The fine structure of fast and slow crustacean muscles.

Known phasic and tonic muscle fibers of the crab Cancer magister were studied by electron microscopy. Phasic fibers have sarcomeres about 4.5 micro long, small polygonal myofibrils, and a well-developed sarcoplasmic reticulum. The thick myofilaments, disposed in hexagonal array, are each surrounded by six thin filaments. The tonic fibers have a sarcomere length of about 12 micro, larger myofibrils, a poorly developed sarcoplasmic reticulum, and a disorderly array of myofilaments. Each thick myofilament is surrounded by 10-12 thin filaments. The same morphological type of slow muscle has been found in the crustaceans, Macrocyclops albidus, Cypridopsis vidua, and Balanus cariosus, in each case in an anatomical location consistent with tonic action. A search of the literature indicates that this type of muscle is found in all classes of arthropods and is confined to visceral and postural muscles or specializations of these.

Microscopic anatomy of pycnogonida: II. Digestive system. III. Excretory system.

The digestive system of several species of sea spiders (Pycnogonida, Arthropoda) was studied by electron microscopy. It is composed of the foregut inside a long proboscis, a midgut and a hindgut. Lips near the three jaws at the tip of the proboscis receive several hundred ductules originating from salivary glands. These previously undetected glands open on the lips, a fluted, projecting ridge at the external hinge line of the jaws, i.e., to the outside of the mouth. This disposition suggests affinities to the chelicerate line. The trigonal esophagus within the proboscis contains a complex, setose filter device, operated by dedicated muscles, that serves to reduce ingested food to subcellular dimensions. The midgut has diverticula into the bases of all legs. Its cells differentiate from the basal layer and contain a bewildering array of secretion droplets, lysosomes and phagosomes. In the absence of a hepatopancreas, the midgut serves both digestive and absorptive functions. The cuticle-lined hindgut lies in the highly reduced, peg-like abdomen. Traditionally, pycnogonids have been claimed to have no excretory organ at all. Such a structure, however, has been located in at least one ammotheid, Nymphopsis spinosissima, in which a simple, but standard, excretory gland has been found in the scape of the chelifore. It consists of an end sac, a straight proximal tubule, a short distal tubule, and a raised nephropore. The end sac is a thin-walled and polygonal chamber, about 150 microm in cross section, suspended in the hemocoel of the appendage, its edges radially tethered to the cuticle at more than half a dozen locations. This wall consists of a filtration basement membrane, 1-4 microm thick, facing the hemocoel, and internally of a continuous carpet of podocytes and their pedicels. The podocytes, measuring maximally 10 by 15 microm, have complex contents, of which a labyrinthine system of connected intracellular channels stands out. These coated cisternae open into a central vacuole that often rivals the nucleus in size. The design of the organ closely approximates that of the primitive crustacean Hutchinsoniella macracantha.

Surface adaptations of the vertebrate epidermis to friction.

Epidermal surfaces in representative vertebrates specialized for lowered or increased friction were studied with the scanning electron microscope. Microvillous and microridged patterns predominate in aquatic vertebrates. In squamate reptiles, the complex and varied ornamentation of the Oberhäutchen functions both in adhesive modifications and in modulating surface reflectivity. Frictional surfaces in birds and mammals are characterized more by anatomical than by cytologic specializations, the detailing of surface cells being mostly a function of turnover rate.

Immunocytochemical localization of progestin receptors in monkey hypothalamus: effect of estrogen and progestin.

The increase in PRL secretion which follows progesterone (P) administration to estradiol (E)-primed women and monkeys cannot be due to an action of P at the pituitary level because lactotropes do not contain progestin receptors (PR). To further the hypothesis that P increases PRL secretion by an action in the hypothalamus, PR-expressing neurons were studied in free-ranging and steroid-manipulated monkeys using immunocytochemistry with a monoclonal antibody to human PR. Specific PR immunoreactivity is localized in the nucleus of individual hypothalamic neurons. Male and female adult and juvenile macaque hypothalami contain significant populations of PR-positive neurons throughout the anterior and medial basal hypothalamus. Ovariectomy decreases, but does not abolish, the number of neurons expressing PR. PR expression was not altered in the supraoptic nucleus (SON) by ovariectomy. Estrogen treatment for 28 days caused a significant increase in the number of PR-positive neurons in the medial preoptic area, the ventro-medial nucleus, the arcuate nucleus, and the median eminence, but not in the SON. P treatment added to the E treatment from day 14 to day 28 did not alter the number of PR-positive neurons in any area. These data suggest that PR may be constitutively expressed in the magnocellular neurons of the SON and in certain other cells throughout the hypothalamus. E induces PR in a large proportion of neurons in the medial basal hypothalamus and this action is not blocked by subsequent P treatment. The inability of P to down-regulate PR in the hypothalamus differs from the reproductive tract and pituitary. Indeed, this observation is consistent with the fact that PRL secretion remains elevated during chronic P administration.

