Ultrastructure of sympathetic ganglion cells and granule-containing cells in the paracervical (Frankenhäuser) ganglion of the newborn rat

Summary A study was made of the ultrastructure of the paracervical (Frankenhäuser) ganglion of the newborn rat, using immersion fixation by glutaraldehyde (2.5%) followed by OsO₄ (1%), or KMnO₄ (3%) fixation. The cells containing dense—core vesicles were divided into three groups: (1) primitive sympathetic cells, (2) cells containing some dense-core vesicles 700–1100 Å in size and structurally resembling sympathetic neurons, called principal neurons, and (3) cells containing many dense-core vesicles with a larger, darker dense core, 800–2000 Å in diameter, called granule-containing cells. Using glutaraldehyde-osmium fixation, the principal neurons were further divided into dark and light cells on the basis of electron opacity of the cytoplasmic matrix. The granule-containing cells were believed to correspond to the small, intensely fluorescent cells (SIF-cells) previously described using the formaldehyde-induced fluorescence technique. On the basis of the amount of granules, the granulecontaining cells were classified as mature or maturing SIF-cells and as more primitive SIF-cells, and developing sympathicoblasts. The development of synapses in autonomic ganglia was discussed.Grant: The Finnish Medical Foundation.     

Fine structure of the small,granule-containing cells in the superior cervical ganglia of hydrocortisone-treated early postnatal and adult rats

Summary Hydrocortisone injections into rats on postnatal days 3–9 caused an increase in the number of small granulecontaining cells in the superior cervical ganglia. These cells, corresponding to the small, intensely fluorescent cells, showed an extensive rough endoplasmic reticulum, a large Golgi apparatus and a very large number of granular vesicles. In addition to the granular vesicles, 70–160 nm in diameter, in which the dense core filled most of the vesicle, most cells of the hydrocortisone-injected rats contained also larger granular vesicles, up to 350 nm in diameter, in which the dense core was eccentrically located. A minority of the cells contained only granular vesicles 70–100 nm in diameter, which was the only type seen in the saline-treated control rats.Thirty days after discontinuation of the hydrocortisone treatment, most of the cells with large granular vesicles had disappeared, and only two profiles of such cells were seen on day 40. The other small cells contained only granular vesicles 70–160 nm in diameter, and these cells could not be distinguished from the small granule-containing cells of 40-day-old control rats treated early postnatally with saline.Hydrocortisone treatment, first on days 3–9 and subsequently on days 40–46, caused reappearance of the small granule-containing cells with large granular vesicles up to 350 nm in diameter, the dense core of which was eccentrically located. Hydrocortisone treatment on days 40–46 only was not followed by appearance of such cells in rats treated with saline on days 3–9.Abbreviations used in the Text PNMT phenylethanolamine-N-methyltransferase - SIF cell small intensely fluorescent cell - SGC cell small granule-containing cell The author is grateful to Professor Olavi Eränkö and Dr. Seppo Soinila for constructive criticism. Expert technical assistance by Miss Hanna-Liisa Alanen, Mrs. Marja-Leena Piironen and Mrs. Anne Reijula is gratefully acknowledged. This study has been supported by a grant from the Finnish Medical Foundation.     

On different types of nerve endings in the frog median eminence after fixation with permanganate and glutaraldehyde-osmium

Summary In the frog median eminence, fixed with glutaraldehyde and osmium tetroxide, four types of nerve endings can be generally distinguished. These endings are in contact with the pericapillary spaces of primary portal vessels and can be identified by the internal structure and the size of their granules and vesicles. Type 1 contains large granules (1500–2400 Å in diameter) and small clear vesicles (300–500 Å in diameter), type 2 intermediate granules (about 1100–1700 Å in diameter) and small clear vesicles, type 3 small granules (about 600–1000 Å in diameter) and small clear vesicles, type 4 only numerous small clear vesicles. The mixed types containing the large, intermediate and small dense granules in the same ending are infrequently found.After KMnO₄ or LiMnO₄ fixation the granules and vesicles mentioned above are observed as follows. The large granules in the type 1 nerve ending appear mostly pale or less-dense. The intermediate granules in the type 2 also appear mostly pale or less-dense, but some frequently show granules of high density. The small granules in the type 3 consistently contain the dense substance and these endings can be subdivided into two different types according to the populations of different sizes of dense granules [type 3a (900–1000 Å) and type 3b (500–800 Å)]. Dense-cored and cleared-synaptic vesicles are frequently present with together in the type 3 endings. The small vesicles (300–400 Å), in the type 4, appear generally pale (type 4a), but some nerve endings contain small dense cored-vesicles (type 4b).The author wishes to thank Prof. H. Fujita for his advice and criticism.     

