A cells and be related to the secretory activity of A cells but not NA cells in the mouse adrenal medulla. GABAB receptor immunoreactive ganglion cells were shown to be nNOS immunopositive by using a double immunostaining method. Weak GABA immunoreactivity was visible in some chromaffin cells and in the numerous nerve fibers of the medulla. By using the FIF method, weak GABA-immunoreactive chromaffin cells were shown to be in the NA cells showing blue-white fluorescence. GABA-immunoreactive nerve fibers were in dense contact in A cells, but not NA cells. GABA-immunoreactive nerve fibers closely contacted a few ganglion cells. Numerous GABA-immunoreactive nerve fibers in the medulla showed ChAT immunoreactive. This result suggests that GABA and acetylcholine may be released from the same nerve fibers and may have a secretory effect on the A cells of the medulla.

Quantitative measurements of formalin-induced fluorescence for differential diagnostics of melanomas and lesions of human skin

The usefulness of formaldehyde-induced fluorescence (FIF) for detection of melanoma cells has been suggested by several investigators during the last 40 years. FIF can be easily excited and observed in microscopic sections of formalin-fixed paraffin-embedded skin samples. However, such an approach has never been widely used in melanoma diagnostics for reasons including lack of clear diagnostic criteria, considerable inconsistencies in both the protocols used and qualitatively analysed results reported by different groups. This study aimed at determination of the spectral bands optimum for detecting melanoma cells. The study involved three sets of the excitation and emission bands: gammaex=366 nm, gammaem>425 nm; gammaex=450-480 nm, gammaem>515 nm; gammaex=450-480 nm, gammaem=510-550 nm. Microscopic digital imaging was used to quantitatively determine the fluorescence intensity of 53 primary melanomas and 32 benign lesions. Best classification of melanomas with algorithm based on fluorescence intensity threshold was obtained for gammaex=450-480 nm, gammaem=510-550 nm. Receiver operating characteristics (ROC) analysis of the algorithm yielded area under the curve=0.84 +/- 0.05 for melanocytic cells present in the stratum corneum. Our results clearly indicate that the FIF emitting molecules (most probably 5-S-cysteinyldopa) are present in melanomas at the concentration significantly higher than in benign lesions. In terms of the ROC analysis, the diagnostic performance of the test based on the FIF intensity is as good as of many other commonly used diagnostic tests.

A synaptic DEG/ENaC ion channel mediates learning in C. elegans by facilitating dopamine signalling

An important component of learned behaviour is the ability to forecast positive or negative outcomes based on specific sensory cues. Predictive capacity is typically manifested by appropriate behavioural patterning. However, the molecular mechanisms underlying behavioural plasticity are poorly understood. Caenorhabditis elegans displays experience-dependent behavioural responses by associating distinct environmental signals. We find that ASIC-1, a member of the degenerin/epithelial sodium channel family, which localizes at presynaptic terminals of dopaminergic neurons, is required for associative learning in C. elegans. ASIC-1 functions in these neurons to amplify normal dopaminergic signalling, necessary for associative learning. Our results reveal a novel role of DEG/ENaC ion channels in neuronal communication by enhancing the activity of dopaminergic synapses. Similar mechanisms may facilitate synaptic plasticity in vertebrates.

P.1.c.023 Effects of a glucocorticoid receptor antagonist and an SSRI on 5-HT transporter binding and 5-HT1A receptor mRNA

Three continuous cell lines isolated from small cell carcinoma of the lung (SCCL) have been examined for their ability to take up and metabolize the biogenic amine precursors, L-DOPA and 5-hydroxytryptophane (5-HTP). In two of the three lines formaldehyde-induced fluorescence (FIF) and mass-spectrographic analysis indicated specific uptake and metabolism of L-DOPA and 5-HTP.

The Pigmented Life of a Redhead

As a redhead I have had a personal interest in red hair, freckles and sunburns since childhood. An observation of a formaldehyde-induced fluorescence in human epidermal melanocytes initiated my scientific interest in these cells. Prota and Nicolaus demonstrated that oxidation products of cysteinyldopas are the main components of pheomelanin. Our identification of 5-S-cysteinyldopa as the source of formaldehyde-induced fluorescence of normal and pathological melanocytes started a series of investigations into this amino acid, enzymatic and non-enzymatic oxidation of catecholic compounds and the metabolism of thiols. All melanocytes with functioning tyrosinase produce cysteinyldopas and the levels of 5-S-cysteinyldopa in serum and urine are related to the size and pigment forming activity of the melanocyte population. The determination of 5-S-cysteinyldopa in serum or urine is a sensitive diagnostic method in the detection of melanoma metastasis. Some non-specific formation of cysteinyldopa is present in the body, as demonstrated by 5-S-cysteinyldopa in individuals with tyrosinase-negative albinism.

