Development of the endothelium.

Our understanding of the regulation of vascular development has exploded over the past decade. Prior to this time, our knowledge of vascular development was primarily based on classic descriptive studies. The identification of stem cells, lineage markers, specific growth factors and their receptors, and signalling pathways has facilitated a rapid expansion in information regarding details of the mechanisms that govern development of the vascular system.

Interleukin-1beta regulates nitric oxide production and gamma-glutamyl transpeptidase activity in sertoli cells.

Several cytokines have been involved in the regulation of Sertoli cell function. Further investigations are required to elucidate the role of interleukin-1beta (IL1beta) in Sertoli cell physiology. Twenty-day-old rat Sertoli cell cultures were used to investigate a possible role of IL1beta in the regulation of gamma-glutamyl transpeptidase (gammaGTP) and to elucidate the signaling pathway utilized by this cytokine. GammaGTP is a membrane-bound enzyme that has been involved in amino acid transport across the plasma membrane and in protection from oxidative stress through its importance in the regulation of glutathione levels. Previous studies suggested that IL1beta stimulates NO biosynthesis in other cell types. Therefore, we investigated whether IL1beta modified the level of nitrite, a stable metabolite of NO, in Sertoli cells. Dose-response curves to IL1beta for gammaGTP activity and nitrite production were observed. The increments observed in gammaGTP activity and nitrite production were partially and completely blocked by simultaneous treatment with the NO synthase inhibitor aminoguanidine. Treatment of Sertoli cell cultures with the NO donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine resulted in an increase in gammaGTP activity. The presence of neural, endothelial, and inducible isoforms of NO synthase (NOS) was investigated by a immunohistochemical technique using specific antibodies. The 2 constitutive isoforms were present under basal conditions, and the inducible protein appeared in IL1beta-treated cultures. Finally, translocation of NF-kappaB p65 subunit to the nucleus in IL1beta-treated cultures was observed. These findings suggest that the action of IL1beta on Sertoli cell gammaGTP activity is partially mediated via activation of NF-kappaB and increments in iNOS and cellular production of NO.

Lipopolysaccharide directly affects pancreatic acinar cells: implications on acute pancreatitis pathophysiology.

We have explored whether lipopolysaccharide (LPS, endotoxin) induces pancreatic injury on pancreatic acinar cells both in vivo and in vitro. Wistar male rats were treated with four intraperitoneal injections of 10 mg/kg LPS, and AR4-2J cells were exposed to increasing doses of LPS. Expression of pancreatitis-associated-protein (PAP) mRNA was strongly induced in AR4-2J cells exposed to LPS, while amylase mRNA was reduced. LPS also induced apoptosis and expression of TNF-alpha, IL-1beta, and IL-8 mRNA in AR4-2J cells. The in vivo effect of LPS showed structural signs of cellular damage, including numerous cytoplasmic vacuoles, severe nuclear alterations, and high expression of PAP mRNA. This study demonstrated that LPS induced pancreatic damage by directly affecting the pancreatic acinar cells. The role of LPS in the pathophysiology of acute pancreatitis may be partly due to the effect LPS has on the acinar cell.

Structural specializations of the eye in the vizcacha (Lagostomus maximus maximus).

Vizcachas (Lagostomus maximus maximus, Chinchillidae) are nocturnal rodents living in burrows in many regions of Argentina, Bolivia, and Chile. We have studied the eye of the vizcacha using several light and electron microscopic procedures, with the purpose of understanding the role of vision in the behavior of this species. Our observations demonstrated an avascular, rod-rich retina, with a specialized region spanning through most of the equator of the eye. In this central band, all neural retinal layers exhibited a high cell density, whereas the photoreceptor layer was characterized by the presence of very long rods. In addition, the central region was associated with a distinct pigmentation pattern, including scarce granulation of the pigment epithelium, low pigmentation of the choroid, and the selective attachment of suprachoroidal cells to the inner scleral surface. These central modifications probably form the structural basis of a reflecting tapetum. The eye of the vizcacha received both long and short ciliary vessels, and a specialized cilio-sclero-choroidal vascular network appeared at the equatorial region. Our findings suggest that the equatorial region of the eye of the vizcacha could be a highly sensitive light detector related to foraging behaviors during crepuscular or nocturnal hours.

Differential effects of superior and inferior spermatic nerves on testosterone secretion and spermatic blood flow in cats.