Immuno-electron-microscopic study of the prolactin cells in the pituitary gland of male Wistar rats during aging

Summary Prolactin cells were identified by means of immunocytochemistry with protein-A gold as a marker on ultrathin sections of the pituitary gland of young (3–4 months), middle-aged (16–19 months), and aged (26–30 months) male Wistar rats. Point-counting volumetry revealed that the prolactin (PRL) cell-volume density in middle-aged rats was significantly increased in comparison to the volume densities in young and aged rats. Within the PRL-cell population, four types of PRL cells were distinguished on the basis of the shape and size of their secretory granules. During aging, dramatic changes occurred in the relative volumes of the four cell types. The volume percentage of cells with round granules (type I, granule diameter 150–250 nm, and type IIA, granule diameter 250–350 nm) increased from ±30% in young rats to ±90% in old rats. The volume percentage of cells with round and polymorphic granules (type IIB; granule diameter 350–400 nm and type III; granule diameter 500–600 nm) decreased from ±70% in young rats to ±7% in old rats. Age-related changes in serum PRL levels were not found. It is concluded that although during the life span of the male Wistar rat considerable changes in PRL-cell volume densities and in the ratios of PRL-cell types occur serum, PRL levels remain more or less constant.     

Ultracytochemical identification of catecholamine-containing vesicles in the ligated sciatic nerve of the rat. Comparison with sympathetic nerve terminals

Summary Using the fixation procedure of Tranzer, three kinds of granular vesicles were identified in certain unmyelinated fibres of rat sciatic nerves proximal to a ligature: (1) small vesicles (SGV: 30–60 nm in diameter), (2) large vesicles (LGV: 60–100nm in diameter), and (3) large elongated vesicles (LEV: 60–100nm in diameter). A comparative study concerning the distribution of these granular vesicles was carried out using a cytopharmacological method (reserpine) and employing different fixatives (aldehydes + OsO₄, or OsO₄ alone) in periarterial nerve plexus of the femoral artery, vas deferens and the pineal organ.Use of Tranzer's method allows preservation in almost all granular vesicles of a strongly electron-dense core, while with the other fixatives mainly small, eccentric dense cores occur in the vesicles. Two main features were observed in ligated sciatic nerves: (i) a clear increase in the number of LGV, and (ii) the presence of LEV, considered as a variety of LGV rather than a new population of granular vesicles. Reserpine caused the cores of SGV to disappear almost completely, while LGV and LEV remained only partly depleted. The original method combining Tranzer's fixation procedure with radioautography revealed radioautographic labelling only in the unmyelinated fibres of ligated sciatic nerves and mainly superimposed over SGV, LGV and LEV. It is suggested that (i) SGV, LGV and also LEV represent possible storage sites of catecholamines, and (ii) a local morphogenesis of SGV from the large vesicles occurs in ligated sympathetic nerve fibres.     

Effect of hydrocortisone on the ultrastructure of the small,intensely fluorescent,granule-containing cells in cultures of sympathetic ganglia of newborn rats

