EphA7 isoforms differentially regulate cortical dendrite development.

The shape of a neuron facilitates its functionality within neural circuits. Dendrites integrate incoming signals from axons, receiving excitatory input onto small protrusions called dendritic spines. Therefore, understanding dendritic growth and development is fundamental for discerning neural function. We previously demonstrated that EphA7 receptor signaling during cortical development impacts dendrites in two ways: EphA7 restricts dendritic growth early and promotes dendritic spine formation later. Here, the molecular basis for this shift in EphA7 function is defined. Expression analyses reveal that EphA7 full-length (EphA7-FL) and truncated (EphA7-T1; lacking kinase domain) isoforms are dynamically expressed in the developing cortex. Peak expression of EphA7-FL overlaps with dendritic elaboration around birth, while highest expression of EphA7-T1 coincides with dendritic spine formation in early postnatal life. Overexpression studies in cultured neurons demonstrate that EphA7-FL inhibits both dendritic growth and spine formation, while EphA7-T1 increases spine density. Furthermore, signaling downstream of EphA7 shifts during development, such that in vivo inhibition of mTOR by rapamycin in EphA7-mutant neurons ameliorates dendritic branching, but not dendritic spine phenotypes. Finally, direct interaction between EphA7-FL and EphA7-T1 is demonstrated in cultured cells, which results in reduction of EphA7-FL phosphorylation. In cortex, both isoforms are colocalized to synaptic fractions and both transcripts are expressed together within individual neurons, supporting a model where EphA7-T1 modulates EphA7-FL repulsive signaling during development. Thus, the divergent functions of EphA7 during cortical dendrite development are explained by the presence of two variants of the receptor.

Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization.

Rats are effective model animals and have contributed to the development of human medicine and basic research. However, the application of reproductive engineering techniques to rats is not as advanced compared with mice, and genome editing in rats has not been achieved using embryos obtained by in vitro fertilization (IVF). In this study, we conducted superovulation, IVF, and knock out and knock in using IVF rat embryos. We found that superovulation effectively occurred in the synchronized oestrus cycle and with anti-inhibin antiserum treatment in immature rats, including the Brown Norway rat, which is a very difficult rat strain to superovulate. Next, we collected superovulated oocytes under anaesthesia, and offspring derived from IVF embryos were obtained from all of the rat strains that we examined. When the tyrosinase gene was targeted by electroporation in these embryos, both alleles were disrupted with 100% efficiency. Furthermore, we conducted long DNA fragment knock in using adeno-associated virus and found that the knock-in litter was obtained with high efficiency (33.3-47.4%). Thus, in this study, we developed methods to allow the simple and efficient production of model rats.

Pronuclear DNA injection for the production of transgenic rats.

In the absence of germ-line-competent ES cells in the rat, pronuclear microinjection remains an essential tool to generate transgenic rat models. However, DNA microinjection procedures first developed for mouse do not provide scientists with satisfying results when applied to rat. Here we describe optimized procedures for rat with a special focus on rat embryo production, in vitro incubation and culture, DNA pronuclear injection, and the transfer of embryos into foster females.

Effect of cryopreservation on proliferative features of neural progenitor cells derived from olfactory bulb of embryonic rat.

OBJECTIVE: Stem cell research offers unique opportunities for developing new medical therapies for devastating diseases and a new way to explore fundamental questions of biology. The use of olfactory bulb neural progenitor cells for transplantation requires efficient recovery methods and cryopreservation procedures. The purpose of this study was to determine cryopreservation techniques for neural progenitor cells derived from olfactory bulb (OB NPCs) of embryonic rat. METHODS: Initially, we compared the survival rates of cryopreserved OB NPCs using three cryoprotectants: dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol with or without 10% FBS. Cells were held at liquid nitrogen (-186 degrees C) for 7 days ("short-term storage") or 6 months ("long-term storage"). We assessed OB NPCs recovery efficiency after freezing and thawing by viability testing, colony-forming ability and immunocytochemistry under different conditions. RESULTS: The survival rate of cryopreserved-thawed OB NPCs was estimated by counting colony numbers under a stereomicroscope. No significant difference in survival rate was observed between cryoprotectants. CONCLUSIONS: These observations indicate that cryopreservation of OB NPCs is possible for up to 6 months in optimal conditions without losing proliferation activity.