It has been postulated that testosterone secretion is partially regulated by signals from the spermatic nerves. To further examine this hypothesis in vivo, the superior (SSN) or the inferior (ISN) spermatic nerves were stimulated electrically (varying intensity, 25 Hz, 0.2 msec, 10 min) in anesthetized cats, determining the testosterone concentration and the blood flow in the spermatic vein. In some additional experiments arterial blood was sampled, and norepinephrine (NE) output was calculated. Stimulation of the SSN (25-35 V) increased the testosterone concentration in spermatic vein blood (P < 0.01 compared with prestimulation levels). The response varied among animals, reaching a 50-100% increase in some animals, whereas in others it ranged from almost undetectable to more than 10 ng/100 g x min. Under the same experimental conditions, the NE output increased from 135.4 +/- 99 to 1614.2 +/- 347 pg/ml (P < 0.01), and spermatic blood flow decreased from 24.1 +/- 1.42 to 20.2 +/- 1.65 ml/min x 100 g (P < 0.05) during nerve stimulation. By contrast, stimulation of the ISN (25-35 V) modified neither the testosterone concentration, the NE output, nor the blood flow in the spermatic vein. High intensity stimulation (36-70 V) of each spermatic nerve evoked different vascular and hormonal effects. SSN activation induced a marked decrease in spermatic blood flow during stimulation and an increase in the testosterone response, whereas ISN activation resulted only in an enhanced spermatic blood flow. Our results suggest that testosterone secretion, although mainly dependent on gonadotropin secretion, could be further regulated by neural inputs from the SSN acting directly or alternatively through changes in blood flow. It would appear that the SSN mainly supplies the vasoconstrictor fibers to the testis, whereas the ISN provides vasodilator fibers.

Innervation of blood vessels in the vomeronasal complex of the rat.

We have made an immunohistochemical study of the vomeronasal (VN) complex of 12-day-old rats to characterize the innervation of its blood vessels. The VN complex can be subdivided into rostral, middle and caudal segments, each one with a particular vascularization pattern. Several small vessels were associated with the rostral segment, whereas a large venous sinus ran along the middle and caudal segments. Immunostaining for alpha-smooth muscle actin demonstrated that the muscular sheath was asymmetric, with more cells layers in its lateral than in its medial walls. Nerves were demonstrated with antisera against protein gene product 9.5 (PGP), and against several molecules associated with specific classes of nerve fibers: the C-terminal peptide of neuropeptide Y (CPON), calcitonin gene-related peptide (CGRP), substance P (SP), galanin (GAL), vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (NOS). The latter, was also studied with NADPH-diaphorase. Vascular associated fibers exhibited NOS-, CPON-, GAL-, CGRP-, SP- and VIP-immunoreactivity. Only the vessels of the rostral segment showed VIP-immunoreactive fibers. Each wall of the venous sinus exhibited different types of nerve fibers. CPON-, GAL-, CGRP- and SP-immunoreactive fibers concentrated in the medial wall, whereas NOS-immunoreactive ones concentrated in the lateral wall. This distribution of vascular fibers, plus the presence of sensory fibers exhibiting CGRP-, SP- and GAL-immunoreactivity within the pseudostratified epithelium of the VN tube, would be relevant to understand the operation of the pumping mechanism regulating influx and efflux from the VN tube.

Role of nitric oxide in salivary secretion.