Steroid action on estrogen and progestin receptors in monkey pituitary cell cultures.

Estradiol (E) treatment of spayed macaques induces progestin receptors (PR) in pituitary gonadotropes, but not lactotropes. In contrast, levels of pituitary estrogen receptors (ER) remain constant in spayed or E-treated monkeys. In monkey pituitary cultures, the number of PR-positive cells varies depending on the donor status, whereas the percentage of ER-positive cells in similar. We sought to determine whether E can directly induce PR in monkey pituitary gonadotropes in culture and to provide further evidence that monkey pituitary ER are constitutively expressed. Dispersed pituitary cells from intact or gonadectomized male and female macaques were cultured on extracellular matrix with and without E, phenol red, high or low insulin, insulin-like growth factor-I, or 5% ovariectomized monkey serum, ER- and PR-positive parenchymal cells were immunohistochemically detected with monoclonal antibodies H222 and D75 (against human ER) and B39 (against human PR). ER levels were also measured with a gradient shift assay incorporating H222. Neither the percentage of ER-positive cells nor the levels of nuclear ER were altered by donor status or by 8 days of culture in phenol red or E. In contrast, cultures from gonad-intact donors exhibited the highest average percentage of PR-positive cells. The lower number of PR-positive cells in cultures from spayed donors did not vary for 8 days on extracellular matrix without phenol red, E, insulin, or serum. E directly increased the percentage of PR-expressing cells in serum-free cultures. Addition of serum also increased the percentage of PR-positive cells. Addition of E to serum-containing cultures further increased the percentage of PR-positive cells. Neither insulin nor insulin-like growth factor-I directly affected the number of PR-positive cells, but a high level of insulin blunted the action of E on PR induction in serum-free culture. We conclude that E treatment has no obvious effect on ER expression in macaque pituitary. However, the induction of pituitary PR by E treatment of spayed monkeys can be accounted for by a direct action of E on PR gene expression in gonadotropes.

Immature rat ovaries become revascularized rapidly after autotransplantation and show a gonadotropin-dependent increase in angiogenic factor gene expression.

When the ovaries of 23-day-old juvenile rats are transplanted to an ectopic site, they recover within 1 week the ability to control gonadotropin secretion via steroid negative feedback. Vascular corrosion casting followed by scanning electron microscopy revealed that the transplanted ovary becomes profusely revascularized within 48 h after transplantation. Vascular ingrowth was accompanied by a 40- to 60-fold increase in expression of the genes encoding two angiogenic factors, vascular endothelial growth factor (VEGF) and transforming growth factor-beta 1 (TGF beta 1), as assessed by RNA blot hybridization of the corresponding mRNAs. Although TGF beta 3 mRNA levels also increased, no changes in the levels of mRNAs encoding other putative angiogenic factors, such as TGF alpha, basic fibroblast growth factor, and TGF beta 2, were observed. Hybridization histochemistry demonstrated that in intact ovaries, VEGF mRNA is mainly expressed in granulosa cells of the cumulus oophorus and thecal cells of large antral follicles. Transplantation is followed by an increase in mRNA abundance and a dramatic shift in cellular localization, so that the mRNA becomes predominantly expressed in cells of the outer ovarian cortex. In intact ovaries, low levels of TGF beta 1 mRNA were detected in thecal-interstitial cells; after transplantation, its expression also became more predominant in the ovarian outer cortex, but this change was not as marked as in the case of VEGF. Because ovarian autotransplantation is followed by a rapid increase in serum gonadotropin levels, experiments were conducted to determine the importance of this rise in the activation of VEGF and TGF beta 1 gene expression. After transplantation, some animals were treated with the LHRH antagonist Nal-Glu LHRH (50 micrograms/rat, once a day for 2 days) to prevent the posttransplantation rise in serum gonadotropins. Quantitation of VEGF and TGF beta 1 mRNA by RNase protection assay 48 h later showed that suppression of gonadotropin secretion diminished the increase in both VEGF and TGF beta 1 gene expression. Concomitant treatment with PMSG (8 IU/rat, single injection), which mainly bypasses the suppression of endogenous FSH levels, restored the TGF beta 1 mRNA response, but had no effect on VEGF mRNA. The results suggest that the increase in gonadotropin secretion following ovarian transplantation contributes to revascularization of the graft by up-regulating the gene expression of two major angiogenic factors.