Adrenergic and acetylcholinesterase-positive innervation of palatine tonsils in mammals

Innervation of human, feline and rabbit palatine tonsils was investigated. Adrenergic nerve components were visualized by formaldehyde-induced fluorescence of catecholamines and 5-HT, or by glyoxylic acid fluorescence, whereas acetylcholinesterase (AChE)-positive nerve structures were demonstrated by the direct thiocholine method. The largest density of adrenergic and AChE-positive nerve profiles was found in the adventitia of arterial branches in the fibrous capsula and septa, mainly in the form of periarterial nerve plexuses of different density. Fine nerve fibres lined the wall of small arteries which penetrated into extrafollicular lymphoid tissue and marginal layers of follicles. It is concluded that there are significant species-specific differences related to density, nature and topographic relations of adrenergic and AChE-positive nerve fibres in the various structural parts of palatine tonsils.

Anatomical distribution of serotonin‐containing neurons and axons in the central nervous system of the cat

By using a monoclonal antibody to serotonin (5-HT), an immunohistochemical study was undertaken to provide a comprehensive description of the 5-HT-containing neurons and of the distribution of their axonal processes in the cat brain and spinal cord. The localization of cell bodies was comparable to that previously reported in studies using formaldehyde-induced fluorescence and other 5-HT antibodies, with a large proportion of labeled neurons in the raphe nuclei and a minor, yet not negligible number, in the ventral, lateral, and dorsal reticular formation. The ascending efferent non-varicose axons were best visualized in sagittal sections and mainly seen taking a rostroventral direction through the tegmentum. The varicose axons could be grossly classified into thin and large fibers, according to the size and shape of the immunoreactive varicosities, which were elongated (up to 2 microm in length and 1 microm in width) or round (2-4 microm in diameter). Varicose axonal arborizations invaded almost every region of the gray matter and avoided large myelinated bundles except in the spinal cord. Variations in the density of the plexuses of immunoreactive fibers generally followed the anatomical divisions and were also observed within nuclei, especially in laminated structures. Only the superior olivary complex could be regarded as devoid of 5-HT-containing axons. A few areas contained extremely rich fiber plexuses. These were the olfactory tubercle, nucleus accumbens, ventral mesencephalon, periventricular gray from the hypothalamus to the pons, facial nucleus, subdivisions of the inferior olive, and the intermediolateral nucleus in the spinal cord. Varicose axons formed tight pericellular arrays in the neocortex, mainly the ectosylvian gyrus, and in the lateral septum and medullar magnocellular nucleus. These data, combined with those of the literature concerning the synaptic versus non-synaptic mode of termination of the 5-HT-immunoreactive varicosities and the high number of distinct receptors, are indicative of the multiple possible actions of serotonin in the central nervous system.

Formaldehyde Induced Fluorescence (F.I.F.)

Formaldehyde Induced Fluorescence (F.I.F.)

The Distribution of Noradrenergic Nerves in the Human Lower Urinary Tract

The purpose of this presentation is to describe the distribution of noradrenergic nerves in the human genitourinary system. The techniques which have been employed include formaldehyde-induced fluorescence and immunocytochemical methods to demonstrate dopamine β-hydroxylase and tyrosine hydroxylase. These methods have been applied to human fetal, neonatal, infant, child and adult tissues removed either at post mortem examination or by surgical excision. The innervation of the fetal urinary bladder is well established by 13 weeks and, as in older specimens, the detrusor receives a sparse noradrenergic nerve supply. In contrast the smooth muscle of the terminal ureter is well supplied by this type of autonomic nerve. An additional incomplete muscle layer has been identified as a nomal component of the terminal ureter which is richly innervated by noradrenergic nerves. In some cases this muscle forms a complete collar which may be responsible for ureteric obstruction. By comparison with the detrusor, bladder neck smooth muscle receives a dense noradrenergic nerve supply particularly in the male. Unlike the detrusor, the structure and innervation of the vas deferens, seminal vesicle and prostate are poorly differentiated in the fetus. In the infant and child, the structure of the intramural smooth muscle of these organs remains immature although a rich noradrenergic nerve supply resembing the adult has been established in the fetus by 30 weeks. In the fetus, autonomic ganglia occur in association with noradrenaline rich paraganglia and surprisingly, with sensory nerve endings resembling pacinian corpuscles. Shortly after birth paraganglia are no longer associated with the autonomic ganglia of the genitourinary system. On the basis of size at least two types of autonomic neuron populate these autonomic ganglia. One type is relatively large and devoid of catecholamines but is closely associated with pericellular noradrenergic nerve fibres. The second type of neuron is small, contains noradrenaline and is arranged in clusters closely related to the capsule of the prostate gland. The significance of these observations will be considered with respect to the neurological control of the genitourinary system.