Since nitric oxide has been found to control the function of many organs of the body by the non-adrenergic, non-cholinergic branch of the autonomic nervous system, we hypothesized that it might play a role in salivary secretion. Therefore, we investigated the distribution of nitric oxide synthase (NOS) throughout the submaxillary gland and also studied the ability of inhibitors of NOS to interfere with salivation induced by a cholinergic agonist, metacholine, and by a polypeptide, substance P. The secretory responses were determined in rats anesthetized with chlorolose following intravenous injection of the various pharmacological agents. There was no basal flow of saliva and dose-response curves were obtained by sequential intravenous injection of increasing doses of the drugs. Then, in the same animal, the same dose-response curves were performed in the presence of NOS inhibitors. L-Nitro-arginine-methyl-ester (L-NAME; 20 mg/kg) produced an over 50% inhibition of the dose-related salivation induced by metacholine. Similar results were produced with L-NG-monomethyl-L-arginine (L-NMMA; 5 mg/kg). The salivation induced by much lower molar doses of substance P was dramatically greater than that obtained with metacholine. The response to substance P was almost completely inhibited by L-NMMA at the lowest dose (0.3 mg/kg), but at higher doses (1 mg/kg), the inhibition was only around 60% and at the highest dose (3 mg/kg) only about 20%. In control rats, there were roughly equal amounts of calcium-dependent and calcium-independent NOS in the gland at this time. At the end of the experiment, the effect of the inhibitor of NOS, L-NMMA, on the NOS activity in the submandibular gland was determined. At this time, the Ca2+-dependent NOS was decreased and the Ca2+-independent NO was increased. The prior injection of L-NMMA reduced calcium-dependent NOS activity by approximately 70% but calcium-independent activity by only 30%. These results indicate that, at least at the end of the experiment, the blockade of NOS imposed by NMMA was incomplete. This could account in part for the failure of the inhibitors to block completely the stimulatory effect of the two secretagogues. Analysis of the distribution of NOS in the salivary gland revealed that it was not present in the acinar cells, but in neural terminals within the gland and also in the ductile system which contained neural (n) NOS in the apical membrane of the excretory and striated ducts, the cytoplasm of granular convoluted tubules and, to a lesser extent, in the cytoplasm of excretory and striated ducts. Macrophage (inducible) NOS was also found not only in the macrophages, but also in the tubules and ducts. Since drugs were used that would act on the receptors in the gland, the role of NO in our conditions is probably mediated by nNOS and iNOS in the ductile and tubular structures. Since iNOS would already be active, it is unlikely to play a role in this acute secretory activity. Rather the nNOS in these non-neural cells is probably activated by muscarinic or K1 receptors by metacholine and substance P, respectively, leading to an increase in intracellular free calcium that activates NOS leading to the generation of cGMP that opens ion channels to initiate the secretory process.

Innervation of nasal turbinate blood vessels in rhinitic and nonrhinitic children.

An immunohistochemical study of the nasal mucosa was done in pediatric patients attending an otorhinolaringology (ORL) clinic. The goal was a comparison between vascular innervation in patients with or without symptoms of chronic rhinitis. All patients had an indication for tonsillectomy prior to their inclusion in this study. Samples were obtained under general anesthesia at the time of programmed surgery and fixed in a paraformaldehyde-picric acid mixture. Cryostat sections were immunostained for the following neuronal markers: protein-gene product 9.5 (PGP), calcitonin gene- related peptide (CGRP), substance P (SP), and C-terminal peptide of neuropeptide Y (CPON). The following classes of vessels were identified: arteries, sinusoids, veins, and arteriovenous anastomoses (AVAs). As shown by immunostaining with the general neuronal marker PGP, each vessel type had a characteristic innervation pattern, differing in the amount of fibers and their distribution within the adventitial and muscle layers. Evaluation of PGP, CPON, and CGRP immunoreactivity patterns indicated that rhinitic arteries and AVAs displayed a richer innervation than did nonrhinitic blood vessels. Quantification of vascular PGP immunostaining confirmed the difference of vascular innervation between nonrhinitic and rhinitic patients. Fibers immunostained by CPON partially accounted for the rhinitic arterial hyperinnervation.

Monoaminergic and peptidergic contributions of the superior and the inferior spermatic nerves to the innervation of the testis in the rat.

Sections of the rat testis and whole-mounts of the testicular capsule were studied microscopically using the glyoxylic acid-induced fluorescence method, to detect monoamines, and immunohistochemical procedures for the detection of immunoreactivities to protein gene-product 9.5 (PGP 9.5), the C-terminal accompanying peptide of neuropeptide Y (CPON), and vasoactive intestinal polypeptide (VIP). Monoaminergic nerves were only observed around the intracapsular blood vessels: the initial segment of the testicular artery and the superior venous plexus, and in the anterior aspect of the upper and lower testicular poles. These capsular nerve networks were associated with the superior and inferior ligaments of the testis. Nerves displaying PGP 9.5 and CPON immunoreactivity appeared in the same sites and followed the same distribution as monoaminergic nerves. By contrast, VIP-immunoreactive fibers were only found in the nerve network of the lower pole. Observations done after different surgical denervation procedures demonstrated that the superior spermatic nerve was the source of fibers for testicular vessels and for the nerve network of the upper pole. On the other hand, fibers from the inferior spermatic nerve were restricted to the nerve network of the lower pole.

Distribution of the inositol 1,4,5-trisphosphate receptor, P400, in adult rat brain.