Neural and glial-mediated effects of growth factors acting via tyrosine kinase receptors on luteinizing hormone-releasing hormone neurons.

It is becoming increasingly evident that the secretory activity of LHRH neurons is regulated not only by transsynaptic inputs but also by trophic molecules of glial and neuronal origin. The present experiments were undertaken to gain insights into the potential cell-cell mechanisms by which basic fibroblast growth factor (bFGF) and transforming growth factor-alpha (TGF alpha), two growth factors produced in the hypothalamus, may affect LHRH neuronal function. Northern blot analysis showed that the LHRH-producing cell line GT1-7 contains the messenger RNA (mRNA) encoding the type 1 fibroblast growth factor receptor (FGFR-1) but not that encoding the epidermal growth factor (EGF) receptors, which mediates the biological actions of both TGF alpha and EGF. Ligand-induced receptor phosphorylation experiments demonstrated that GT1-7 cells possess biologically active FGFR-1s but not EGF receptors. Exposure of the cells to bFGF resulted not only in FGFR-1 tyrosine phosphorylation, but also in tyrosine phosphorylation of phospholipase C gamma, one of the initial enzymes in the intracellular signaling cascade initiated by FGFR activation. GT1-7 cells proliferated in response to this activation. Despite the presence of biologically active receptors, bFGF did not significantly stimulate release of the mature LHRH decapeptide. Instead, bFGF increased the steady-state levels of the mRNA encoding the LHRH precursor processing endoprotease PC2, with a time course comparable to that of phorbol esters, suggesting that, as shown in the companion paper, the actions of the growth factor on LHRH neurons involve facilitation of the initial step in LHRH prohormone processing. The increase in PC2 gene expression was not accompanied by changes in LHRH mRNA levels. Unlike these direct actions of bFGF on GT-1 cells, TGF alpha appears to act indirectly via astroglial intermediacy. Exposure of GT1-7 cells to TGF alpha or EGF failed to affect several parameters of cellular activity including LHRH release, LHRH and PC2 mRNA levels, and cell proliferation. In contrast, astrocyte culture medium conditioned by treatment with TGF alpha led to sustained stimulation of LHRH release with no changes in LHRH gene expression and a transient increase in PC2 mRNA levels. Although no definitive evidence for the presence of FGFR-1 in normal LHRH neurons could be obtained by either double immunohistochemistry or double in situ hybridization procedures, fetal LHRH neurons in primary culture responded to bFGF with neurite outgrowth. Thus, normal LHRH neurons may have an FGFR-1 content too low for detection by regular histochemical procedures, and/or detectable expression of the receptor may be confined to a much earlier developmental stage. The mitogenic effect of bFGF on GT1-7 cells supports this possibility and suggests a role for FGF in the cell proliferation events that precede acquisition of the LHRH neuronal phenotype. It appears that once this phenotype is established, bFGF may promote the differentiation of LHRH neurons. The results also suggest that the secretory capacity of LHRH neurons develops under a dual trophic influence, one on peptide processing exerted directly by bFGF on early neurons, and another on LHRH release, exerted by TGF alpha via the intermediacy of astroglial cells.

Harlequin ichthyosis with epidermal lipid abnormality.

An infant with phenotypic harlequin ichthyosis survived for nine months, then died a crib death. At autopsy, an enlarged, but structurally normal, thymus was found. Light microscopically, the epidermis showed massive hyperkeratosis and variable parakeratosis, and a stain for neutral fat was positive in the upper epidermis and stratum corneum. Electron microscopic study disclosed crystals resembling cholesterol and masses of autophagic vacuoles, many of them glutted with lipid, deposited within cells of the stratum corneum. Biochemically, cholesterol and triglyceride levels in the stratum corneum were sharply elevated (19.8 and 32.0 mg/g of dry weight, respectively). A defect in epidermal lipid metabolism is postulated.

Ciliary vasoconstriction after topical adrenergic drugs.

