Formaldehyde Fluorescence Technique Research Articles

Beta adrenergic receptors in the parathyroid glands.

The location of beta-adrenoceptors in human parathyroid gland was studied using an immunohistochemical method. Frozen sections of human parathyroid glands, taken from surgical samples, were treated with (-)-alprenolol, washed and exposed to (-)-alprenolol antibodies conjugated with fluorescent dyes. The (-)-alprenolol was bound to the parathyroid principal cells and to the main blood vessels. On the contrary, adrenergic nerve fibres, demonstrated with formaldehyde fluorescence technique, were only located within the walls of main blood vessels. The findings are discussed.

Transmitter histochemistry of the rat olfactory bulb. II. fluorescence histochemical, autoradiographic and electron microscopic localization of monoamines

The rat olfactory bulb was studied with the Falck-Hillarp formaldehyde fluorescence technique, including recent modifications, autoradiography and electron microscopic cytochemistry (permanganate fixation). Some periglomerular cells and few superficial tufted cells take up and accumulate catecholamines and precursors. They probably represent dopamine cells. In the glomeruli, probable 5-hydroxytryptamine nerve terminals could be identified. In the granular and external plexiform layers, noradrenaline axon terminals were present. At the ultrastructural level, monoamine boutons were in synaptic contact with dendritic spines of granule cells and in the glomeruli, probable 5-hydroxytryptamine boutons formed synapses with periglomerular cell dendrites. Therefore, monoamine afferents to the bulb may exert their influence via interneurons.

Catecholaminergic structures in the nervous system of three nematode species, with observations on related enzymes

The formaldehyde fluorescence technique has been used to demonstrate amine‐specific fluorescence in the nervous system of three nematode species, Prionchulus punclatus Cobb, Panagrellus redivivus Goodey, and Aphelenchus avenae Bastian. Examination of some of the physical and chemical properties of this fluorescence has shown it to be due principally to the primary catecholamine dopamine. Dopamine and dopa decarboxylase were also detected biochemically in A. avenae. Dopamine has now been proposed as a putative neurotransmitter in a number of nematode species and the role of this and other biogenic amines in nematodes is discussed. Of the two principal enzymes involved in the metabolism of monoamines, catechol‐O‐methyltransferase was detected in both A. avenae and P. redivivus but monoamine oxidase could not be detected in these or other nematode species.

The effect of intraspinal microinjections of 6-hydroxydopamine on the inhibitory influence exerted on spinal sympathetic activity by the baroreceptors.

1. The inhibition of spinal somato-sympathetic reflex activity, following electrical stimulation of the carotid sinus nerve in cats has been studied before and after chronic intraspinal microinjections of 6-hydroxydopamine into the cervical spinal cord. 2. The effect of these microinjections on bulbospinal catecholaminergic neurones was assessed using the formaldehyde fluorescence technique for the demonstration of intraneuronal monoamines. 3. In 5 6-hydroxydopamine-treated animals, inhibition of the spinal sympathetic reflex response following stimulation of the sinus nerve was reduced compared to control untreated animals. When damage to the bulbo-spinal catecholamine system in the dorso-lateral funiculi was extensive, the inhibition was almost completely abolished; with more limited destruction a lesser effect was observed. 4. These effects were not seen in a group of control animals, which received intraspinal microinjections of the 5-hydroxytryptamine neurotoxin 5,6-dihydroxytryptamine and which showed damage to the indoleaminergic neuronal system but no damage to the bulbo-spinal catecholaminergic system. 5. It is concluded that the inhibitory effects of the baroreceptors on spinal sympathetic activity are mediated by bulbo-spinal catecholaminergic neurones.

Recent developments in monoamine histochemistry

Publisher Summary This chapter discusses the recent developments in the formaldehyde fluorescence technique of Falck and Hillarp to study monoamine histochemistry. With the routine procedure of the Falck–Hillarp technique, that is treatment of freeze dried tissues with formaldehyde vapors, it is extremely difficult to visualize very fine noradrenaline nerve terminals such as those present in cortical areas. Furthermore, the dopamine (DA), nerve terminals, for example, in the neostriatum do not appear as distinct dot-like fluorescent structures but as a diffuse fluorescence indicating a diffusion of the amines out of the nerve endings during the processing procedures. This technique has recently been improved6 by the introduction of glyoxylic acid, which can be used instead of formaldehyde at the perfusion and partly also at the vapour reaction step. The latter procedure is advantageous especially for the visualization of DA. Moreover, the combination of different histochemical techniques, for example, formaldehyde fluorescence with immunofluoresence, performed either on consecutive sections or on the same section, may be valuable for studying the morphological interrelationships between various neuron systems.