Development of serotoninergic chick retinal neurons

Numerous neurotransmitters have been studied in detail in the developing retina. Almost all known neurotransmitters and neuromodulators were demonstrated in vertebrate retinas using formaldehyde-induced fluorescence, uptake autoradiography or immunohistochemistry procedures. Serotoninergic (5HT) amacrine neurons were described in the inner nuclear layer (INL) of the retina with their dendrites spreading within the inner plexiform layer (IPL). The present work describes the morphological pattern of development of serotoninergic amacrine neurons with a stratified dendritic branching pattern in the chick retina from embryonic day 12 to postnatal day 7. Serotoninergic-bipolar neurons are also described. 5HT-amacrine neurons have round or pear-shaped somata and primary dendritic trees oriented toward the IPL that runs through the INL, showing several varicosities. Secondary dendrites then go through the INL, without any collateral branch. At the outer and inner margin of the IPL the primary and secondary dendrites originate an outer and an inner serotoninergic network, respectively. When the primary dendritic tree reaches the IPL it deflects laterally in sublayer 1—the outer serotoninergic network. Tertiary branches then arise from the secondary dendrite and deflect in the innermost sublayer of the IPL— the inner serotoninergic network. The final pattern of branching of 5HT amacrine cells was present at embryonic day 14 and was completely developed at hatching. Serotoninergic (5HT) bipolar neurons were also present in the INL at hatching. They are weakly immunoreactive and are probably a subset of bipolar cells that accumulate serotonin from the intersynaptic cleft and are not ‘‘true’’ 5HT neurons.

Neurogenetics of Synaptic Transmission in Caenorhabditis eleguns

Publisher Summary Caenorhabditis elegans are widely used as powerful tools for the analysis of mutants and genes. From the perspective of a mammalian neurobiologist, there are two basic strategies that may profitably be employed using C. elegans . Firstly, the basic information derived from C. elegans biology and molecular genetics is applied to mammalian systems; that is, genes first characterized in C. elegans are used to identify mammalian homologues. The other powerful use of C. elegans is to start with known mammalian biology and genes and then identify the C. elegans homologues of these genes. The availability of a large collection of C. elegans mutants has permitted careful in vivo analysis of cholinergic regulation and the behavioral consequences of cholinergic hypofunction. Animals completely lacking the vesicular acetylcholine transporter (VAChT) protein cannot grow or survive after hatching. Animals with milder mutations in the unc-17 gene are small, slow-growing, and display a number of neurornuscular deficits. They are also quite strongly resistant to inhibitors of acetylcholinesterase. All of these phenotypes are shared by cha-1 mutants that argue that vesicular transport is necessary for cholinergic function. Three amine neurotransmitters have been identified thus far in C. elegans : Dopamine (DA), formaldehyde-induced fluorescence (FIF), and serotonin (5HT). The C. elegans Genome Sequencing Project has identified a genomic sequence that appeared to encode part of a vesicular monoamine transporter (VMAT)-like protein. The C. elegans VMAT homologue appears to be encoded by the previously identified cat-2 gene. Using these transporter genes and mutants, together with standard C. elegans tools, it can be now possible to analyze the differentiation of particular neuronal cell types and also the contribution of particular protein domains to cellular and overall behavioral function.

Dopamine efflux from the carotid body during hypoxic stimulation.