Summary Sympathetic chain ganglia of newborn rats were cultured in Rose chambers with or without hydrocortisone. After one week, the cultures were examined by light microscopy for formaldehyde-induced catecholamine fluorescence and by electron microscopy after fixation in 5% glutaraldehyde solution and thereafter in 1% osmium tetroxide. Hydrocortisone (10 mg/l) caused a great increase in the number of the small, intensely fluorescent (SIF) cells in the ganglion explants, and the fluorescence intensity of these cells was also increased. The SIF cells corresponded to small, granule-containing (SGC) cells in the electronmicros copic preparations, and in addition to an increase in their number there was also an increase in the size and number of granular vesicles in the presence of hydrocortisone. In control cultures the granular vesicles were round (about 100 nm in diameter) or elongated (40–150 nm in cross section and 150–250 nm in length); both types of vesicles contained electron dense cores. In hydrocortisone-containing cultures round granular vesicles up to 200 nm in diameter were also observed; the cores of these vesicles were of variable electron density. It is concluded that in tissue culture, hydrocortisone causes an increased formation of catecholamine-containing granular vesicles in SIF-SGC cells and their precursors and an increase in the number of these cells.This work was supported by grants from the National Heart Foundation, the Australian Research Grants Committee and the Sigrid Juselius Foundation.University of Melbourne Senior Research Fellow, September, 1971 – August, 1972; present address: Department of Anatomy, University of Helsinki, Siltavuorenpenger, Helsinki, Finland, 00170.Holder of a grant from the National Health and Medical Research Council of Australia.Sunshine Foundation and Rowden White Research Fellow in the University of Melbourne, September, 1971 – August, 1972; present address: Department of Anatomy, University of Helsinki, Siltavuorenpenger, Helsinki, Finland, 00170.     

Ultrastructural changes in the neurohypophysis of the aged male rat

Summary The neurohypophyses (neural lobes of the pituitary) of young adult (3–6 months) and aged (12–30 months) male rats were studied by means of electron microscopy. Stereomorphometric analyses were performed to determine the size, number and relative volume of the hormone-containing vesicles. The principal observations included: 1) the conspicuous depletion in aged rats of the granular hormone-containing vesicles from the axon terminals and the Herring bodies, with a decrease in the relative volume fraction of the vesicles from 4.8 Vv % in the control animals to 1.1 Vv % in the aged rats; 2) a change in the size-distribution of the hormone vesicles; 3) an increase in the extracellular space around the nerve terminals, axons and capillaries; and 4) lipid accumulations and signs of activation in the pituicytes. The possible physiological significance of the findings is discussed in the light of several regulatory functions known to be altered during the process of ageing.     

Morphological evidence for a direct innervation of the mouse vomeronasal glands

Summary The morphological evidence for a direct autonomic innervation of the mouse vomeronasal glands is presented. Axonal varicosities containing a few densecore vesicles and numerous clear vesicles (36–60 nm in diameter) make synaptic contacts with the secretory cells at the base of the glandular acini. The axonal presynaptic membrane is associated with a distinct dense material and it is separated from the secretory cell by a synaptic cleft of about 12–14 nm. At the postsynaptical level, coated vesicles can be found. Additional postsynaptical specializations have not been observed.     

Fine structure of neurons of the arcuate nucleus and median eminence of the hypothalamus of the golden hamster following immobilization

Summary The fine structure of arcuate neurons of the arcuate nucleus, the ependymal tanycytes and the contact zone of the median eminence was examined following immobilization, an acute stress which significantly activated the hypothalamo-pituitary-adrenal (HPA) axis. Arcuate neurons of immobilized adult male hamsters displayed morphological indications of heightened activity; the number of lysosomes and dense core vesicles (80–120 nm) was increased. A markedly greater number of dense core vesicles was present in axon terminals of the contact zone of the mid-central median eminence and the ventral proximal stalk.Tanycytes of the median eminence exhibited an augmented number of electron dense bodies in both perikarya and end processes. These results indicate that the arcuate neurons, the axons of the contact zone, and the ependymal tanycytes of the hamster medial basal hypothalamus (MBH) may be involved in the response to immobilization.This work was supported by Program Project Grant #NS-11642     

The formaldehyde-induced fluorescence of the developing hypogastric (main belvic) ganglion of the rat

Summary The development of the hypogastric ganglion of normal and testosterone-treated rats was studied using formaldehyde-induced fluorescence (FIF) method. The fluorescence intensities were recorded microspectrofluorimetrically. In normally developing rats cytoplasmic FIF decreases and cell size increases with age. In testosterone-treated animals FIF increases during 2–6 weeks compared to the controls. The differences between control and experimental rats were significant. The diameters were significantly longer in treated animals in three and four week old groups. Vacuolated neurons were seen earlier in testosterone-treated rats. No changes in FIF or in cell size were noticed in the superior cervical ganglion. The male sex steroid, testosterone evidently influences the catecholamine turnover and cellular growth during development in the male pelvic ganglion.     

