H. G. Baumgarten

Auerbach's plexus of mammals and man: Electron microscopic identification of three different types of neuronal processes in myenteric ganglia of the large intestine from rhesus monkeys, guinea-pigs and man

SummaryGanglia from Auerbachs plexus of the large intestine (caecum, appendix vermiformis, colon transversum and rectum) in man, rhesus monkey and guinea-pig are composed of nerve cells and their processes, typical Schwann cells and a vast neuropil. The neuropil consists of dendrites and axons of intrinsic nerve cell perikarya and axons of extrinsic neurons. Axonal profiles in large nerve fibre bundles are of uniform size and appearance, embedded in infoldings of Schwann cell cytoplasm and contain occasional large granular vesicles, mitochondria and neurotubules. Preterminal axons widen into vesicle filled varicosities, some of which establish synaptic contact with intrinsic nerve cell bodies.At least three different types of neuronal processes can be distinguished in the myenteric neuropil according to the size, appearance and commutual proportion of vesicles present in axonal varicosities, and their ability to accumulate exogenous 5- and 6-hydroxydopamine and 5-hydroxydopa: 1. Axonal enlargements containing a major population of small electron lucent “synaptic” vesicles (350–600 Å in diameter) together with a small number of membrane-bound, opaque granules (800–1,100 Å). These profiles have been identified as “cholinergic” axons. The boutons establish synaptic contacts with dendritic processes of intrinsic nerve cell bodies; membrane specializations are found at the preand postsynaptic sites. 2. Axonal beads of sometimes very large diameter, containing an approximately equal amount of large granular vesicles (850–1,600 Å) and small, electron lucent or faintly opaque vesicles (400–600 Å). The granular core of the large vesicles is of medium electron density and may either fill the entire vesicle or is separated from the limiting membrane by a more or less clear interspace. The fibres probably belong to intrinsic neurons, and because of the similarity of the large, membrane-bound vesicles with neurosecretory elementary granules, they have been designated “p-type fibres” (polypeptide fibres). The granular core of the vesicles in these fibres becomes more electron dense after treatment with 5-OH-dopa. The accumulation of an amine precursor analogue in combination with a possible storage of a polypeptide substance (or an ATP-like substance) resembles the situation in several diffusely distributed endocrine cell systems. 3. Varicosities of axons equipped with small (400–600 Å) empty or sometimes granular vesicles, medium sized (500–900 Å) vesicles with highly electron dense cores and occasional large (900–1,300 Å) granular vesicles. Pretreatment with 5-OH-dopamine increases the electron density in almost all medium-sized granular vesicles and some of the large granular vesicles; an osmiophilic core develops in some small vesicles. 6-hydroxydopamine results in degenerative changes in the varicosities of this type of neurons. Concomitantly, both catecholamine analogues markedly reduce neuronal noradrenaline in the large intestine, as demonstrated by fluorescence histochemistry and in fluorimetric determinations. The ultrastructural features of these varicosities and their reaction to 5- and 6-OH-dopamine indicate that they belong to adrenergic, sympathetic nerves. No membrane specializations could be detected at sites of close contact of the adrenergic boutons with dendrites and cell bodies of intrinsic nerve cells.

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

SummaryThe 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 μ 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.

Arrangement, ultrastructure, and adrenergic innervation of smooth musculature of the ductuli efferentes, ductus epididymidis and ductus deferens of man

