Postnatal development of autonomic and sensory innervation of thoracic hairy skin in the rat

Summary Histochemical, immunocytochemical, and radioenzymatic techniques were used to examine the neurotransmitter-related properties of the innervation of thoracic hairy skin in rats during adulthood and postnatal development. In the adult, catecholamine-containing fibers were associated with blood vessels and piloerector muscles, and ran in nerve bundles throughout the dermis. The distribution of tyrosine hydroxylase (TH)-immunoreactive (IR) fibers was identical. Neuronal fibers displaying neuropeptide Y (NPY) immunoreactivity were seen in association with blood vessels. Double-labeling studies suggested that most, if not all, NPY-IR fibers were also TH-IR and likewise most, if not all, vessel-associated TH-IR fibers were also NPY-IR. Calcitonin gene-related peptide (CGRP)-IR fibers were observed near and penetrating into the epidermis, in close association with hair follicles and blood vessels, and in nerve bundles. A similar distribution of substance P (SP)-IR fibers was evident. In adult animals treated as neonates with the sympathetic neurotoxin 6-hydroxydopamine, a virtual absence of TH-IR and NPY-IR fibers was observed, whereas the distribution of CGRP-IR and SP-IR fibers appeared unaltered. During postnatal development, a generalized increase in the number, fluorescence intensity, and varicose morphology of neuronal fibers displaying catecholamine fluorescence, NPY-IR, CGRP-IR, and SP-IR was observed. By postnatal day 21, the distribution of the above fibers had reached essentially adult levels, although the density of epidermal-associated CGRP-IR and SP-IR fibers was significantly greater than in the adult. The following were not evident in thoracic hairy skin at any timepoint examined: choline acetyltransferase activity, acetylcholinesterase histochemical staining or immunoreactivity, fibers displaying immunoreactivity to vasoactive intestinal peptide, cholecystokinin, or leucine-enkephalin. The present study demonstrates that the thoracic hairy skin in developing and adult rats receives an abundant sympathetic catecholaminergic and sensory innervation, but not a cholinergic innervation.     

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.     

Adrenergic innervation of the oesophagus in the cat (Fellis domestica) and rhesus monkey (Macacus rhesus)

Summary The distribution of monoamines in the pharynx and oesophagus of the rhesus monkey (Macacus rhesus) and the cat (Felis domestica) was investigated by means of fluorescence microscopical and chemical methods. Fluorimetric determinations reveal the presence of varying amounts of noradrenaline in the pharynx and oesophagus of the rhesus monkey. The lowest amount (0.05 (g/g) was found in the lower part of the oesophagus, the so-called sphincter-segment. The middle and upper part of the oesophagus contain medium amounts of noradrenaline (0.06–0.09 g) whereas the highest concentration was detected in the pharynx (0.14 (g/g). Neither dopamine nor adrenaline occurred in the tissue pieces analyzed. Fluorescence microscopically noradrenaline was found to be located in varicose intramural nerve fibre plexus which innervate mucous glands and blood vessels in the pharynx of both species. In the rhesus monkey, the lamina muscularis mucosae of all parts of the oesophagus is supplied by a well developed noradrenergic ground-plexus. Preterminal and terminal varicose nerve fibres are distributed in myenteric and submucous ganglia of the oesophagus; the number of such ganglia decreases towards the lower segment. The density of the adrenergic innervation is higher in myenteric when compared to submucous ganglia. The arrangement of the intraganglionic terminals suggests that both axosomatic and axodendritic contacts occur in Auerbach's ganglia whereas axodendritic contacts seem to predominate in Meissner's ganglia. Myenteric ganglia situated close to the submucosa as well as true submucous ganglia may be occasionally seen to be traversed by faintly fluorescent non-varicosed fibres which do not establish any synaptic contacts. The fluorescence intensity of intraganglionic varicosities varies considerably; accordingly the transmitter content of individual varicosities seems to be very variable. The adrenergic innervation of the lamina muscularis is restricted to single contorted fibres being sparsely distributed throughout the longitudinal smooth muscle layer. The circularly arranged smooth musculature of the sphincter-segment lacks an adrenergic nerve supply. The vagus nerve carries sympathetic adrenergic fibres to the lower oesophagus and the cardia. Species differences between the innervation pattern in rhesus monkeys and cats are outlined: No adrenergically innervated ganglia occur in the submucosa of the cat. However, part of the myenteric ganglia in cats exhibit an adrenergic innervation pattern similar to that seen in submucous ganglia of the rhesus monkey. They might therefore be regarded as morphologically equivalent to the plexus submucosus which is, however, present in the whole gut. The density of the noradrenergic ground-plexus in the muscularis mucosae of the cat's oesophagus is less than that of the corresponding plexus in rhesus monkeys.The influence of noradrenaline upon the smooth musculature and the neurons from myenteric as well as submucous ganglia is discussed. From the point of view of the adrenergic innervation there is no structure corresponding to the sphincterlike lower oesophageal segment.Supported by the Deutsche Forschungsgemeinschaft and the Joachim-Jungius-Gesellschaft zur Förderung der Wissenschaften, Hamburg.     