Efecto de la terapia láser de baja potencia sobre el hueso alveolar dañado / The low level laser therapy effect on alveolar bone damaged

La terapia con láser de baja potencia ha demostrado tener propiedades analgésicas antiinflamatorias, bioestimulantes y promotoras de la respuesta tisular al daño. El propósito de este estudio fue determinar el efecto que el láser de baja potencia tiene sobre el hueso alveolar dañado. Se utilizaros 13 ratas Sprage Dawley, en las cuales se realizó una lesión estandarizada del hueso alveolar, posterior a lo cual una muestra aleatoria de 7 ratas fue sometida a un protocolo de irradiación de 6 J/cm2, tres veces por semana durante cuatro semanas. Las muestras obtenidas del sitio lesionado en ratas expuestas y no expuestas a la terapia fueron procesadas para hematoxilina eosina, contabilizándose el número de osteonas al microscopio óptico con aumento de 40x. Los resultados muestran un aumento en el número de osteonas en el grupo irradiado, diferencia que resultó estadísticamente significativa (p<0,01), con una alta fuerza de asociación estadística (O.R=5,6 ). Estos resultados sugieren que la terapia láser de baja potencia favorece la respuesta del hueso alveolar dañado.

The Low Level Laser Therapy (LLLT) has demostrated to have analgesic, antiinflamatory, bioestimulant and promoters from the tissues responses properties to the damage. The purpose of this study was determinate the Low Level Laser Therapy effect in the damaged alveolar bone. Thirteen Sprage Dawley rats were used. Total number of animals alveolar bones a standarized lesion was made, later an aleatory sample of seven rats was subjected to the irradiation protocol 6 J/cm², three times per week during four weeks. The obtained samples of the injured area, of exposed and not exposed rats to the laser therapy were processed for hematoxilin & eosin, being the osteon number count by optic microscope with increase of 40x. The result show an increase in the osteon number in the irradiated group, this differentiated was statistically significant (p<0.01), whit a high strength of statistical association (OR=5.6). These result suggest that the therapy laser of low power favors the answer of the damaged alveolar bone.

A comparative ontogenic study of urinary bladder: impact of the epithelial differentiation in embryonic and newborn rats.

The present study aimed to show the cellular and subcellular distribution of glycogen content during the differentiation of urothelial cells from simple cuboidal to stratified transitional epithelium. Bladder samples were taken from rat embryos on the 15th to 19th days and newborn at 21st day. During the development of the bladder, the formation of fusiform vesicles, asymmetric unit membrane (AUM) and microridges were examined with staining with haematoxylin-eosin and periodic acid Schiff for light microscope and periodic acid-thiocharbohydrazide-silver proteinate for transmission electron microscope. The topographical changes of luminal differentiation were examined with the scanning electron microscope. The urothelium was simple cuboidal from 15th till the 17th days of gestation. Glycogen content was present in the cytoplasm till the 18th day of gestation. At the early stage (16th day) of gestation, the apical surface contains microvilli that points the undifferentiated cells. The density of microvilli decreased and ropy microridges appeared at the 17th day of gestation. The small discoid vesicles lined with AUM developed at the apical cytoplasm of the surface cells at the 17th day of gestation. After this stage, both the density of microridges and large and elongated fusiform vesicles increased. The differentiation of the urothelium begins with the formation of the round and small vesicles, continues with the formation of the AUM and at the final stage there is a decrease in both glycogen content and the appearance of the microridges at the luminal surface of the urothelial cells.

LIS1 RNA interference blocks neural stem cell division, morphogenesis, and motility at multiple stages.