Summary1.There is a gradual proximo-distal increase in the thickness of the muscle coat of the human ductuli efferentes, duetus epididymidis and ductus deferens. Circularly arranged smooth muscle bundles predominate in the ductuli efferentes and ductus epididymidis of the caput section. Scanty strands of longitudinally and obliquely oriented smooth muscle bundles form an additional, incomplete outer muscle layer around the ductus epididymidis of the corpus. Small smooth muscle-like cells constitute the muscle elements of the upper sections of the excretory ducts (from the ductuli efferentes to the midcauda). At the transition of the corpus and cauda epididymidis ordinary large smooth muscle cells join the small contractile cells to form—in more distal sections of the cauda—a composed, thick subepithelial muscle coat. In most distal portions of the cauda, the two-layered muscle coat of the ductus epididymidis is transformed into a three-layered coat, a pattern of construction which is retained in the vas deferens.2.Electron microscopically, three types of contractile cells are distinguished in the human ductuli efferentes and ductus epididymidis: a) contractile cells of medium transparency containing exclusively thin myofilaments (60 Å in diameter), b) dark contractile cells containing bundles of thin myofilaments (60 Å in diameter) and single coarse filaments (140 Å in diameter), c) light contractile cells with loosely dispersed, interweaving thin and thick myofilaments. Commutual diameter changes at regular intervals are seen in individual myofilaments, giving the impression of structural periodicity not unlike that of filaments of striated muscle. Ordinary smooth muscle cells of the cauda epididymidis and vas deferens are characterized by uniformly sized, closely packed but evenly distributed thin myofilaments with numerous dense patches.3.Fluorescence microscopy performed on formaldehyde treated freeze dried tissues reveals that the contractile cells of the ductuli efferentes in man and monkey receive a low number of single adrenergic terminal fibres penetrating the depth of the muscle coat. The adrenergic innervation of the ductus epididymidis is restricted to small peritubular nerve fascicles running contiguous to the most superficially located bundles of smooth muscle-like cells. The adrenergic ground plexus is rather wide-meshed in the proximal cauda, becomes increasingly dense in more distal cauda sections and in initial, funicular portions of the vas deferens, and reaches maximum density in abdominal parts of the ductus. Perivascular and adventitial adrenergic plexuses are well developed at arteries of the caput and corpus epididymidis in man, monkey, rabbit, guinea-pig and rat.4.Electron microscopically, noradrenergic nerves have been identified by the presence of small granular vesicles in preterminal varicose axon dilatations. Nerve fibre swellings filled with small empty spherical vesicles have been considered to belong to “cholinergic” neurons whereas occasional varicosities equipped with some large membrane bound granules and abundant mitochondria may represent local expansions of sensory axons.5.Neuromuscular relationships in the upper sections of excretory ducts comprise adrenergic synapses by distance (more than 1000 Å), and a few intimate, ensheated close contacts, whereas the main type of contact of nerves to ordinary smooth muscle cells in the lower duct section is by means of close but not intimate approach (500–2000 Å).6.Adrenergic synapses in the ductus epididymidis and ductus deferens of the monkey resemble—what concerns their morphology, relationship to effectors and distribution pattern—those of man.7.In accordance with the total number of vascular and non-vascular adrenergic nerves, visualized by fluorescence microscopy, the amount of noradrenaline varied considerably in different sections of the human male internal genital organs: The lowest amounts were estimated in the testis (0.12±0.03 μg/g). Medium to high concentrations were detected in various sections of the caput and corpus epididymidis (ductuli efferentes 0.60±0.09 μg/g; ductuli efferentes and caput 0.72±0.13 μg/g; corpus epididymidis 1.04±0.25 μg/g; proximal cauda 0.95±0.17 μg/g; distal cauda 0.97±0.19 μg/g). The highest noradrenaline content was found in the human vas deferens (prox. vas deferens 1.11±0.21 μg/g; interm. vas deferens 1.20±0.42 μg/g; distal portion 1.43±0.39 μg/g).8.For comparison, the noradrenaline content of the testis and epididymis of the rhesus monkey, the epididymis of the rabbit and the vas deferens of the rabbit, mouse, guinea-pig and rat has been determined.9.Adrenaline of exogenous origin was detected in the vas deferens, cauda epididymidis and plexus pampiniformis of two cases who received this catecholamine as part of the local anaesthetic drug mixture. Due to methodological reasons, the presence of small amounts of adrenaline of endogenous source in adrenergic nerves of the human and monkey internal male genital organs cannot be excluded.10.The differences in motility behaviour of the ductus epididymidis (spontaneous, rhythmic contractions) and ductus deferens (absence of any spontaneous movements under conditions at rest) in vivo and in vitro have been correlated with the occurrence of specialized contractile cells in the upper segment (ductuli efferentes, ductus epididymidis of the caput, corpus and initial cauda) and ordinary large smooth muscle cells in the lower segment (ductus epididymidis of the distal cauda and the vas deferens) and furthermore correlated with differences in the pattern of the adrenergic innervation; the concept is advanced that progressive cytological differentiation of smooth muscle cells and the development of a dense direct adrenergic innervation suppresses autocontractility and, that the reverse condition may favour spontaneous motility of smooth muscle elements.