Concentration of dopaminergic fibres in the marginal zone of the bird retina

Summary Nerve fibres containing dense core vesicles of 100–160 nm diameter are concentrated in a narrow zone (near the ora serrata) of the marginal retina in the pigeon and the chicken. Synaptic junctions exist between terminals containing dense core vesicles and the most marginal ganglion cells; however, numerous fibres seem to end freely near the vitreous body or even penetrate the pars plana of the ciliary body. Histofluorescence demonstrates the aminergic nature of these fibres. Determination of catecholamines with the dansylation technique reveals six times more dopamine in the marginal than in the central retina, whereas the noradrenalin level is unchanged. The dopaminergic fibres originate in neurons of the innermost zone of the inner nuclear layer. The marginal localisation of these fibres apparently excluding any visual function seems rather to indicate a possible photoreceptor system involved in neuro-endocrine regulation.     

Precursors of monoamine-storage organelles in developing megakaryocytes of the rat

Summary Identification and distribution of the precursors of aminestorage organelles in rat megakaryocytes during cell maturation were studied, using the uranaffin reaction for adenine nucleotide. The precursors of the amine-storage organelles appeared as 200–300 nm vesicles having an uranaffin electron dense granule, whereas they appeared as empty vesicles by conventional glutaraldehyde-OsO₄ fixation. X-ray probe microanalysis confirmed the existence of U and P in the uranaffin reaction positive vesicles. The precursors appeared in the immature megakaryocytes, especially at the trans(mature) face of the Golgi apparatus, and rapidly increased in number in the maturing cells. The size of the uranaffin granules in the precursory organelles increased gradually during cell maturation and became almost equivalent to the dense body of blood platelets in the final stage of cell maturation.     

Electron microscopic study of the giant cells in the olfactory bulb of labyrinth fish (Belontiidae,Perciformes)

Summary The giant cells in the olfactory bulb of labyrinth fish, most likely belonging to the nervus terminalis, show ultrastructural features of active synthesis of secretory material. Dense core vesicles (70–100 nm in diameter), found in the perikaryon as well as in the axon, are the possible storage sites of the secretory substance. Its chemical nature is unknown. In some of these vesicles acid phosphatase is demonstrable. Large membrane-bound bodies (up to 2000 nm in diameter), either containing an electron dense matrix or debris of cytoplasmic organelles, are also acid phosphatase-positive, suggesting their lysosomal nature. Some other ultrastructural characteristics of these cells are also described.     

5-HT nerve terminals in the fourth ventricle of the rat brain: Their identification and distribution studied by fluorescence histochemistry and electron microscopy

Summary The occurrence and distribution of supra-ependymal nerve terminals storing serotonin (5-HT) are described for the fourth ventricle of the rat brain. The nerve terminals were identified as monoaminergic 1) fluorescence-histochemically, by the presence of a varicose, formaldehyde-induced fluorescence (FIF) on the free surface of the ependyma, 2) electron microscopically, by the presence of electron dense (chromaffin) cores in small (50 nm) and large (100 nm) vesicles found within the varicose regions of supra-ependymal nerve fibres, and 3) by the absence of both the FIF and chromaffin dense cores after treatment with reserpine. Moreover, the serotonergic nature of these nerve fibres could be concluded from 1) the yellow colour of the FIF, 2) the increased FIF after treatment with nialamide or reserpine+nialamide, 3) the diminished FIF and absence of chromaffin dense cores after treatment with p-CPA, and finally 4) the persistence of the FIF and chromaffin dense cores after treatment with -MPT.A high density of 5-HT nerve terminals occurred throughout the floor of the fourth ventricle and on the floor and roof of the lateral recess. Few 5-HT nerve terminals occurred only on the roof of the fourth ventricle (velum medullare, lamina epithelialis of the tela chorioidea), and the surface of the choroid plexus epithelia was devoid of such nerves. Virtually all nerve terminals in the fourth ventricle appear to be serotonergic.Results presented in part at the Autumn Meeting of the British and Italian Pharmacological Societies, Bristol, 1974 (Lorez et al., 1974). The skilful assistance of Mr. R. Wybrecht, Mr. R. Reese and Mrs. M. Gschwind is gratefully acknowledged.     