Mutations in the human LIS1 gene cause the smooth brain disease classical lissencephaly. To understand the underlying mechanisms, we conducted in situ live cell imaging analysis of LIS1 function throughout the entire radial migration pathway. In utero electroporation of LIS1 small interference RNA and short hairpin dominant negative LIS1 and dynactin cDNAs caused a dramatic accumulation of multipolar progenitor cells within the subventricular zone of embryonic rat brains. This effect resulted from a complete failure in progression from the multipolar to the migratory bipolar state, as revealed by time-lapse analysis of brain slices. Surprisingly, interkinetic nuclear oscillations in the radial glial progenitors were also abolished, as were cell divisions at the ventricular surface. Those few bipolar cells that reached the intermediate zone also exhibited a complete block in somal translocation, although, remarkably, process extension persisted. Finally, axonal growth also ceased. These results identify multiple distinct and novel roles for LIS1 in nucleokinesis and process dynamics and suggest that nuclear position controls neural progenitor cell division.

Optimal conditions for successful in vitro fertilization and subsequent embryonic development in Sprague-Dawley rats.

The present study was conducted to determine the optimal conditions for successful in vitro fertilization (IVF) in Sprague-Dawley (SD) rats. The IVF of oocytes from SD and Wistar rats was compared in different fertilization media (mR1ECM, IVF-20, and modified Krebs-Ringer bicarbonate solution [mKRB]), and IVF conditions were then optimized for oocytes of the SD strain. Results showed that in mR1ECM medium, fertilization rates were markedly lower in SD rats (15%) than in the Wistar strain (73%), although this response was significantly improved by increasing the NaCl concentration. In addition, fertilization rates in SD rats were higher in modified IVF-20 (73%) than in IVF-20 (18%) and mKRB (53%). In contrast, fertilization rates in Wistar rats were higher in IVF-20 and modified IVF-20 than in mKRB (78%, 74%, and 36%, respectively). Further investigation concerning the effects of the NaCl supplementation (10- 40 mM) in IVF-20 on the fertilization of oocytes in the SD strain indicated that significantly higher percentages of oocytes were fertilized in IVF-20 supplemented with 30 mM NaCl (66%) and developed to the blastocyst stage (47%) in vitro. After transfer, embryos derived from this IVF system developed to term at a percentage comparable to that of in vivo-fertilized controls. In conclusion, differences exist in optimal IVF conditions between rat strains, and a modified culture medium has been successfully developed for assessment of the developmental competence of oocytes in SD rats.

Efficiency of transgenic rat production is independent of transgene-construct and overnight embryo culture.

The aim of the present work was to study factors affecting the efficiency of transgenic technology in rats. We investigated the possible effects of pronuclear microinjection of buffer or different DNA-constructs on survival and development of rat zygotes in vitro and in vivo as well as the influence of overnight culture of these embryos before transfer into pseudopregnant foster mothers. The survival rate of zygotes and their development to the two-cell and morula stage was not affected by pronuclear microinjection with different DNA-constructs or buffer. However, the development to the blastocyst stage was impaired. Nevertheless, there was no difference in blastocyst development between zygotes injected with DNA-constructs or with buffer. Neither was there a difference in cell number in in vitro cultured blastocysts resulting from pronuclear microinjection of a transgene compared with non-injected controls. The survival rate to term was about 30% irrespective of whether microinjected embryos were transferred immediately after microinjection or after overnight culture in vitro. However, a reduction in the survival to term was observed for non-injected zygotes when they were developed in vitro to the two-cell stage before transfer to a pseudopregnant female. The percentage of transgenic rats that resulted from microinjected zygotes was similar in all groups regardless of the DNA-construct used (2.7-10.0%). In conclusion, the main detrimental factor in the microinjection of rat zygotes is the introduction of solution in the pronucleus. Overnight culture of zygotes between microinjection and oviduct transfer does not decrease the efficiency of transgenic rat generation.

