Observations on the cellular localization of dopamine in the caudate nucleus of the rat

Summary The cellular localization of dopamine in the caudate nucleus of the rat hat been studied with the highly sensitive and specific fluorescence method of Falck and Hillarp, and by electron microscopy. The histochemical studies provided strong support for the view that the dopamine is concentrated within very fine nerve fibres which have abundant varicosities with an intense fluorescence. The electron microscopical studies revealed the presence of a tightly packed plexus built up i.a. of abundant synaptic nerve terminals, many of which had a diameter below 0.4 . The terminals made synaptic contact mainly with processes that seemed to belong to an extensive dendrite net.The investigation was supported by research grants from the United States Public Health Service (02854-04), The Swedish Medical Research Council and the Knut and Alice Wallenberg Foundation.     

Neuronal nitric oxide synthase immunoreactivity in the respiratory tract of the frog,Rana temporaria

Summary Physiological and histochemical studies have recently supported the notion that nitric oxide (NO) is the transduction signal responsible for the non-adrenergic, non-cholinergic relaxation of the vasculature as well as the airways of the mammalian lung. We report the presence of immunoreactivity to NO synthase (NOS) in nerve cell bodies and nerve fibres in the neural plexus of the buccal cavity and lungs of the frog, Rana temporaria, using the indirect immunocytochemical technique of avidin-biotin and the NADPH-diaphorase technique. The neural ganglia located next to the muscle layer and within the connective tissue of the buccal cavity were partially immunoreactive for NOS. In the lungs, NOS immunoreactivity occurred in nerve cell bodies, as well as in both myelinated and unmyelinated nerve fibres. Fine nerve fibres immunoreactive to NOS were observed within the muscle fibre bundles and next to the respiratory epithelium. Both the presence of NOS immunoreactivity and the positive histochemical reaction for NADPH-diaphorase in the neural plexus of amphibian respiratory tract suggests a broad evolutionary role for NO as a peripheral neurotransmitter.     

The monoamines in molluscs. II. Dopamine and neurotransmission. Cardiac dopaminergic innervation in Helix pomatia (author's transl)]

In the molluscs, dopamine is very probably a chemical transmitter at the level of both the central nervous system and certain peripheral structures. The heart of Helix pomatia does not have any intrinsic innervation, but it receives extrinsic innervation from fibres coming from the visceral nerve. Formaldehyde fluorescence histochemistry localizes the cardiac catecholamines in some of these fibres and in their endings. However, dopamine, which dominates, does not seem to be a transmitter involved in cardioregulation in the same way as 5-hydroxytryptamine. The quantities of active dopamine (stimulants) cannot be compared with those required for a neurotransmitter. This is also true for noradrenaline. Dopamine more certainly plays a role at the metabolic and tropic level by acting within a more or less short period as a regulator of cellular activity and contractility. The Helix heart is a suitable model for future research in this field.     

Histochemical localization of cholinesterases and monoamines in the central heart of Sepia officinalis L. (Cephalopoda)

Summary The central heart of the coleoid cephalopod, Sepia officinalis, was studied using acetylcholinesterase and fluorescence histochemistry. Using histo- and cytochemical reactions, acetylcholinesterase was localized in the axolemma and axoplasm of specific cardiac nerve fibres, as well as in the sarcolemma and within the sarcotubular system of the muscle cells. Butyrylcholinesterase exhibited a different distribution, being found only in the luminal trabecular muscle layer. Glyoxylic-acid-induced fluorescence indicated the presence of catecholamines (emission maximum 470 nm) in cardiac nerve axons. These histochemical findings support the hypothesis that noradrenaline and/or dopamine and acetylcholine act antagonistically as natural transmitters. Fluorophores indicating the presence of serotonin were not observed. The present results are discussed in the light of previous pharmacological findings.     

Nerves and nerve endings in the skin of tropical cattle.