Somato-social development in the rhesus monkey following olfactory bulbectomy

Bilateral olfactory bulbectomy was performed on the rhesus macaque (M. mulatta) at 6 to 8 months of age. Social interaction and plasma testosterone levels obtained at approximately 2-1/2 years of age indicated that early olfactory bulb ablation resulted in higher levels of aggressivity concomitant with lower levels of social facilitory behavior. Bulbectomy did not interfere with the development of normal sexual posturing and orientation at the juvenile stage. Lower testosterone levels in the anosmic females indicated possible delay in the onset of puberty, while no differences were found in testosterone between the anosmic and control males. This investigation was supported by Behavioral Science Foundation, Los Angeles, California.     

Androgen metabolism in the rhesus monkey.

A mixture of 3H-testosteron (T) and 14C-4-androstene-3, 17-dione (A) was injected intravenously into 2 (I and II) rhesus monkeys (Macaca mulatta). A third monkey (III) was injected with 3H-T only. Urine and bile samples were collected at intervals for 6 hours following the injection. The excretion, conjugation and aglycone metabolites of the steroids injected were studied using these samples. Of the injected dose, animal I (male) excreted 32% 3H and 23% 14C in the bile and 30% 3H and 21% 14C in the urine in 6 hours. Animal II (female), however, had a comparatively higher biliary excretion (66% 3H, 40% 14 C), but a urinary excretion (18% 3H, 13% 14C) comparable to that of animals I and III. The averages in the bile of the 3 animals were: unconjugated compounds 3%, glucosiduronates 78%, sulfates 9%, sulfoglucosiduronates 5% and disulfates 3%; and in urine, 5% unconjugated, 92% glucosiduronates and 3% sulfates. The aglycones obtained following hydrolysis were separated gy chromatography on Lipidex 5000, further purified by thin layer and paper chromatography and identified by co-crystallization. The major matabolites from 3H-T were androsterone and 5beta-androstane-3alpha,17beta-diol, whereas that from 14C-A was androsterone. Other metabolites identified were: etiocholanolone (3beta-hydroxy-5-beta-androstan-17-one); T, epitestosterone (epi-T), (17alpha-hydroxy-4-androsten-3-one); epiandrosterone (3-beta-hydroxy-5alpha-androstan-17-one) and 5alpha-androstane-3alpha, 17beta-diol. The results indicate that while androgen metabolism in the rhesus monkey is similar to that of the baboon and human in conjugate and metabolite formation, the rate of excretion was significantly different, resembline more closely that of the baboon than the human.     