Classical conditioning in the rat fetus. III. Retention, extinction, and re-activation of the conditioned response (CR).

A single paired presentation of an artificial nipple (conditioned stimulus, CS) and milk (unconditioned stimulus, US) resulted in classical conditioning. When re-exposed to the artificial nipple CS after conditioning, fetuses showed fewer oral grasp responses compared to control fetuses exposed to the milk (US) and artificial nipple (CS) in an unpaired fashion. The reduction in oral grasping was evident when the test of oral grasping was administered 18 min after conditioning but not 21 to 30 min after conditioning (Experiments 1a and 1b). Experiments 2a and 2b confirmed that nine or more exposures to the CS after conditioning, without a milk infusion, resulted in experimental extinction of the conditioned reduction in oral grasping. One re-exposure to milk during the period after classical conditioning was sufficient to re-activate memory for the association between artificial nipple CS and milk US (Experiment 3a). Fetuses exposed to one or two re-activation treatments that comprised an intraoral infusion of milk showed evidence of the conditioned reduction in oral grasping when tested after a delay sufficient to produce forgetting. Experiment 3b showed that the orosensory properties of milk were sufficient to re-activate fetal memory for the association between artificial nipple and milk. Experiment 3c indicated that central injection of 10 ng of dynorphin A 1-13 re-activated fetal memory for the classically conditioned association. Experiments 3b and 3c suggested that the orosensory properties of milk activated a kappa opioid system in the fetal brain that reduced oral grasping of the artificial nipple during the test for oral grasping.

Granule neurons generated during development extend divergent axon collaterals to hippocampal area CA3.

Most excitatory intrahippocampal pathways are characterized by significant, highly ordered projections into the long, or septotemporal, hippocampal axis. However, the mossy fiber system, the excitatory projection by which the dentate gyrus projects to hippocampal area CA3, is considered an exception, being organized to innervate exclusively transversely oriented cortical layers of the hippocampus. In the present study, the anatomy of the rat mossy fiber system was investigated using axonal tracing techniques, with an emphasis on determining its projection pattern into the long hippocampal axis. To this end, we used dual ipsilateral retrograde tracer injections to determine whether individual granule cells extend divergent axon collaterals to septotemporally distinct levels of hippocampal area CA3. We combined this technique with the fluorescent immunohistochemical detection of 5-bromo-2'-deoxyuridine (BrdU), a marker of granule cell precursors and their progeny, to address whether the divergence of mossy fiber collaterals within area CA3 might by related to ontogenic gradients in granule cell genesis. We observed single granule neurons that had retrogradely transported both tracers, indicating that they had axon collaterals passing through or terminating within the distinct levels of area CA3 into which tracer had been injected. By using BrdU labeling, we identified divergent granule neurons that were produced during embryonic and postnatal development. We observed no adult-generated granule neurons with significantly diverging mossy fiber collaterals. However, many fewer cells were labeled with BrdU in adult-exposed animals. Because of this smaller sample, we cannot rule out the possibility that small numbers of divergent adult-generated granule cells exist. We conclude that a proportion of the hippocampal mossy fiber projection extends septotemporally divergent axon collaterals to hippocampal area CA3.

Guidance of dopaminergic neuritic growth by immature astrocytes in organotypic cultures of rat fetal ventral mesencephalon.