The distribution of the inositol 1,4,5-trisphosphate receptor protein, P400, was investigated in adult rat brain by immunocytochemistry with the monoclonal antibody 4C11 raised against mouse cerebellar inositol 1,4,5-trisphosphate receptor protein. Immunoreactive neuronal cell bodies were detected in the cerebral cortex, the claustrum, the endopiriform nucleus, the corpus callosum, the anterior olfactory nuclei, the olfactory tubercle, the nucleus accumbens, the lateral septum, the bed nucleus of the stria terminalis, the hippocampal formation, the dentate gyrus, the caudate-putamen, the fundus striatum, the amygdaloid complex, the thalamus, the caudolateral part of the hypothalamus, the supramammillary nuclei, the substantia nigra, the pedunculopontine tegmental nucleus, the ventrotegmental area, the Purkinje cells in the cerebellum, the dorsal cochlear nucleus, the subnucleus oralis and caudalis of trigeminal nerve, and the dorsal horn of the spinal cord. Immunoreactive fibres were found in the medial forebrain bundle, the globus pallidus, the stria terminalis, the pyramidal tract, the spinal tract of trigeminal nerve, and the ventral horn of spinal cord. Nerve fibres forming a dense plexus ending in terminal-like boutons were detected in relation to nonimmunoreactive neurons of the dentate, interpositus, and fastigial nuclei of the cerebellum and around neurons of the vestibular nuclei. This receptor protein binds a specific second messenger, inositol 1,4,5-trisphosphate, which produces a mobilization of intracellular Ca2+ and a modulation of transmitter release.

Immunohistochemical localization of the inositol 1,4,5-trisphosphate receptor in the human nervous system.

A monoclonal antibody raised against the mouse cerebellar inositol trisphosphate receptor was used to study the immunohistochemical localization of this protein in the human central nervous system. As in the brain of rodents, strong immunoreactivity was found in dendrites, axon and cell bodies of Purkinje cells, as well as in nerve endings in the cerebellar and vestibular nuclei. Cerebellar efferent fibres were the only positive structures demonstrated in the brainstem and no immunostaining could be detected in the spinal cord or dorsal root ganglia. By contrast, numerous immunoreactive neurons were present in several telencephalic and diencephalic structures, including the brain cortex, hippocampus, basal ganglia, basal forebrain, amygdala and thalamus. Immunostaining of these brain neurons was weaker than that found in Purkinje cells and was evident in cell bodies and dendrites. Thus, the human brain contains a molecule cross-reacting with the mouse inositol trisphosphate receptor protein that is expressed in a pattern similar to that found in rodents. These findings can be of great importance for understanding the function of this protein in normal brain and its modifications in neuropathological disorders.

Redistribution of secretory granule components precedes that of synaptic vesicle proteins during differentiation of a neuronal cell line in serum-free medium.

Incubation of the rat sensory neuron-derived cell line ND7 in serum-free medium results in the arrest of mitosis and the appearance of numerous neuronal processes. During this differentiation event, secretory granule components such as chromogranins, neuropeptide Y and the C-flanking peptide of pro-neuropeptide Y move to the tips of the majority of the neuronal processes regardless of process length. In contrast, the synaptic vesicle component, synaptophysin, is found only at the tips of the very long processes which appear following prolonged periods of culture in serum-free medium. A similar restriction of synaptophysin to long processes is also observed following differentiation and process formation induced by other treatments such as incubation in reduced serum or treatment with cyclic AMP or phorbol myristate acetate. Hence the regulated secretory pathway associated with the chromogranins and neuropeptides appears to be segregated into the processes at an earlier stage of ND7 differentiation than the synaptophysin-associated synaptic vesicle pathway. ND7 cells therefore provide a model system for studying the processes regulating these pathways and the redistribution of their components during neuronal differentiation.

Developmental pattern and distribution of nerve growth factor low-affinity receptor immunoreactivity in human spinal cord and dorsal root ganglia: comparison with synaptophysin, neurofilament and neuropeptide immunoreactivities.