Adrenergic Innervation of the Human Testis, Epididymis, Ductus Deferens and Prostate : A Fluorescence Microscopic and Fluorimetric Study*

Summary1.The localization of catecholamines and 5-hydroxytryptamine has been investigated in the human testis, epididymis, ductus deferens and prostate obtained at operations on patients aged 26–79 years.2.In fluorimetric determinations noradrenaline was found to be present in varying concentrations in the genital tract. The lowest amount (0.07 μg/g) was found in the testis. In proximal direction along the ejaculatory duct system there was a progressive increase in the noradrenaline level, reaching a maximum of 1.43 μg/g in ductus deferens. The prostatic tissue (obtained from hyperplastic glands) contained only 0.15 μg/g noradrenaline.No dopamine was present. Adrenaline occurred, by all probability as an artefact, only in tissues from one single case to which this amine had been administered as part of the local anaesthetic mixture given before operation.3.Fluorescence microscopic analysis of formaldehyde-treated genital organs revealed the presence of adrenergic nerves, the number of which well agreed with the noradrenaline concentrations.Only vascular nerves were found in the testis. The bulk of the adrenergic nerve terminals in the epididymis, ductus deferens and prostate innervated the smooth muscle coats, whereas only a smaller portion of the adrenergic nerve population was of vasomotor nature.4.Microspectrophotometric characterization of the formaldehyde-induced fluorescence in the tissue sections confirmed that the adrenergic nerves contained noradrenaline.5.The finding of fluorescent ganglion cells in the prostate indicated that part — if not all — of the male genital tract also in humans is innervated by way of so-called short adrenergic neurons, originating in peripherally located sympathetic ganglia.6.Flask-shaped cells, morphologically resembling the enterochromaffin cells and emitting a yellow indole fluorescence, were present in the epithelium of the prostatic mucosa.7.Apart from the specific, formaldehyde-induced monoamine fluorescence, a considerable amount of autofluorescence of varying colour and related to different structures occurred both in the epithelial layers and in the connective tissue.

Catecholamine im Hypothalamus vom Goldfisch (Carassius auratus)

SummaryThe hypothalamus of Carassius auratus was investigated by means of the method for fluorescence-microscopical detection of catechol- and tryptamines. The hypothalamus was found to contain two hitherto unknown nuclei with specific catecholamine-fluorescence. They are situated in the pars posterior of the lobus medius hypothalami and are termed nucleus recesaus lateralis and nucleus recessus posterions. The unmyelinated axons of these nuclei give rise to four different tracts which run uncrossed in the frontal direction and terminate within the hypothalamus. Their course is described in detail. According to the results of histochemical and pharmacological tests it is assumed that the fluorescent substance in the hypothalamus is noradrenaline. The two nuclei are bordering the ventricular wall and are thought to secrete catecholamines into the cerebrospinal fluid.ZusammenfassungMit der fluoreszenzmikroskopischen Methode zur Lokalisation von Catechol- und Tryptaminen wurden im Hypothalamus von Carassius auratus zwei bisher unbekannte catecholaminhaltige Kerngebiete gefunden. Sie liegen in der Pars posterior des Lobus medius hypothalami und werden Nucleus recessus posterioris und Nucleus recessus lateralis genannt. Aus diesen Kerngebieten gehen vier markarme Bahnen hervor, die ungekreuzt verlaufen und innerhalb des Hypothalamus endigen. Verschiedene pharmakologische und histochemische Befunde legen die Vermutung nahe, daß die fluoreszierende Substanz im Hypothalamus Noradrenalin ist. Eine Abgabe von biogenen Aminen in den Liquor cerebrospinalis wird für möglich gehalten.