The carotid bodies of cats are known to contain high levels of dopamine (DA), persisting even after complete denervation of these organs (Zapata et al, 1969; Mir et al, 1982; Fitzgerald et al, 1983). Thus DA appears to be concentrated in glomus cells, characterized by their chromaffin reaction, abundance in dense-core granules and formaldehydeinduced fluorescence (see Eyzaguirre & Zapata, 1984). Since the DA content and the number of dense-core granules are reduced in direct proportion to the severity and duration of hypoxia in rats (Hanbauer & Hellström, 1978; Hansen, 1981), and radiolabeled DA effluxes from rabbit and cat carotid bodies —in parallel with the frequency of carotid chemosensory discharges (f x)— are also proportional to the intensity of hypoxic superfusions (Fidone et al, 1982; Rigual et al, 1986), the idea that DA serves as the glomus cell to sensory nerve endings transmitter during hypoxia is highly attractive (see Gonzalez et al, 1994).KeywordsCarotid BodyGlomus CellDopamine EffluxHypoxic ChallengeHypoxic StimulationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Instrumentation of ramus circumflexus compromises the innervation of coronary artery and myocardium

A fine non-compressing methacrylate ring was implanted on the ramus circumflexus (RC) in 13 dogs. After the animals survived 7–14 days, the distribution and the rate of the Wallerian degeneration was studied in coronary arteries and myocardium by means of the formaldehyde induced fluorescence (FIF) technique and the transmission electron microscopy. The density of vasomotor innervation in the main arterial trunks was expressed as the number of transsected terminals per 1 mm 2 of the transversally sectioned media; the density of adrenergic terminals in the myocardium was determined by the point-counting method. A compact scar encompassing the ring was found. The scar compressed some perivascular nerves. As a consequence, by about 40% of terminals degenerated in the RC. The innervation of the branching of this artery was compromised as well. Moreover, the density of nerve terminals in myocardium decreased around the whole circumference of the ventricles and in the septum, equally by 29%. The value was lower than that found after the instrumentation of ramus interventricularis anterior (50%). The results indicate (i) a compromise of the innervation of coronary artery and myocardium after the instrumentation of RC; (ii) the different decrease in density of nerve terminals in the myocardium suggests that majority of adrenergic nerve supply for ventricle myocardium is running along ramus interventricularis anterior.

Neuroepithelial endocrine and nervous system in the respiratory tract of Cynops pyrrhogaster with special reference to the distribution of nitric oxide synthase and serotonin.

The respiratory tract of urodeles harbours an intramural nerve network comprising an innervated system of neuroepithelial endocrine (NEE) cells. However, striking differences have been noted between phylogenetically closely related species. Zamboni- or formaldehyde-fixed whole-mount preparations and sections of the saclike lungs of a Japanese salamander, Cynops salamander, Cynops pyrrhogaster, have been investigated for the immunocytochemical detection of nitric oxide synthase (NOS), serotonin (5-HT), VIP, somatostatin, calcitonin, and bombesin; for the enzyme-cytochemical demonstration of NADPH diaphorase (NADPHd); and for formaldehyde-induced fluorescence. In addition, the ultrastructural morphology has been examined by using glutaraldehyde/osmium tetroxide fixed lung tissues. Ovoid 5-HT-immunoreactive (IR) NEE cells occur singly or grouped in the ciliomucous epithelium of the trachea and lungs of Cynops, and a few somatostatin-, calcitonin-, and bombesin-like IR NEE cells are also observed. These cells exhibit a characteristic neuroendocrine morphology as seen with the electron microscope. In addition, large numbers of 5-HT-IR interstitial cells, with round to oval cell bodies and two or three long, slender, sometimes branching processes, are located preferentially along large blood vessels in the connective tissue capsule of the lung and trachea. Immunoelectronmicroscopy shows that 5-HT is localized over large dense granules in the cell bodies and processes of these interstitial cells. NOS-like immunoreactivity occurs in a nerve plexus composed of thick nerve bundles and nerve cells, and in a fine varicose nerve network that originates at least partly from intrapulmonary NOS-containing nerve cells. VIP-like immunoreactivity appears to be colocalized with NOS in the latter network. All NOS-positive nerve fibres in the lungs of Cynops pyrrhogaster and Ambystoma mexicanum stain for NADPHd. It is concluded that the pulmonary NEE cells observed in Cynops pyrrhogaster are similar to those described in other vertebrate species and that the 5-HT-IR interstitial cells resemble mast cells. In addition, nitric oxide is likely to be a bioactive substance involved in nonadrenergic, noncholinergic inhibitory neurotransmission in the pulmonary nervous system of urodeles, where it may be colocalized with VIP.