Postnatal ontogenic development of the adrenergic innervation pattern in the rat portal vein

Summary The postnatal development of the adrenergic innervation pattern in the rat portal vein has been studied with the histochemical fluorescence method of Hillarp and Falck.Stretch preparations and transverse freeze-dried sections of intact portal veins were studied from rats during the first 5 weeks of life and from adult rats. Orientation of undifferentiated smooth muscle cells into two layers was observed at 4 days of age. Dominance of the thick outer longitudinal muscle layer was apparent at two weeks of age. A terminal adrenergic nerve plexus with some varicosities was restricted outside the media at the end of the first week. Ingrowth of penetrating non-terminal adrenergic nerve fibers through the longitudinal muscle layer occurred during the second week of age when the main terminal nerve plexus was developing between the two muscle layers. After 3 weeks of age the adult pattern of a two-dimensional adrenergic plexus between the muscle was established. In the adult rat pharmacological treatment with nialamide and noradrenaline revealed the thin, penetrating non-terminal adrenergic nerve fibers in the longitudinal muscle layer which were poorly visible otherwise.The present observations strongly indicate that the main adrenergic plexus between the two muscle layers emanates directly from the outer axonal plexus. These findings are discussed regarding possible trophic interactions between ingrowing sympathetic adrenergic vasomotor nerves and maturing vascular smooth muscle.Supported by grants from the Swedish Medical Research Council (grants No. 14X-2207, O4P-4173, 3884), Magn. Bergwall's Foundation, G. & M. Lindgren's Foundation, the Medical Faculty of University of Göteborg. The technical assistance of Miss Serney Bööj, Mr. Pär-Anders Larsson and Miss Ann Kjellstedt is gratefully acknowledged     

The formation and distribution of intercellular junctions in the rhesus monkey optic cup: The early development of the cilio-iridic and sensory retinas

The eyes of prenatal monkeys from 30 to 102 ± 2 days old were examined by light microscopy, conventional electron microscopy, and the freeze-fracturing technique. At 30 days, invagination of the optic vesicle has begun, and the inner and outer walls of the forming optic cup are closely apposed anteriorly; invagination is complete at 45 days. By 58 days, the rudiment of the ciliary body and iris has appeared; at 71 days, primitive ciliary processes are present and retinal photoreceptors begin to differentiate. The distribution of intercellular junctions varies both in different regions of the optic cup and at different stages of development. At 30 days, adjacent ventricular and adjacent pigmented cells are joined throughout the optic cup by zonulae adhaerentes and gap junctions. The anterior region of the cup, however, contains two additional junctional specializations: (1) fasciae occludentes between ventricular cells and (2) intermediate and gap junctions between the apposing luminal surfaces of ventricular and pigmented cells. By 36 days the fasciae occludentes between ventricular cells in the anterior optic cup become zonular, signaling the morphological development of the blood-aqueous barrier. In the posterior optic cup, zonulae occludentes appear between adjacent pigmented cells at 36 days; furthermore, with the continuing obliteration of the optic ventricle, luminal junctions spread toward the optic stalk but do not reach the optic disc until 45 days, when invagination is complete. Between 58 and 102 days there are no further changes in the distribution of the junctions anteriorly between the primitive cilio-iridial epithelial cells, whereas in the posterior optic cup the luminal gap and intermediate junctions between pigmented cells and differentiating photoreceptors decrease in number and finally disappear. Two main conclusions can be drawn from this study. (1) In the optic cup, intermediate junctions are consistently present in regions of the plasma membrane which later contain junctional complexes. The temporal and spatial pattern of junctional development suggests that intermediate junctions are necessary for the establishment of tight and gap junctions. (2) Twenty days before the ciliary body-iris anlage becomes visible in the light microscope, the distribution of junctions in the anterior part of the optic cup is identical to that in the adult cilio-iridic retina. The time-honored view that the cilio-iridic retina appears late in development is, therefore, no longer tenable. In the monkey, the optic cup is divided into a cilio-iridic and a sensory region soon after the onset of invagination.     

The development of sympathetic innervation and the functional state of the cardiovascular system in newborn dogs.

The present study in dogs indicates that the peripheral sympathetic fibers develop mostly after birth and reach a full maturity at about 2 months of life. The norepinephrine content of the heart, spleen, intestine, salivary glands, and adrenal glands increased from birth to 56 days of age. In contrast, the content of the stellate ganglia decreased during this period. In most of the organs studied, the uptake of [3H] norepinephrine developed in parallel with the norepinephrine content, except in the right atrium and salivary glands where it was fully developed soon after birth. During development, the systemic blood pressure increased from 40 to 100 mm Hg. Bilateral adrenal vessel clamping failed to induce a fall in blood pressure in growing dogs which indicates that the adrenal medulla or the baroreceptors did not fully compensate for the lack of peripheral sympathetic fibers and for the lower blood pressure in newborn animals. Although cardiac norepinephrine content was still very low in 10-day-old animals, cardiovascular responses to direct and reflex sympathetic stimulation were similar to those observed in 56-day-old animals. These results indicate that the sympathetic nervous system becomes functional before the fibers reach their full maturity.     