Rabbits underwent the single-dose or long-term therapeutic administration of the adrenergic drugs phenylephrine hydrochloride, timolol maleate, and betaxolol hydrochloride. After a single dose, all three drugs caused substantial, localized constriction in the arterioles that supply the ciliary processes but did not affect the downstream bore of the same vessels. After seven weeks of a daily dose, tolerance reduced the response to betaxolol to insignificant levels and that to phenylephrine substantially, whereas timolol maleate continued to produce identical levels of vasoconstriction to those seen with single-dose administration. In addition to the consequent lowering of perfusion of the ciliary processes and presumptive impact on aqueous humor production, vasoconstriction also reinforces concerns about impaired vascular perfusion of eyes undergoing long-term ocular therapy.

Microscopic anatomy of Pycnogonida: I. Cuticle, epidermis, and muscle.

The integument of Pycnogonida (Arthropoda) consists of an epicuticle decorated with tubercles and a filamentous coat, an exocuticle with a small number of ill-defined layers, and an endocuticle whose numerous layers are composed of conspicuously cross-banded fibrils. This cuticular periodicity, attributable to cross-linked chitin, has been observed previously in uncalcified and untanned cuticle of many lower crustaceans, especially branchiopods and copepods, and in scattered examples of thin respiratory or excretory cuticles of other arthropods. It is uniformly present in all representatives of all nine pycnogonid families examined to date. Stomodeal, proctodeal, and arthrodial cuticles are devoid of the endocuticular periodicity. The cuticle is decorated with sensory filaments and setae, but is more noteworthy for a dense coverage by glands, up to 1,400/mm2. Myocuticular junctions have desmosomal fine structure previously found only in chelicerates. Muscle fine structure is that of slow fibers with long sarcomeres and a high actin to myosin filament ratio, except for cardiac muscle, which has short sarcomeres. Among the arthropods, only merostomates resemble the pycnogonids in the lack of fast somatic muscle fibers. Pycnogonids display a hybrid array of fine structural features that variously serve to relate them to some arthropod subphyla and distance them from others.

Root dentin morphology after different modes of citric acid and tetracycline hydrochloride conditioning.

The purpose of this study was to assess the effect of citric acid and tetracycline HCl application to dentin surfaces by a "passive dripping" or an "active burnishing" technique. Twenty dentin blocks were prepared from freshly extracted non-diseased human impacted third molars. The blocks were root planed and randomly assigned to two groups for treatment with either citric acid or tetracycline HCl. The duration of treatment was 30, 60, 120, or 240 seconds. Control blocks were treated with distilled water. After treatment the blocks were processed for observation and measurements in the scanning electron microscope (SEM). Application of either of the acid solutions resulted in removal of the smear layer. Measurements indicated a time dependent increase in the mean dentinal tubule orifice diameter ranging from 1.05 microns in control specimens to 3.18 microns after 4 minutes treatment (citric acid group). The increase in tubule diameter was significantly greater (P less than or equal to 0.01) for both citric acid treatment modalities than tetracycline HCl treatment. There was also a time dependent increase in the depth of penetration as measured by a trumpeting of the tubule profiles, and this penetration was significantly greater (P less than or equal to 0.01) after citric acid treatments. Passive or active application of the acids did not seem to have any major impact on the measurements or on the surface morphology. It was concluded that citric acid causes more extensive changes than tetracycline HCl and that the mode of application of the agent is probably not critical.

The brain of the horseshoe crab (Limulus polyphemus). III. Cellular and synaptic organization of the corpora pedunculata.

The large, hemispherical mass of the Limulus corpora pedunculata consists of two highly branched lobes, each connected to the protocerebrum by a narrow stalk. About 10(4) afferent fibers enter through the stalks and make diverse, profuse, and often reciprocal contacts with several million Kenyon (intrinsic) cells and one another. The Kenyon cell axonal arborizations converge on a few hundred efferent dendrites. The afferent fiber types can be classified into five types. Type A forms the club-shaped core of glomeruli and circumglomerular annuli, and contains small flat vesicles, suggesting an inhibitory function. Type B terminates with bushy endings in glomeruli and is presynaptic to both Kenyon cells and to Type A terminals. It has clear round vesicles and is the presumptive excitatory input. Type C terminates on other afferents, in glomeruli, and rarely on Kenyon cell bodies, contains angular (neurosecretory) granules and is postulated to impart circadian rhythm. Type D terminates on Kenyon cell somata and the initial neurite segment (but not in glomeruli), and contains dense-cored vesicles. Type E terminates in peduncles on other afferents and Kenyon cell telodendria. It contains dense vesicles. The C, D, and E afferents have reciprocal synaptic connections with Kenyon cell axon terminals. Glomeruli thus receive three different inputs of presumptive inhibitory (A), excitatory (B), and neuromodulatory nature (C). Kenyon cells, increasing in number up to about 1 x 10(8) in the adult, show minor variations in their dendritic pattern and have only one rare variant cell type. Interactions between them occur primarily at their axonal boutons as they crowd around efferent fibers. The latter have large receptive fields, some of their large somata are located within the confines of the corpora pedunculata, and they receive input almost only from Kenyon cells. Numerical and directional details of the circuitry in the corpora pedunculata have been extracted by a combination of light and electron microscopy, serial sectioning, silver staining, and stereology. The corpora pedunculata appear to process primarily the voluminous chemosensory input from the appendages, an assumption that is supported by the major connections of the organ.