Ontogeny of catecholamine systems in the central nervous system of anuran amphibians: An immunohistochemical study with antibodies against tyrosine hydroxylase and dopamine

To get more insight into developmental aspects of catecholamine systems in vertebrates, in particular anuran amphibians, these systems were studied immunohistochemically in embryos and larvae of Xenopus laevis and Rana ridibunda. Antisera against tyrosine hydroxylase (TH) and dopamine (DA) revealed that catecholamine systems are already present at early embryonic stages. The first dopamine group to be detected was found ventral to the central canal of the spinal cord of Xenopus, soon followed by DA cell groups in the posterior tubercle, the hypothalamic periventricular organ, the accompanying cell group of the periventricular organ, and the suprachiasmatic nucleus. Although weakly TH-immunoreactive cells were found in the olfactory bulb at about the same embryonic stages, DA immunoreactivity was not detected until premetamorphic stage 49. Dopamine cell groups in the caudal brainstem, midbrain, and pretectum appeared at late premetamorphic and prometamorphic stages, whereas the preoptic group was first observed at the metamorphic climax stage. Rana showed an almost similar timetable of development of catecholamine cell groups, except for the caudal brainstem group which was already present at the end of the embryonic period. When compared with previous studies by means of formaldehyde-induced fluorescence technique, it becomes clear that TH/DA immunohistochemistry enables an earlier detection of catecholamine cell groups and fiber systems in anuran amphibians. The present study also revealed that the DA-immunoreactive cells of the hypothalamic periventricular organ never stained with the TH antiserum during development, thus supporting their putatively DA accumulating nature. Another notable result is the site of origin and rather late appearance of the midbrain dopaminergic cell group. It is suggested that the latter cell group only partly corresponds to the ventral tegmental area and substantia nigra of amniotes.

The visual fields of American horseshoe crabs: two different eye shapes in Limulus polyphemus.

The optical alignment of individual cuticular cones in the dioptric array of the lateral eye of Limulus polyphemus was determined with a precision two-circle goniometer constructed and mounted to the stage of a compound microscope and using a new formaldehyde-induced fluorescence procedure. All measurements were made from the corneal surface of the excised eye mounted in seawater through an air/water interface perpendicular to the optic axis of the microscope. Our results revealed two variants of visual field and eye curvature which can actually be discriminated in casual examination of adult animals. We call animals possessing these two variants "morlocks" and "eloi." Adult male and female morlocks about 25 cm across the carapace have eyes which are relatively elongated, often darker in pigmentation, smaller, and relatively flatter in curvature. Morlocks have a monocular field of view of about 3.13 steradians or 50% of a hemisphere. The coverage averages 115 deg along the vertical axis and 168 deg along the horizontal axis of the eye, with maximum resolution in the anteroventral quadrant. Adult male and female eloi of comparable size have eyes which are relatively more round, often lighter in pigmentation, larger with more ommatidia, and relatively more bulged. Eloi have a monocular field of view of approximately 3.83 steradians or 61% of a hemisphere that covers 145 deg vertically and 185 deg horizontally. Eloi have more uniform resolution than morlocks with best resolution in the posteroventral quadrant. All horseshoe crabs examined, whether morlocks or eloi, have an identical orientation of the margin of the eye relative to the animals' coordinates.

The role of noradrenaline in the differentiation of amphibian embryonic neurons.