The nerves and nerve endings in the skin of tropical cattle were studied using histological and histochemical techniques. Many nerve trunks and fibres were present in the reticular and papillary dermis in both hairy and non-hairy skin sites. In non-hairy skin locations such as the muzzle and lower lip, encapsulated endings akin to Krause and Ruffini end bulbs, which arise from myelinated nerve trunks situated lower down the dermis were observed at the upper papillary layer level. Some fibre trunks seen at this level extended upwards to terminate within dermal papillae as bulb-shaped longitudinally lamellated Pacinian-type endings, while other onion-shaped lamellated nerve structures were located either within dermal papillae or near the dermo-epidermal area. Intraepidermal free-ending nerve fibres, appearing non-myelinated were observed in areas with thick epidermis. Intraepidermal free-ending nerve fibres, appearing non-myelinated were observed in areas with thick epidermis. On hairy skin sites, however, organized nerve endings or intraepidermal nerve endings were not readily identifiable.     

Combination of non-specific cholinesterase histochemistry and immunofluorescence staining for the study of the sensory innervation of skin and muscle

Summary In the present study we describe the application of the non-specific cholinesterase (nChE) histochemical method for the detection of encapsulated sensory nerve endings prior to immunofluorescence staining of the sensory nerve fibres. The nChE staining of Schwann-derived structures surrounding sensory terminals allowed us to identify unequivocally the sensory corpuscles in the skin and the muscle proprioceptors (muscle spindles and Golgi tendon organs) in longitudinal sections of muscle tissue. The nChE staining of sensory nerve endings and immunofluorescence-labelled nerve fibres and their terminals could be viewed and photographed in the same section using appropriate filters. Since nChE activity persists in terminal Schwann cells for a long time after loss of the sensory axons, this combined enzyme- and immunohistochemical approach is also useful for experimental studies involving denervation and re-innervation of sensory nerve endings.     

Histochemical and Ultrastructural Features of the Biceps Brachii of the African Chameleon (Chamaeleo senegalensis)

Abstract Characteristics of reptilian muscle fibres were investigated in the biceps brachii of the African chameleon, Chamaeleo senegalensis. Fibres were classified as slow and fast. These types of fibre were distinguished on the basis of histochemical staining for myofibrillar ATPase (mATPase). Fast fibres stained dark for mATPase while slow fibres stained light. The patterns of innervation of slow and fast fibres were investigated by staining nerve endings for acetylcholinesterase activity. Slow fibres have a pattern of multiple innervation, whereas fast fibres are associated with individual endplates. The organization of the myofibrils and the sarcoplasmic reticulum in slow muscle fibres from the chameleon biceps brachii was compared with that in fast fibres. Slow fibres lacked an M-line and the Z-lines were uneven. They had fibrils that were not clearly separated from each other and the sarcoplasmic reticulum was poorly developed. These features are in sharp contrast to those of fast fibres which had straight Z-lines, clear M-lines and well-developed sarcoplasmic reticulum.     

Evidence for exclusive adrenergic innervation of feather muscles (mm. pennati) in the chicken

Summary Feather follicles in the avian skin are interconnected by well-defined bundles of smooth muscle cells, which are responsible for the erection and depression of feathers and thus play an important role in thermoregulation. The depressing and erecting muscle bundles were found to receive a very dense supply of unmyelinated nerve fibres that displayed ultrastructural and histochemical characteristics of noradrenergic axons (formaldehyde- and glyoxylic acid-induced catecholamine fluorescence; uptake to 5-hydroxydopamine). No nerve fibres were encountered showing histochemical acetylcholinesterase activity. There was no indication of the presence of peptidergic or purinergic nerve endings.The neuromuscular space usually ranged from 40–60 nm in width and contained a basal lamina. Occasionally, this space was reduced to approximately 20 nm. At such close neuromuscular contacts a basal lamina was lacking, and focal densities beneath the pre- and postsynaptic plasma membrane were observed. Since no gap junctions between muscle cells were detected, the dense supply with noradrenergic nerve fibres indicates a high amount of directly innervated smooth muscle cells.An additional finding of the present study was the observation that high local concentrations of 5-hydroxydopamine led to degeneration of noradrenergic nerve endings.Supported by a grant from the Deutsche Forschungsgemeinschaft (Dr. 91)     

The catecholaminergic system of an annelid (Ophryotrocha puerilis,Polychaeta)