Immunocytochemical expression of the low-affinity nerve growth factor receptor was studied in human fetal and adult tissues using the monoclonal antibody ME20.4. In dorsal root ganglia, a few immunoreactive neurons were first detected in nine-week-old fetuses and many more were found in the following weeks of gestation. However, none was present in adult ganglia. The ME20.4-positive cells were larger than neurons immunostained by substance P, calcitonin gene-related peptide or galanin antibodies. In the spinal cord, fibres immunostained by ME20.4 appeared in a characteristic pattern that differed from the spatial and temporal distributions of synaptophysin- and neurofilament-immunoreactive fibres. Those expressing the low-affinity nerve growth factor receptor were only detected in regions containing collaterals of primary sensory axons: (i) in the dorsal funiculus between seven and 18 weeks of gestation; (ii) in a ventrodorsal bundle reaching the ventral horn from weeks 12-14; (iii) in the medial region of the dorsal horn between weeks 12 and 20; (iv) in the superficial layers and lateral portion of the dorsal horn after the 14th week of gestation and also in adult spinal cord. During the fetal period, ME20.4 immunoreactivity was also found in motoneurons and peripheral nerve fibres in the skin, myotomes and gut. Sheaths of peripheral nerves and the adventitia of blood vessels were stained both in fetal and adult tissues. Thus, the low-affinity nerve growth factor receptor is: (i) strongly expressed in the developing human nervous system; (ii) transiently associated with a subset of large primary sensory neurons and with motoneurons; (iii) transiently and sequentially expressed by various groups of sensory afferents to the spinal cord; (iv) permanently expressed by fibres in the superficial layers of the dorsal horn, Clarke's column, nerve sheaths and the adventitia of blood vessels.

Transient expression of neuropeptide Y and its C-flanking peptide immunoreactivities in the spinal cord and ganglia of human embryos and fetuses.

An immunohistochemical study of spinal cord, dorsal root and sympathetic ganglia of human embryos and fetuses demonstrated that neuropeptide Y and its C-flanking peptide could be detected in seven-week-old embryos but were absent or difficult to demonstrate after the 17th week of gestation. The peptides were found in several structures of the spinal cord, e.g. fibres in the dorsal portion of the lateral funiculus, cell bodies and fibres in the dorsal horn, and motoneurons, and also in numerous primary sensory neurons of dorsal root ganglia. They were also present in sympathetic neurons and since these are the only structures expressing neuropeptide Y and its C-flanking peptide in the adult, it must be concluded that their presence in other neurons is a transient developmental feature. To assist in understanding the relationship of these transient structures with other spinal and sensory neurons, a comparison was made with other neuronal structures showing immunoreactivity for two general neuronal markers, neurofilaments and protein gene product 9.5, and two neuropeptides present in primary sensory afferents, somatostatin and substance P. In the dorsal root ganglia, numerous neuropeptide Y- and C-flanking peptide-immunoreactive neurons were observed before substance P- or somatostatin-immunoreactive cells could be detected. Therefore, neuropeptide Y and its C-flanking peptide could represent a primitive peptidergic system appearing before primary sensory neurons express their characteristic adult phenotype. The fibres of the lateral funiculus showing immunoreactivity for neuropeptide Y and its C-flanking peptide were longitudinally orientated and could be detected at all cephalocaudal levels of the spinal cord. Comparison with the other immunohistochemical markers indicated that they were not primary sensory afferents. At least some of them probably originated from neuropeptide Y- and C-flanking peptide-immunoreactive neurons of the dorsal horn, that may be considered to be a subset of early-appearing interneurons.

Intracellular redistribution of neuropeptides and secretory proteins during differentiation of neuronal cell lines.

We have demonstrated that the mouse neuroblastoma N18Tg2 cell line and several clones of hybrid ND cells (ND7, ND9 and ND21), derived from the fusion of neonatal rat sensory neurons with that neuroblastoma, show immunostaining to protein gene product 9.5, neuropeptide Y, C-flanking peptide of neuropeptide Y, tyrosine hydroxylase and chromogranins. Synaptophysin could only be detected in ND cells. Immunoreactivities to substance P, calcitonin gene-related peptide, galanin and somatostatin could not be detected in any of these cell lines. After three days of incubation in a differentiation medium, cell processes of various lengths were observed both in neuroblastoma and ND cell cultures. In ND7 cells there was also a redistribution of neuropeptide Y and its C-flanking peptide to the tips of cell processes. The differentiation of cell processes was also accompanied by the appearance of immunostaining to rat chromogranins in their tips. In contrast, synaptophysin expression was found mainly in cell bodies. Neuropeptide Y, its C-flanking peptide and chromogranins have been associated with secretory granules, whereas synaptophysin is a marker for small synaptic-like vesicles. Therefore, our morphological findings further support and expand the view that these markers are primarily associated with different subcellular structures. Moreover, they indicate that the regulated secretory pathway associated with chromogranins is segregated into nerve processes at an early stage of differentiation, when the synaptophysin-associated pathway is not yet mature. ND7 cells thus provide a useful model system for studying changes in the distribution of neuropeptides, cytoskeletal elements and proteins associated with cell secretion during neuronal differentiation.

The eye of the magellanic penguin (Spheniscus magellanicus): structure of the anterior segment.