The possibility that monoamines might act as signalling molecules during the early development of the nervous system has been examined in embryos of the amphibian Xenopus laevis. The distributions of 5-hydroxytryptamine, dopamine, noradrenaline and their precursor, dopa, were determined from the fertilized egg up to the late neurula stages using High Performance Liquid Chromatography, formaldehyde-induced fluorescence and antibody staining. 5-hydroxytryptamine was not detected until the tail bud stage. The fertilized egg contained significant concentrations of dopa (10(-6) M) and dopamine (10(-7) M). Both monoamines persisted with little change in concentration up to the late neurula stage. Early neurula stage embryos contained very low levels of noradrenaline. Aldehyde-induced fluorescence showed that monoamines are localized in dorsal regions of the embryo, in ectoderm and mesoderm cells. Monoamines were not present in endoderm cells. Immunocytochemical staining showed dopamine predominantly in the ectoderm, except in future neural regions where it was found also in the mesoderm. Dopamine staining was always most intense in dorsal regions of the embryo. The consequences for subsequent neuronal differentiation of interfering with the biosynthesis and receptor binding of monoamines during neurulation was assayed. Neuronal differentiation was monitored quantitatively in cultures set up as the neural tube closed and qualitatively in intact tadpoles that were left to develop for two days after washout of test reagent. The number of neurons, the number of muscle cells and the total number of differentiated cells were counted after 18-24 hours of culture. Comparison of the number of neurons that differentiated from control and treated embryos showed that inhibition of dopamine beta-hydroxylase, the enzyme catalysing the conversion of dopamine to noradrenaline, during the neural plate stages reduced substantially subsequent neuronal differentiation. The differentiation of myocytes and the total number of differentiated cells were not affected. Exogenous noradrenaline (10(-6) M) or dopamine (10(-6) M) could increase the number of neurons that differentiated subsequently in culture. Interfering with noradrenaline binding to receptors with receptor antagonists during neurulation showed that alpha-adrenergic receptor antagonists reduced substantially the subsequent differentiation of neurons. The differentiation of myocytes and the total number of differentiated cells were not affected. The effect of alpha-adrenergic receptor antagonists was overcome by the simultaneous inclusion of noradrenaline or alpha-receptor agonists, but not agonists at beta-adrenergic receptors. The quantitative reduction in the differentiation of neurons was paralleled by defects in the Central Nervous System of intact tadpoles.(ABSTRACT TRUNCATED AT 400 WORDS)

Sympathetic neurons outlive the original host after transplantation to the rat submandibular gland

Sympathetic ganglion tissue of aged (36 months) Wistar rats was allotransplanted into the submandibular gland (SMG) of young (3 months) animals to study whether sympathetic neurons can outlive the original host. The visbility of the transplants was evaluated one yeat postgrafting, using the formaldehyde-induced fluorescence technique (FIF) for histochemical demonstration of catecholamines, tyrosine hydroxylase (TH) immunohistochemistry, and morphometry. One year after transplantation, grafted superior cervical ganglion (SCG) cells demonstrated catecholamine fluorescence and tyrosine hydroxylase immunoreactivity. The transplants consisted of groups of sympathetic neurons dispersed in a fibrous matrix. After long postgrafting time, the sympathetic neurons of aged rats showed several signs of enhanced degeneration; increased autofluoroscent lipopigment, decreased neuronal density and reduced catecholamine fluorescence. The mean diameter of the transplanted aged neurons was significantly decreased. The histograms of grouped diameter values showed a shift to smaller cells in ganglion transplants. A subpopulation of small and medium-sized grafted neurons sent out fluorescent fibers, which were located in a fibrous scar area but did not extend into submandibular host tissue. The results indicate that a long postgrafting time induced degeneration which is comparable to normal aging changes in grafted very old neurons. Thus, aged sympathetic neurons maintain plasticity to survive as transplants, and under favourable conditions these neurons outlive the original host.

Organization of catecholaminergic systems in the hypothalamus of two elasmobranch species, Raja undulata and Scyliorhinus canicula. A histofluorescence and immunohistochemical study.

We examined the organization of catecholaminergic neurons in the hypothalamus of the painted ray, Raja undulata, and the small-spotted dogfish, Scyliorhinus canicula, with the use of formaldehyde-induced fluorescence (FIF) methods and tyrosine hydroxylase (TH) immunohistochemistry. In both species we identified distinct populations of catecholamine-containing neurons differing in a) their immunoreactivity to antibodies against the enzyme tyrosine hydroxylase (TH), b) their fluorescence in response to FIF methods for the detection of catecholamines, and c) their relationship with the third ventricle. One population is made up of FIF-positive and TH-negative neurons (most of which are CSF [cerebrospinal fluid]-contacting) and located in two circumventricular organs, the preoptic recess organ and the organon vasculosum hypothalami. Another population comprises TH-immunoreactive (TH-IR), FIF negative neurons that are located in the suprachiasmatic nucleus and the posterior tuberculum and are not related to the third ventricle recesses. A third population of TH-IR, CSF-contacting neurons is also present in the organon vasculosum hypothalami. The existence of three catecholaminergic populations suggests differences in the metabolism of catecholamines and/or different functions. The circumventricular neurons are not associated with the hypophysis and appear to accumulate catecholamine (dopamine) obtained from exogenous sources. In both Raja and Scyliorhinus the neurointermediate lobe is innervated by TH-IR fibres originating from dopamine-synthesizing neurons of the second catecholaminergic population.