Summary The complex catecholaminergic (CA) nervous system of the polychaete Ophryotrocha puerilis is documented using glyoxylic acid induced fluorescence (GIF) and immunohistochemistry. CA-neurons are found both in the central and peripheral nervous system. In the brain, about 50 CA-neurons are present in the perikaryal layer together with numerous CA fibres. Two pairs of CA perikarya are characteristic for each ganglion of the ventral nerve cord. CA-neurites in the ventral nerve cord are mainly arranged in 4 strands paralleling the longitudinal axis of the worm. Fluorescent neurons with receptive ciliary structures are present in body appendages (antennae, palps, urites, parapodial cirri), in the body-wall, and within the oesophageal wall. Furthermore, a subepidermal nerve net of free CA nerve endings has been found. After incubation of specimens with dopamine prior to the development of GIF more fluorescent perikarya could be observed; the fluorescence was also intensified. Pre-incubation with reserpine reduced the intensity of GIF. Results of high pressure liquid chromatography and immunostaining with a polyclonal antibody against a dopamine-glutaraldehyde-complex suggest that dopamine is the major CA transmitter. It is thought that dopaminergic neurons together with ciliary receptive structures act as mechano- and/or chemoreceptors.     

Fine structure of synapses in the hippocampus of the rabbit with special reference to dark presynaptic endings

Summary The stratum radiatum of h ₃ and h ₄ in the hippocampus of the rahbit, where the mossy fiber endings are distributed, was investigated under the electron microscope. These regions contain a certain number of electron dense presynaptic endings. These are characterized by highly dense synaptic vesicles and mitochondrial matrices. The dense endings are not considered as degenerated. Electron dense silver particles, substituted for zinc, occurred on the synaptic vesicles of these dense terminals as well as the mossy fiber endings after the application of Timm's histochemical method modified for electron microscopy. It is concluded that the dark synaptic endings observed might represent mossy fiber terminals in a special functional phase, or might be the result of structural alteration in the course of tissue preparation. The zinc localized in the synaptic vesicles is thought to be associated with the neurotransmitter present in these endings.     

Intrinsic innervation of the rat knee joint articular capsule and ligaments

In spite of the practical importance of having a detailed knowledge of knee joint innervation to understand the pathophysiologic aspects, little information is now available concerning the density and pattern of the nerve fibres which are distributed to it. The present study has been designed to investigate the density and distribution of nerve fibres and receptor corpuscles in the knee joint articular capsule, cruciate and collateral ligaments in the rat, using the acetylcholinesterase (AChE) histochemical in toto staining technique. The investigation was performed on male Wistar rats of 3 months of age, some of which had been treated with capsaicin to deplete their afferent 'C' fibres of their content of neuropeptides. AChE-positive nerve fibres and different types of receptor corpuscle endings were found within articular capsule and ligaments. The highest density of AChE-positive nerve fibres was noticeable in the fibular collateral ligament followed by the tibial collateral ligament, the posterior cruciate ligament, the anterior cruciate ligament and the articular capsule. In the articular capsule the number of type I endings was higher than in the ligaments. The opposite is true for the other type of receptor corpuscles found as well as for nerve endings. Capsaicin treatment significantly reduced the density of AChE-positive nerve fibres in knee joint ligaments but did not affect nerve fibres in the articular capsule. Moreover, it caused the disappearance of some kind of receptor corpuscles within the collateral and cruciate ligaments. The above data collectively suggest that the AChE in toto staining technique may represent a good method for investigating joint innervation and that a significant percentage of nerve fibres supplying knee joint ligaments is represented by C fibre afferents.     