Autoradiographic localization of aromatic hydrocarbon receptor (AHR) in rhesus monkey ovary

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic congener of a large class of manmade pollutants that persist in the environment. TCDD exerts its toxic effects, in part, by binding to its receptor known as the aromatic hydrocarbon receptor (AHR). TCDD is estrogen modulatory and in some systems its receptor associates directly with estrogen receptors via co-activator molecules. TCDD inhibits steroid synthesis in human ovarian granulosa cells and AHR is found in these cells. We have previously shown that AHR is found in whole rhesus monkey ovary, but have yet to establish its location. In the present study, we set out to show that radiolabeled TCDD binds to monkey ovarian follicles and that this binding is receptor mediated. Ovaries from Macaca mulatta were sectioned on a cryostat at 10 micro m; and sections were incubated with either control vehicle, (3)H-TCDD, or (3)H-TCDD plus alpha-naphthoflavone (ANF), a known receptor-blocking agent. Here, we show for the first time specific binding of TCDD to the granulosa cells of antral follicles and other regions of the rhesus monkey ovary. Our data indicate a 60-fold increase in binding with (3)H-TCDD over that of control, and that this binding is reduced to the levels seen in controls with the addition of the competitive antagonist ANF. These findings support the hypothesis that TCDD directly affects primate ovarian function via the AHR.     

Observations on trichinosis in the rhesus monkey.

Monkeys infected with 2.0 larvae/g body weight died 31-41 days post-infection (PI): two of three monkeys infected with 1.0 larva/g body weight became moribund and were sacrificed at 50 days, but six of six monkeys infected with 1.0 larva/g body weight were healthy 225 days PI. Periorbital and facial edema and eosinophilia were observed in all groups during the second week PI, and myalgia and stiffening of joints was observed during the third week. High numbers of encyse, biceps brachii and deltoideus. Adult worms were recovered from the intestine 49 days PI. The morphological changes were essentially similar to those seen in humans and a generalized lymphoid hyperplasia was not observed. Thus, rhesus monkeys develop trichinosis which show similarities clinically, pathologically and morphologically to human disease.     

Differential preimplantation development of rhesus monkey embryos in serum-supplemented media

Several media, some augmented with amino acids, have been formulated recently, based on simplex optimization, to support the preimplantation development of mouse embryos. For the highly limited studies on preimplantation development of nonhuman primate embryos, a complex medium (CMRL-1066) has been employed. Our objective was to compare the developmental ability of rhesus monkey embryos in a simple medium containing amino acids, KSOM/AA, with the complex media used previously. Zygotes (99) were recovered following in vitro fertilization (IVF) from six monkeys, allocated to either CMRL or KSOM/AA both containing 10% fetal calf serum (FCS), and monitored daily until reaching the expanded or hatched blastocyst stage. The distribution of cells between the inner cell mass (ICM) and trophectoderm was determined at the end of culture by differential nuclear staining. Although a greater number of embryos cultured in KSOM/AA vs. CMRL developed to the morula stage (80%) and beyond (66% to expanded blastocyst), the differences were not significant. Such embryos in KSOM/AA did, however, develop at a significantly faster rate, on average, reaching the expanded blastocyst stage 26 hr earlier than did embryos cultured in CMRL. KSOM/AA embryos hatched in less time and had a higher percentage (43 vs. 34) of cells allocated to the ICM. These results indicate that a simple medium, KSOM/AA, in the presence of serum, supports the development of rhesus monkey embryos at high efficiency and at a faster rate than that observed for embryos cultured in the complex medium, CMRL-1066. © 1996 Wiley-Liss, Inc.