Glutamate-Glutamine Cycle and Aging in Striatum of the Awake Rat: Effects of a Glutamate Transporter Blocker

This study investigated the effects of aging on the actions of a specific glutamate reuptake blocker, L-trans-pyrrolidine-2, 4-dicarboxylic acid (PDC), in extracellular glutamate and glutamine in striatum of the awake rat. Microdialysis experiments were performed on young (2–3 months), middle-aged (12–14 months), aged (27–32 months) and very aged (37 months) male Wistar rats. Local infusion of PDC (1–4 mM) in striatum increased the dialysate concentration of glutamate and decreased dialysate concentration of glutamine in all the age-groups. In young rats, decreases of dialysate glutamine were correlated with increases of dialysate glutamate. The same profile glutamine/glutamate as in young rats was found in middle-aged, aged and very aged rats, which suggests that the action of glutamate on the glutamate-glutamine cycle in striatum of the awake rat is not modified as a consequence of aging. We also found a significant correlation between the increases of glutamate produced by PDC and the basal dialysate concentration of glutamine, a relationship that did show a significant change with age. Although the significance of this latter finding remains to be elucidated, it may be important to understand the changes in glutamate-glutamine cycle during aging.     

Electron microscopic analysis of tyrosine hydroxylase,dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase immunoreactive innervation of the hypothalamic paraventricular nucleus in the rat

Summary The catecholaminergic innervation of the hypothalamic paraventricular nucleus (PVN) of the rat was studred by preembedding immunocytochemical methods utilizing specific antibodies which were generated against catecholamine synthesizing enzymes. Phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive terminals contained 80–120 nm dense core granules and 30–50 nm clear synaptic vesicles. The labeled boutons terminated on cell bodies and dendrites of both parvo- and magnocellular neurons of PVN via asymmetric synapses. The parvocellular subnuclei received a more intense adrenergic innervation than did the magnocellular regions of the nucleus. Dopamine--hydroxylase (DBH)-immunopositive axons were most numerous in the periventricular zone and the medial paryocellular subnucleus of PVN. Labeled terminal boutens contained 70–100 nm dense granules and clusters of spherical, electron lucent vesicles. Dendrites, perikarya and spinous structures of paraventricular neurons were observed to be the postsynaptic targets of DBH axon terminals. These asymmetric synapses frequently exhibited subsynaptic dense bodies. Paraventricular neurons did not demonstrate either PNMT or DBH immunoreactivity. The fibers present within the nucleus which contained these enzymes are considered to represent extrinsic afferent connections to neurons of the PVN.Tyrosine hydroxylase (TH)-immunoreactivity was found both in neurons and neuronal processes within the PVN In TH-cells, the immunolabel was associated with rough endoplasmic reticulum, free ribosomes and 70–120 nm dense granules. Occasionally, nematosome-like bodies and cilia were observed in the TH-perikarya. Unlabeled axons established en passant and bouton terminaux type synapses with these TH-immunopositive cells. TH-immunoreactive axons terminated on cell bodies as well as somatic and dendritic spines of paraventricular parvocellular neurons. TH-containing axons were observed to deeply invaginate into both dendrites and perikarya of magnocellular neurons.These observations provide ultrastructural evidence for the participation of central catecholaminergic neuronal systems in the regulation of the different neuronal and neuroendocrine functions which have been related to hypothalamic paraventricular neurons.Supported by NIH Grant NS 19266 to W.K. Paull     