Ultrastructural identification of catecholamine neurones in the hypothalamic periventricular-arcuate nucleus-median eminence complex with special reference to quantitative aspects

Summary The basal hypothalamus of the rat, with special reference to the external and internal layer of the median eminence, the arcuate nucleus and the periventricular area, was studied with the formaldehyde fluorescence technique of Falck and Hillarp and with electron microscopy. The presence in boutons of so-called small granular vesicles,i.e., sinaptic vesicles with a diameter of about 50 nm containing an electron-dense core, was used as a criterion for identification of monoamine boutons at the ultrastructural level. After a routine glutaraldehyde-OsO4 fixation procedure no such boutons could be identified. However, after KMnO4 fixation small granular vesicles were found in boutons in the internal layer of the median eminence, the arcuate nucleus and in the periventricular region,i.e., in regions assumed to contain mainly NA axon terminals. However, after administration of exogenous amines such as 5-hydroxydopamine (5-OH-DA) or 6-hydroxydopamine (6-OH-DA), eitherin vivo by intraventricular injections or to brain slicesin vitro, it was also possible to identify boutons with small granular vesicles in the external layer of the median eminence, a region containing predominantly DA terminals. When exogenous amines were used such boutons were also demonstrated in all regions studied after glutaraldehyde-OsO4 fixation although in our hands this procedure gave less distinct dense cores as compared with KMnO4 fixation. The quantitative study revealed that the density of monoamine boutons is highest in the superficial, lateral parts of the median eminence, where about 33% of all boutons, corresponding to a density of about 120 such boutons/500 sq.μm area, belong to monoamine neurones. In the arcuate nucleus, on the other hand, the corresponding values were 2.6% and 2/500 sq.μm. These results demonstrate that in the external layer,i.e., the neurohaemal contact zone, about two-thirds of all boutons belong to non-monoamine neurones, which tentatively may be supposed to store and release the releasing and/or inhibitory factors controlling the hormone secretion from the anterior pituitary. Several studies have indicated an important functional role of DA in the external layer of the median eminence, mainly in the control of gonadotropin secretion. However, the present results do not give a definite answer as to the site of action of the amine. With the techniques used, including ethanolic phosphotungstic acid impregnation, no typical ‘synapses’ between axon profiles could be identified. Such synapses would strongly have supported the view of an axo-axonic influence of DA on the axon terminals containing the releasing and/or inhibitory factors.

The histochemical fluorescence method for the demonstration of catecholamines. Theory, practice and application.

The formaldehyde fluorescence technique is a highly sensitive and specific method for the demonstration of catecholamines (CA) at the cellular level. It permits the demonstration of as little as about 5 x l0–4 pg noradrenaline, present in a single varicosity. The method can also be used for quantitative microfluorimetric studies on the CA stores at the cellular level, although a concentration-dependent quenching of the fluorescence in locations where the CA concentration is very high may impose some limitations. The sensitivity of the technique has been increased by using sections of unembedded tissue from unfixed or formalin-fixed tissue (Hökfelt and Ljungdahl). A glyoxylic acid technique, recently introduced by Axelsson and his co-workers, may prove to be an additional reliable alternative method for the demonstration of CA. Examples of neurobiologic problems that could be approached by using the formaldehyde fluorescence technique are briefly reviewed.

Modification of the Falck-Hillarp formaldehyde fluorescence method using the Vibratome: simple, rapid and sensitive localization of catecholamines in sections of unfixed or formalin fixed brain tissue.

Two modifications of the original Falck-Hillarp formaldehyde fluorescence technique are presented, both based on a recently introduced instrument, the Vibratome®, which permits cutting of unembedded tissue with a section thickness down to 10 μ,.