Evidence for a degeneration of indoleamine containing nerve terminals in rat brain, induced by 5,6-dihydroxytryptamine

Summary5,6-dihydroxytryptamine (5,6-DHT) has been found to induce a substantial degree of “chemical degeneration” of indoleamine containing nerve terminals in the rat brain following a single intraventricular injection of 75 μg 5,6-DHT per animal. The disintegration of varicose terminal portions of serotonin containing neurons is reflected1.by a loss of yellow fluorescent varicosities in certain defined parts of the rat CNS, despite a mild inhibition of the serotonin catabolizing enzyme monoamine oxidase with nialamide in the pretreated animals,2.by a significant drop of the chemically measurable 5-hydroxytryptamine content in nearly all parts of the rat brain and spinal cord,3.by the appearance of highly, orange or brownish fluorescent axons provided with numerous unusually large, distorted and intensely fluorescent swellings (“droplets”), resembling proximal stumps of mechanically severed indoleamine containing axons,4.a temporary increase in the amount of indoleamine fluorophores stored in some neuronal pericarya, and5.the electron microscopical demonstration of degenerating synaptic swellings of unmyelinated axons at all sites investigated. The selectivity of the effect of 5,6-DHT on indoleamine neurons is indicated by the absence of similar signs of injury in catecholamine containing neurons of the rat CNS.

Chemical degeneration of indolamine axons in rat brain by 5,6-dihydroxytryptamine

SummaryEvidence has been obtained by electron microscopy of a direct cytotoxic effect of intraventricularly administered 5,6-dihydroxytryptamine (5,6-DHT) on unmyelinated axons in the rat brain. Ultrastructural signs of axonal damage were observed in areas rich in indolamine nerve terminals as early as 2 hrs after injection. By 6–24 hrs, characteristic and more dramatic signs of degeneration developed, involving coalescence of all axonal constituents—often in combination with a uniform osmiophilic impregnation of the axoplasm—accompanied by engulfment of the dystrophic structures by glial processes. During the next five days, the degenerating axons and axon terminals appeared to be removed by glial cell phagocytosis, whose equivalents were the inclusion of axonal residues into membrane-bound lysosome-like bodies. Concomitantly, there was a progressively increasing number of extremely large and dilated axons in all regions analysed. These axonal swellings, which have an ultramorphology similar to that of dilated stumps of mechanically severed monoamine axons, correspond most probably to proximal, dilated portions of drug-damaged axons.The present results, in combination with biochemical and fluorescence microscopical data, indicate that within a proper dose range the 5,6-DHT-induced degeneration is largely restricted to indolamine axons and axon terminals. However, unselective effects on other unmyelinated axons, on myelin, and on glial cells were observed in narrow subependymal zones close to the lateral ventricles, i.e. close to the injection cannula.

MODE AND MECHANISM OF ACTION OF NEUROTOXIC INDOLEAMINES: A REVIEW AND A PROGRESS REPORT

The first description of the rather selective reduction in regional brain 5-hydroxytryptamine (5-HT) content after injection of 25-75 pg of 5,6-dihydroxytryptamine (5,6-DHT) into the lateral ventricle of the adult rat was provided by Baumgarten et a1.l in 1971. These results were soon confirmed by two one of which pointed to serious limitations in the applicability of 5,6-DHT as a tool for selective degeneration of serotonin (5-HT) neurons by demonstrating that there was a partial atrophy of the septum and caudate on the injection side.* Electron microscopic findings of Baumgarten et al.4 revealed that, in addition to injuring nonmyelinated presumptive monoaminergic axons and terminals, 5,6-DHT also lesioned glial cells and myelinated fibers in the head of the caudate and the septum on the injection side. The potential of 5,6-DHT to nonselectively damage both catecholamine-containing and serotonin-containing neurons was substantiated by four findings: the reduction of striatal dopamine (DA) and forebrain noradrenaline (NA) content after a high intraventricular dose of 5,6-DHT,2 the destruction of peripheral sympathetic noradrenergic fibers by 5,6-DHT, particularly after prior depletion of NA by reserpine and subsequent inhibition of monoamine oxidase (MAO) ,5 the destruction of subependymally located DA terminals in the head of the left caudate nucleus after left lateral ventricular injections of low to high doses of 5,6-DHT (25-75 pg),s and the destruction of DA-containing axons by small intracerebral injections of 5,6-DHT.7v * The need for a less generally cytotoxic serotonin neurotoxin was thus clearly evident, and in 1972 Baumgarten and Lachenmayer described the effects of intraventricular administration of 5,7dihydroxytryptamine (5,7-DHT) on central 5-HT neurons by use of the conventional Falck-Hillarp method. This dihydroxyindoleamine appears to have