We undertook a light and scanning electron microscopic study of the eye in the Magellanic penguin (Spheniscus magellanicus). The anatomical peculiarities of the eyeball shape in Sphenisciformes have been previously described by others; here, we show that they are accompanied by several modifications in the organization of the anterior segment of the eye. The main change was found in the portion of opaque sclera extending from the cornea to the anterior border of the scleral ossicles, which was much broader than in other avian eyes. This scleral region was made of a very dense fibrous tissue and was as difficult to cut as the ossicles. The corneo-scleral boundary was also different from that of other birds, since the aqueous humor channel and the pectinate ligament were located 1.0-1.5 mm posterior to the cornea. The osseous ring was formed by 13 bones, including three pairs of over- and underplates. There was a single ciliary muscle, with meridionally oriented striated fibers. They were inserted on a circumference along the boundary between the fibrous sclera and the ossicles, far away from the wall of the aqueous humor channel. On their posterior end, the muscle fibers formed a tendinous structure attached to the inner surface of the sclera and to the outer surface of the ciliary body. Only short zonular fibrils were observed. These anatomical features are probably relevant for the adaptation of penguin eyes to vision on land and in the aquatic environment.

Changes in the shape and optical properties of the chicken lens after vitreous and scleral removal.

Ocular dimensions and lens refractive properties were studied in enucleated eyes from 5-day old chicken (Gallus domesticus). Measurements were made in frozen hemisections and in fresh eyes sectioned through a para-equatorial plane. In these specimens, lens properties were examined after systematical removal of the vitreal mass. This procedure caused significant changes in lens diameter and refractive properties. Our data indicate that avian lens shape could be controlled by vitreal pressure and/or scleral support.

Effects of p-bromophenacyl bromide on neurite growth at different levels of nerve growth factor.

We have previously been shown that the phospholipase inhibitor p-bromophenacyl bromide (BPB) modifies neurite morphology and growth rate in rat dorsal root ganglion explants. Neurite extension is stimulated by 2.5 X 10(-7) M BPB but it is prevented by concentrations higher than 10(-5) M. Under our experimental conditions, no neurites appeared in the absence of added nerve growth factor (NGF). Therefore, we have tested the effect of a low BPB concentration at different levels of exogenous NGF. Thoracic and lumbar ganglia were obtained from 19.5-day-old fetal rats and were cultured for 24 h on a polyornithine substrate. Neurite growth and development of a dense halo were NGF-dependent in both ganglionic populations; however, each of them showed a characteristic dose-response curve. Incubation with BPB induced neurites in the absence of added NGF and stimulated their growth when low levels of this factor were added. In lumbar ganglia incubated with higher levels of NGF, BPB had no detectable effects. On the contrary, similarly treated thoracic ganglia showed an inhibition of neurite extension. Our findings suggest a relationship between the effects of BPB and the initial, membrane-activating actions of NGF.

Early changes of surface morphology in monolayer cultures of neural retina cells. The effect of p-bromophenacyl bromide.

Scanning electron microscopy was used to study the surface morphology of neural retina cells from fetal rats. Dissociated cells were plated on polyornithine and examined after 1.5-3.0 hr in vitro. A quantitative analysis of the proportion of cells with and without processes was made and the former were classified according to the length and number of their processes. Treatment with p-bromophenacyl bromide (BPB), a selective inhibitor of phospholipase A2 (PLA2), induced significant changes on the early surface activity of retinal cells. An inhibitory effect on cell process formation was observed in monolayers grown for 2 hr in the presence of BPB: process formation was also inhibited when high concentrations (10(-6) M or more) were applied as a 30 min pulse, whereas a similar pulse of a lower concentration (10(-7) M) stimulated the appearance of cells with short processes. These observations suggest that PLA2 or some other BPB-reactive substance is involved in the extension of neural cell processes.

Silver staining of DNA synthesizing cells in the neural tube of chick embryos.

I report a study by light microscopy of the spinal cord of early chick embryos stained with the ammoniacal silver carbonate solution of Del Rio Hortega. Cell nuclei are stained in a selective fashion and two classes of nuclei - dark and pale - can be distinguished in the neuroepithelium. Neuronal nuclei also show a characteristic staining pattern. A radioautographic study after [3H] thymidine incorporation has shown that it is the dark neuroepithelial nuclei that are engaged in DNA synthesis. Dark nuclei disappear after administration of cytosine arabinoside, supporting the association between DNA synthesis and silver staining of neuroepithelial nuclei.