Histochemical localization of monoamines in the nervous system of Dipylidium caninum (Cestoda) by the formaldehyde fluorescence technique

Shield J. M., 1971. Histochemical localization of monoamines in the nervous system of Dipylidium caninum (Cestoda) by the formaldehyde fluorescence technique. International Journal for Parasitology, 1: 135–138. The Falck and Hillarp formaldehyde fluorescence technique was used to study the localization of biogenic monoamines in the nervous system of the cestode, Dipylidium caninum. Monoamines were found in nerve fibres of the longitudinal nerve cords, muscular body wall, cirrus and copulation canal.

Gastric and pancreatic sympathetic denervation in the rat. Technique and results.

The extirpation of the coeliac and superior mesenteric ganglia was found to be a simple and effective method of achieving complete adrenergic denervation of the stomach and pancreas in the rat. Thus, using the formaldehyde fluorescence technique, no remaining adrenergic nerves in these organs could be found after ganglionectomy. The vagus nerves did not significantly contribute to the adrenergic innervation of the stomach and pancreas. Chemical methods revealed minute amounts of noradrenaline in the pancreatic tissue even after complete ganglionectomy. The cellular source of this catecholamine is unknown. Sympathetic denervation of the stomach did not change the gastric acid secretory pattern, as studied with chronic gastric fistula rats.

Dopamine and Mast Cells in the Adipose Tissue of the Ox and the Effect of Noradrenaline and Dopamine Infusions on the Plasma Unesterified Fatty Acids

SUMMARY The catecholamines of ox adipose tissue were extracted and separated using thin layer chromatography. The predominant catecholamine present has the same R f value as standard dopamine. Using a formaldehyde fluorescence technique it was shown that the tissue mast cells of ox adipose tissue contained a primary catecholamine which was thought to be dopamine. Using acridine orange stain the same cells were then shown to contain heparin. Intravenous infusion of 1 μg./kg. body weight)min. of noradrenaline caused an increase in the plasma unesterified fatty acids (UFA) from 8·2 to 36·0 mg./100 ml. and infusion of dopamine (5 μg./kg. body weight/min.) caused a rise in plasma UFA from 5·4 to 11·1 mg./100 ml. The action of bothnoradrenaline and dopamine on plasma UFA could be blocked by the β-blocking agent propranolol.

FLUORESCENCE HISTOCHEMICAL FINDINGS OF THE STOMACH WALLS IN RESPONSE TO ULCEROGENIC STIMULI IN RATS

Depending upon the origin of the stimuli which signal the secretion of gastric juice, three distinct aspects of secretion have been elucidated: the cephalic, gastric and intestinal. The efferent cholinergic mechanism is involved in the cephalic phase of gastic secretion, because cutting of the vagi just above the stomach completely eliminates the secretion. On the other hand, participation of gastrin to the gastric phase of secretion and its chemical structure have recently been demonstrated (1). However, the gastric secretion mechanism is more complicated. It is possible that the intact cholinergic innervation (2) and the presence of endogenous monoamines in the gastric wall (3) are the prerequisite to the gastrin-induced gastric secretion. There are controversial reports regarding the effect of adrenergic stimuli on the gastric secretion (4-7). In the mucous membrane of the glandular portion of the rat stomach, dopa-decarboxylase activity is high (8), but the high activity is not explained by the number of mast cells (9) and adrenergic nerves (10, 11) in the gastric mucosa. The distribution of the enterochromaffin cells is also different from that of gastric dopa decarboxylase (8). Intraperitoneal injection of dopa or 5-hydroxytryptophan (5-HTP) to rats causes a marked increase in the concentration of dopamine and 5-hydroxytryptamine (5-HT) in the gastric mucosa, and the amines are located to an enterochromaffin-like cell system in the pyloric part of the gastric mucosa (8). However, the physiological significance of the uptake and decarboxylation of dopa or 5-HT remains to be settled. In the present experiments the distribution of the endogenous monoamines in the structures of the gastric wall of the rat was studied using a formaldehyde fluorescence technique (12, 13). The temporal changes of the monoamine fluorescence were also followed in the course of formation of the gastric ulcers in the Shay rat and the restraint, cold-exposed rat.