Organization and ultrastructural identification of the catecholamine nerve terminals in the neural lobe and pars intermedia of the rat pituitary

Summary The central catecholamine innervation of the pituitary neural lobe and pars intermedia of the rat have been identified ultrastructurally and their organization has been investigated in a combined fluorescence histochemical and electron microscopical study. The dopamine analogues, 5-hydroxydopamine and 6-hydroxydopamine, were used to label the catecholamine terminals, and to enable the direct correlation between the fluorescence microscopical and the electron microscopical pictures.The fibre type that was identified as catecholamine-containing was ultrastructurally chiefly characterized by dense-cored vesicles, 500–1200 Å in diameter, intermingled with varying numbers of small empty vesicles. 5-hydroxydopamine was selectively accumulated in these fibres and caused an increased electron density of the granular vesicles as well as of some small normally agranular vesicles, and systemically administered 6-hydroxydopamine caused a selective degeneration of these fibres, most prominently within the neural lobe. The dopaminergic terminals of the neural lobe showed frequent close contacts (80–120 Å), without real membrane thickenings, to neurosecretory axons and to pituicyte processes. It is suggested that these close contacts might signify a direct dopaminergic influence on the neurosecretory axons and/or on the pituicyte processes. The identified central catecholamine fibres were also found to make common synapse-like contacts on the pars intermedia cells, whereas the innervation by neurosecretory fibres was very rare. This suggests that the direct central nervous control of the rat pars intermedia is exerted by the catecholamine neurons. A very special feature of the catecholamine fibres in the pituitary is the occurrence of peculiar, large dopamine-filled droplet-like swellings. Electron microscopically, such large axonal swellings (more than 2 in diameter) were found to contain, in addition to the characteristic vesicles and organelles, strongly osmiophilic lamellated membrane complexes resembling myelin bodies and multivesicular bodies encircling disintegrated vesicles, suggesting that these droplet fibres represent dilated stumps of spontaneously degenerating dopaminergic axons. It is suggested that the dopaminergic neural lobe fibres are undergoing continuous reorganization through degeneration—regeneration cycles, a phenomenon previously suggested for the neurosecretory axons of the neural lobe.Supported by the Deutsche Forschungsgemeinschaft.Supported by Svenska Livförsäkringsbolags Nämnd för Medicinsk Forskning, by The Medical Faculty, University of Lund and by the Ford Foundation.     

Organization of the sympathetic innervation in liver tissue from monkey and man

Summary The sympathetic innervation of the liver of monkey and man has been investigated in a combined fluorescence histochemical, chemical and electron microscopical study. By means of the Falck-Hillarp fluorescence method a dense network of monoamine-containing nerve fibers was visualized in liver tissue of monkey and man. The nerve fibers ran in close contact to both hepatocytes and blood vessels. Chemical quantitations showed high concentrations of noradrenaline in both human and monkey liver. Microspectrofluorometry of the intraneuronal monoamine resulted in spectra characteristic of a catecholamine. For the electron microscopical study the dopamine analogue, 5-hydroxydopamine, was used to label the catecholamine terminals in both human and monkey liver. The nerve profiles, identified as catecholamine-containing, were demonstrated in a perivascular location and in close contact to hepatocytes. No synaptic membrane specializations were present between nerve fibers and hepatocytes. The general ultramorphology and intralobular distribution pattern of nerves in the liver of monkey and man were similar. The present results prove the existence of a sympathetic innervation of hepatocytes and blood vessels in the liver of man and monkey.     

Histochemical localization of cholinesterases and monoamines in the central heart of Sepia officinalis L. (Cephalopoda)

The central heart of the coleoid cephalopod, Sepia officinalis, was studied using acetylcholinesterase and fluorescence histochemistry. Using histo- and cytochemical reactions, acetylcholinesterase was localized in the axolemma and axoplasm of specific cardiac nerve fibres, as well as in the sarcolemma and within the sarcotubular system of the muscle cells. Butyrylcholinesterase exhibited a different distribution, being found only in the luminal trabecular muscle layer. Glyoxylic-acid-induced fluorescence indicated the presence of catecholamines (emission maximum, 470 nm) in cardiac nerve axons. These histochemical findings support the hypothesis that noradrenaline and/or dopamine and acetylcholine act antagonistically as natural transmitters. Fluorophores indicating the presence of serotonin were not observed. The present results are discussed in the light of previous pharmacological findings.     