Effect of monoamines on the taste buds in the mouse

Summary Mouse taste buds were investigated following administration of monoamines and their precursors by fluorescence and electron microscopy. The appearance of fluorescent cells within the taste bud and the ultrastructural changes of vesicles in the gustatory cells were due to the treatment of 5-hydroxytryptophan. Small dense-cored vesicles (30–60 nm in diameter) appeared throughout the cytoplasm and accumulated especially at the presynaptic membranes of afferent synapses. Large dense-cored vesicles (80–100 nm) increased twice in number, and electron densities of their cores became more dense as compared with untreated mice. Fluorescent cells appeared in the taste bud of l-DOPA treated mice, whereas no ultrastructural changes were observed. These results suggest that the gustatory cells of the taste bud are capable of taking up and storing monoamines, which might act as neurotransmitters from the gustatory cells to the nerves.     

Observations on the ultrastructure of nerve cells in the brain of the planarian,Dugesia gonocephala

Summary The submicroscopic structure of the nerve cells in the planarian brain was studied. Close similarities with neurons of other invertebrates were noted. In the cytoplasm of the planarian nerve cells there are at least three types of vesicular inclusions: 1) Clear vesicles (200–800 Å in epon embedded tissue) similar in morphological appearance to classical synaptic vesicles. These have generally some content of extremely low density but occasionally a dense core. 2) Dense vesicles (400–1,200 Å in epon embedded tissue) containing highly osmiophilic granules. Between the limiting membrane of the vesicle and the granule there is always a clear rim of variable width. These vesicles closely resemble synaptic vesicles described in vertebrate adrenergic endings. 3) Neurosecretory vesicles (600–1,300 Å in Vestopal embedded tissue) similar to elementary granules observed in neurosecretory systems in vertebrates and invertebrates. All three vesicle types have the same mode of origin from the Golgi membranes. All are present in the nerve cell processes of the neuropil as well as in the perikarya. Any given perikaryon or axon contains only one of the three vesicle types. All of these vesicles are considered to be discharged into the axons from their site of origin within the perikaryon.     

Electron microscopic studies on the pars intermedia in normal and in mice with hereditary nephrogenic diabetes insipidus

Summary The ultrastructure of the pars intermedia (PI) of the normal VII +/+ and hereditary nephrogenic diabetes inspidus DI Os/+ mice has been studied with particular reference to the morphology of the glandular cells and their innervation. Four types of cells were observed in both the genotypes of mice, 1) the light glandular cell, 2) the dark cell, 3) a type of cell similar to ependymal cells and 4) a small percentage of typical ACTH cells, observed mostly on the PI border of the cleft and rarely in the centre of PI. The predominant light glandular cells contain mainly two types of membrane bound granules: 1) electron dense core granules, which measure 1500–2500 Å and 2) electron lucent vesicles, which measure 3000–4000 Å in diameter. Granules of intermediate size with various density are also present in both types of mice. The electron dense core granules are predominant in DI Os/+ mice, whereas, electron lucent vesicles are predominant in the normal VII +/+ mice. Similar uniform size membrane bound electron dense granules have been observed in ACTH cells of PI and pars distalis. From earlier experimental evidences and the present observations, it is concluded that the dense core granules in PI may be synthesizing ACTH or ACTH-like substance. It is also discussed that these dense core granules may further mature and give rise to MSH in the form of electron lucent vesicles. If it is so, PI light glandular cells may have dual functions, of producing MSH and ACTH. One of the functions of ependymal-like cells, may be the transport of PI secretion.Three types of nerve endings are observed throughout the PI, making synaptic contact with the predominant cell type. The innervation is more in DI Os/+ mice than in normal mice. The classification of these nerves is according to Bargmann and co-workers 1) peptidergic neurosecretory fibers, contain mainly membrane bound dense core granules, measuring 1200 to 1800 Å, and are the classic neurosecretory granules; 2) adrenergic fibers, measuring 700–900 Å; 3) cholinergic fibers, measuring 300–400 Å. Adrenergic and cholinergic fibers are more towards the hypophysial cleft. The increased innervation, the synaptic contact, the extremely hypertrophied PI and the greater activity of its light glandular cells in the DI Os/+ mice show the PI is under the influence of the nervous system.This study was supported by MRC of Canada Grant No. MA-3759.