The brain of the horseshoe crab (Limulus polyphemus). II. Architecture of the corpora penduculata.

The corpora pedunculata, or mushroom bodies, of the horseshoe crab, Limulus polyphemus, form a bulbous ventral hemisphere composed of two internal lobes that are highly branched like a caulifower. This organ is clothed with a deep layer of small association neurons called globuli or Kenyon cells. In an animal that is 50 mm in width, they number 3-7 X 10(6), a value that rises to about 1 X 10(8) in an adult (250 mm width). The neuropil of each corpus peduculatum converges from its peripheral lobules toward several major peduncles, which are in communication with the protocerebral neuropil by a narrow stalk containing about 5000 fibers in a 50 mm animal. The numberical relations suggest that presumptive second-order chemosensory fibers enter the corpora pedunculata and synapse divergently onto Kenyon cells. The axons of Kenyon cells, in turn, converge onto efferent fibers that leave through the stalk.

The brain of the horseshoe crab (Limulus polyphemus). I. Neuroglia.

The brain of the horseshoe crab, Limulus polyphemus, harbors three populations of neuroglial cells, whose distribution and cellular details are best appreciated by a combination of silver impregnation, scanning, and transmission electron microscopy. Stellate astrocytes envelop neurons as satellite cells, permeate the neuropile, and secrete a framework of sustentacular trabeculae throughout the brain. Velate astrocytes are restricted to Kenyon cells, i.e. small association neurons, of which they harbor up to 150 per neuroglial cell. Vascular neuroglia is composed of glycogen and mitochondria-laden, interlocked cells that form an open meshwork in the hemocoelic spaces of the brain. Aside from supportive functions of neuroglia, the vascular neuroglial cells in particular seem to subserve the role of a metabolic reserve cell for the central nervous system.

Gill receptor arrays in the horseshoe crab (Limulus polyphemus).

The branchial warts on the endopodites of the gills are covered with goblet-shaped cuticular appendages, whose internal structure shows them to be chemoreceptors. The innervated goblets have a cuticular tubule that connects an external pore through their hollow interior with the epidermal sensillum. Associated sensory neurons give rise to small axons that pass through a synaptic plexus below the epidermis. The sense organs seem specialized for sampling the exhalent water current.

Anatomical circuitry of lateral inhibition in the eye of the horseshoe crab, Limulus polyphemus.

Lengthy uninterrupted series of sections of the neural plexus in the compound eye of the horseshoe crab, Limulus polyphemus, have been used to reconstruct all the arborizations and their synaptic interconnections in a neuropil knot. This one microglomerulus contains the axons of 19 retinular cells, which pass by without contacts; 13 efferent fibres with 44 synapses to and from eccentric cell collaterals; and arborizations from 54 eccentric cells with 577 synapses. Eccentric cell axons are devoid of synaptic input. Their collaterals ramify in synaptic knots and subserve both pre- and postsynaptic functions simultaneously. Arborizations near the axon of origin have a highly branched pattern (up to 20 bifurcations), a high synaptic input: output ratio (up to about 9:1), and high synaptic density (a maximum of 12 per micrometre of neurite length). The opposite extreme is represented by sparsely branched eccentric cell collaterals distant from their axons of origin with very little synaptic input and sparse output. Spatially graded lateral inhibition is the apparent outcome of a radially decreasing distribution of inhibitory synapses on the arborizations of eccentric cell collaterals combined with possible decremental signal transmission in the plexus. The synaptic analysis has a bearing on most physiological aspects of lateral inhibition that have been studied in the Limulus eye. Implied in the results is the suggestion that synapse formation is an intrinsic property of the presynaptic element, but that the connectivity is governed by the electrical activity of target neurons.

Stereo photomicrography of Golgi preparations.

Photomicrographs of thick sections taken with reciprocal, lateral illumination in a compound microscope produce stereo-pairs. The method is particularly useful with thick celloidin sections of nervous tissue impregnated with the Golgi selective silver technique.