Sensory innervation of the temporomandibular joint in the mouse

The sensory innervation of the temporomandibular joints (TMJs) of 8 STR/IN mice was investigated by means of light and electron microscopy. Through the cutting of complete semithin sections in series it was possible to investigate the joints thoroughly. Additionally, one joint with its nerve supply was reconstructed three-dimensionally with a computerized three-dimensional programme. The reconstruction was based on one complete semithin section series. The joint's nerve supply originates from the nervus auriculotemporalis and additionally from motor branches of the n. mandibularis: n. massetericus, n. pterygoideus lateralis and the nn. temporales posteriores. The greatest number of nerve fibres and endings is located in the dorsolateral part of the joint capsule. They lie only in the stratum fibrosum and subsynovially. Neither the stratum synoviale nor the discus articularis contain any nerve fibres or endings, whereas the peri-articular loose connective tissue is richly innervated. The only type of nerve ending observed within the joint was the free nerve ending, which is assumed to serve not only as a nociceptor but also as a polymodal mechanoreceptor. Merely within the insertion of the musculus pterygoideus lateralis at the collum mandibulae single stretch receptors of the Ruffini type were observed. Ultrastructurally, they correspond to those described in the cat's knee joint. Neither lamellated nor nerve endings of the Golgi or Pacini type were observed in the joint or in the peri-articular connective tissue. The unexpected paucity of nerve fibres and endings in the TMJ itself of the mouse suggests that the afferent information from the joint is less important for position sense and movement than the afferent information from muscles, tendons and periodontal ligaments.     

Electron microscopic and histochemical observations on different types of nerve endings in the extraocular muscles of the rat

Summary Nerve endings in the extraocular muscles of the rat were submitted to histochemical tests for formalin-induced fluorescence and carboxylic esterases. Acetylthiocholine, butyrylthiocholine and -naphthyl acetate were used as substrates and iso-OMPA, 284C51, eserine and E-600 as inhibitors. The ultrastructure of the endings was studied with the electron microscope.Both single and multiple nerve terminals were observed in all six extraocular muscles. The single terminals of myelinated axons were comparable in their light and electron microscopic structure with the typical motor end plates of other striated muscles, and like these they exhibit acetylcholinesterase (AChE), non-specific cholinesterase (ns. ChE) and non-specific esterase (ns. E) activity. These endings were apposed to twitch-type muscle fibres.The multiple terminals were classified with the light microscope into two types. The larger type was 1/3 of the size of the motor end plate; 2–5 endings innervated the same muscle fibre; subneural infoldings were weakly developed and possessed only slight AChE and ns. ChE and probably no ns. E activity. No subneural lamellae were visible under the light microscope in the smaller type, which also possessed AChE and ns. ChE and was composed of 10–20 small dots dispersed along a single muscle fibre. The Schwann cells along nerve fibres leading to these two types of multiple endings exhibited ns. ChE but not AChE and ns. E activity.The ultrastructure of the two types of multiple endings was principally similar. The main difference, compared with the motor end plate, was that these endings were derived from unmyelinated axons which either make synaptic contacts along their course with the muscle fibre at variable distances (smaller-type) or these terminals were grouped closely together (larger-type).A few dense-core vesicles were observed in these unmyelinated nerves and in their terminals which were considerably smaller than those in the motor end plate. They were not always separated from each other by sarcoplasm and teloglia (larger-type) and contained also empty vesicles. The secondary synaptic clefts were often sparse and irregular or even absent, but the typical myoneural postsynaptic electron density was always observed. These multiple endings, in contrast to the motor end plate, were apposed only to muscle fibres with slow contraction.No catecholamine containing nerve endings were observed in the extraocular muscles. These observations indicate that the rat extraocular muscles have a double cholinergic innervation.The author wishes to express his gratitude to Professor Antti Telkkä, M. D., Head of the Electron Microscope Laboratory, University of Helsinki, for permission to avail himself of the electron microscope facilities.     