Evidence for the existence of serotonin-, dopamine-, and noradrenaline-containing neurons in the gut of Lampetra fluviatilis

SummaryMonoamine-containing neurons in the gut of Lampetra fluviatilis are characterized by histochemical, electron microscopical and biochemical methods. Strongly yellow fluorescent, probably serotonin-containing intrinsic neurons are found along the entire length of the intestine. Their processes aggregate to form large bundles of mainly non-terminal axons, constituting a subepithelial fibre plexus. This subepithelial, ganglion cell comprising plexus is connected to a wide-meshed subserosal plexus which has ganglion cells of different size and few varicose, single axons. Intermingled with both plexus there occur — in the anterior and middle but not in the preanal portion of the lamprey intestine — scattered green fluorescent intrinsic perikarya, emanating faintly green fluorescent, poorly varicosed axons.The formaldehyde-induced neuronal fluorophores conform to serotonin (yellow fluorescent compound), noradrenaline, and dopamine (green fluorescent substance), as revealed in microspectrofluorimetric recordings. The electron microscopical analysis of the yellow fluorescent intrinsic neurons in the terminal hindgut shows nerve cell pericarya and axons equipped with a typical population of occasional small granular and many large granular vesicles (750–1600 Å). The number and opacity of cores of the small and the osmiophilia of the cores of the large granular vesicles are significantly increased following short-term treatment with 5,6-dihydroxytryptamine. Long-term treatment with 5,6- or 5,7-dihydroxytryptamine provokes severe signs of ultrastructure impairment and eventual degeneration in the supposed serotonin-containing axons, besides indications of piling-up of organelles in the non-terminal axons due to arrest of axonal transport.Chromatography of acid extracts from the lamprey intestine, gills and kidney reveals the presence of serotonin (besides another unidentified indoleamine) and dopamine and noradrenaline in the gut, but only dopamine in the brain. The detection of serotonin, noradrenaline and dopamine in the lamprey gut is confirmed by chemical determinations.The occurrence of intrinsic serotonin-, noradrenaline- and dopamine-containing neurons in the gut of Lampetra fluviatilis deviates from the established pattern of innervation of the vertebrate intestine and is considered to be a remnant of an autonomic innervation principle common in invertebrates.

De- and regeneration of the bulbospinal serotonin neurons in the rat following 5,6-or 5,7-Dihydroxytryptamine treatment

SummaryIn an attempt to determine the conditions which permit central 5-HT neurons to respond to a chemical injury of their axons by sprouting and regeneration, the pattern and time-course of recovery of 5-HT concentrations and regrowth of bulbospinal 5-HT axons were evaluated in rats subjected to intraventricular treatment with either 75 μg 5,6- or 150 μg 5,7-DHT. While 5,6-DHT treatment is followed by a significant recovery of 5-HT concentrations in the telodiencephalon, brainstem and upper part of the spinal cord within 3 months, there is no significant restoration of the severely depleted 5-HT levels in the telodiencephalon and spinal cord, and only limited recovery in 5-HT content of the brainstem preparation after 5,7-DHT.These differences conform to the observation of widespread and effective regrowth and regeneration of the bulbospinal 5-HT neurons in the 5,6-DHT treated lower brainstem and upper spinal cord but restricted and localized sprouting efforts in the 5,7-DHT treated lower medulla oblongata. This could be explained by a cell body near lesion of the non-terminal indoleamine axons by 5,7-DHT which results in a late retrograde, irreversible degeneration of most of the indoleamine pericarya from group B1 and many of group B3.It is concluded that the preservation of a critical length of the main axon and part of its collaterals is necessary for the neurons survival, and that the individual pattern of the neuropil architecture of brain centres which are invaded by the axonal sprouts may significantly influence their growth characteristics and thus either favour or impede their chance to reestablish connections with their original effector. Aberrant, localized, intense sprouting of drug-damaged axons may in itself reflect the need of the neuron—deprived of most of its axonal tree—to reestablish its original total axonal length by multiple branching.