The subcommissural organ of the frog Rana perezi is innervated by nerve fibres containing GABA

The innervation of the frog subcommissural organ was studied by light-microscopic and ultrastructural immunocytochemistry using antisera against serotonin, noradrenaline, dopamine, gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, different GABA receptor subunits and bovine Reissner's fibre material (AFRU). In the proximity of the organ, serotonin- and noradrenaline-containing fibres were rare whereas dopamine-immunoreactive fibres were more numerous. Many GABA- and glutamic acid decarboxylase-containing nerve fibres were found at the basal portion of the ependymal cells of the subcommissural organ. Under the electron microscope, these GABA-immunolabelled nerve endings appeared to establish axoglandular synapses with secretory ependymal cells of the subcommissural organ. In addition, the secretory ependymal cells expressed high amounts of the beta2-subunit of the GABA(A) receptor. Since GABA-immunoreactive neurons were present in the frog pineal organ proper and apparently contributed axons to the pineal tract, we suggest that at least part of the GABAergic fibres innervating the frog subcommissural organ could originate from the pineal organ.     

RELATIONSHIP BETWEEN THE RATE OF AXOPLASMIC TRANSPORT AND SUBCELLULAR DISTRIBUTION OF ENZYMES INVOLVED IN THE SYNTHESIS OF NOREPINEPHRINE

The net rate of proximo-distal transport of tyrosine hydroxylase, dopamine β-hydroxylase, DOPA decarboxylase and choline acetyltransferase was determined by measuring the accumulation of these enzymes proximal to a ligature of the rat sciatic nerve. The rate of accumulation was constant for at least 12 h. For the enzymes involved in the biosynthesis of norepinephrine the rate of transport was correlated to their subcellular distribution and a close correlation between these two parameters was found. Dopamine β-hydroxylase, an enzyme mainly localized in the particulate fraction of the sciatic nerve, showed the fastest rate of transport (1·94 mm/h) whereas DOPA decarboxylase, exclusively located in the high-speed supernatant fluid, gave the slowest (0·63 mm/h) rate of transport. Tyrosine hydroxylase, predominantly located in the non-particulate fraction of the sciatic nerve was transported much slower (0·75 mm/h) than dopamine β-hydroxylase but still significantly (P < 0.005) faster than DOPA decarboxylase. The subcellular distribution of dopamine β-hydroxylase in ganglia did not differ significantly (0·45 > P > 0·40) from that in the sciatic nerve, but in nerve endings a greater proportion of dopamine β-hydroxylase was localized in particulate fractions. Tyrosine hydroxylase and DOPA decarboxylase were found exclusively in the non-particulate fractions of ganglia. In the nerve endings of the effector organs a small but consistent portion of tyrosine hydroxylase was found in particulate fractions, whereas DOPA decarboxylase was exclusively localized in the high-speed supernatant fluid.     

The innervation of the salivary gland of the moth, Manduca sexta: evidence that dopamine is the transmitter.

1. Using the Falck-Hillarp histochemical technique for monoamines, evidence was found for the presence of a catecholamine in the salivary gland nerves of the moth, Manduca sexta. 2. The innervation was studied with the electron microscope. Only the fluid-secreting region of the gland is innervated and the nerve endings are characteristic of monoamine-containing terminals. 3. Using a sensitive enzymatic-isotopic assay for catecholamines, it was found that whole salivary glands contain 0.33 mug/g dopamine but no noradrenaline. 4. It seems likely that dopamine mediates fluid-secretion in the salivary gland of Manduca as it does a number of other arthropods.     

Light and electron microscopic histochemical observations on cholinesterase-containing sympathetic nerve fibres in the pineal body of the rat

Synopsis Pineal glands of adult albino rats were examined histochemically using, first, formaldehyde-induced fluorescence to study monoamines and, second, copper thiocholine or copper ferrocyanide methods to study acetylcholinesterase and non-specific cholinesterase by light and electron microscopy. Cholinesterase was determined quantitatively by a constant pH titration assay.Fluorescent and acetylcholinesterase-positive nerve nets formed identical patterns. Nonspecific cholinesterase was observed only in nerve trunks outside the pineal. Bilateral removal of superior cervical ganglia resulted in complete disappearance of fluorescence and acetylcholinesterase from nerve fibres. Electron microscopically, acetylcholinesterase was found on sympathetic axons containing small granular vesicles. Quantitative cholinesterase determinations suggested that the pineal activity was mainly due to acetylcholinesterase. Comparison of the incubation times required for equal histochemical acetylcholinesterase reactions suggested that the activity of the sympathetic nerve fibres in the pineal is of the same order of magnitude as that in the nerve fibres of the iris.