Astrocytes, with their many functions in producing and controlling the environment in the brain, are of great interest when it comes to studying regeneration after injury and neurodegenerative diseases such as in grafting in Parkinson's disease. This study was performed to investigate astrocytic guidance of growth derived from dopaminergic neurons using organotypic cultures of rat fetal ventral mesencephalon. Primary cultures were studied at different time points starting from 3 days up to 28 days. Cultures were treated with either interleukin-1 beta (IL-1 beta), which has stimulating effects on astrocytic proliferation, or the astrocytic inhibitor cytosine arabinoside (Ara-C). Tyrosine hydroxylase (TH)-immunohistochemistry was used to visualize dopaminergic neurons, and antibodies against glial fibrillary acidic protein (GFAP) and S100 beta were used to label astrocytes. The results revealed that a robust TH-positive nerve fiber production was seen already at 3 days in vitro. These neurites had disappeared by 5 days. This early nerve fiber outgrowth was not guided by direct interactions with glial cells. Later, at 7 days in vitro, a second wave of TH-positive neuritic outgrowth was clearly observed. GFAP-positive astrocytic processes guided these neurites. TH-positive neurites arborized overlying S100 beta-positive astrocytes in an area distal to the GFAP-positive astrocytic processes. Treatment with IL-1 beta resulted in an increased area of TH-positive nerve fiber network. In cultures treated with Ara-C, neither astrocytes nor outgrowth of dopaminergic neurites were observed. In conclusion, this study shows that astrocytes play a major role in long-term dopaminergic outgrowth, both in axonal elongation and branching of neurites. The long-term nerve fiber growth is preceded by an early transient outgrowth of dopamine neurites.

Ntab, a novel non-coding RNA abundantly expressed in rat brain.

We have identified a novel transcript that is abundantly and specifically expressed in both the adult and developing rat CNS. Within the full-length cDNA sequence we were unable to identify a clear open reading frame. Moreover, we were unable to detect any protein product derived from the full-length cDNA sequence using an in vitro translation assay. Therefore, we suggest this gene is one of a growing number of non-coding mRNA-like RNA transcripts that exert their cellular functions directly as an RNA. We have named this novel gene Ntab for non-coding transcript abundantly expressed in brain (accession number AY035551). In addition, in some regions of the brain we find evidence for RNA accumulation in cellular processes at some distance from the soma. These findings suggest that Ntab is actively transported and may function within cellular processes. Since Ntab is a targeted non-coding RNA, such cellular functions could include the targeting and/or regulation of localised translation of other mRNA species.

SPACRCAN in the developing retina and pineal gland of the rat: spatial and temporal pattern of gene expression and protein synthesis.

SPACRCAN is a hyaluronan-binding proteoglycan that is present in the pineal gland and interphotoreceptor matrix of the retina. Here, we evaluate the pattern of SPACRCAN gene expression and protein appearance during retinal and pineal gland development in the rat. In situ hybridization histochemistry with SPACRCAN riboprobes indicates that hybridization signals are first evident in the retina over developing photoreceptor cells at embryonic day 16 (E16) and in the pineal gland at E21. Immunocytochemistry using a SPACRCAN antibody shows localization of SPACRCAN protein in the developing interphotoreceptor matrix by Postnatal day 5 (P5) and in the pineal gland by P6. These studies suggest that SPACRCAN mRNA expression may occur substantially earlier than the time when SPACRCAN protein is detectable in both the retina and the pineal gland. The period of retinal histogenesis when SPACRCAN is detected first is coincident with the time photoreceptors begin to extend from the outer retinal surface, suggesting that SPACRCAN may participate in the maturation and maintenance of the light-sensitive photoreceptor outer segment.

Expression of the receptor protein-tyrosine kinases Tyro-3, Axl, and mer in the developing rat central nervous system.

Tyro-3, Axl, and Mer are three related receptor protein-tyrosine kinases (RPTKs) characterized by an extracellular domain exhibiting significant amino acid sequence similarity to neural cell adhesion molecules. The molecule Gas6 (for growth arrest-specific gene-6) has been shown to activate each of these receptors. Gas6 is expressed extensively in the central nervous system (CNS), suggesting that interactions between Gas6 and its receptors are likely to have physiologically relevant functions. To identify and localize the relevant Gas6/RPTK pairs, we have characterized the developmental expression of Tyro-3, Axl, and Mer in rat CNS using blotting and mRNA in situ hybridization analyses. Throughout development, Tyro-3 was the most widely expressed of the three receptors in the CNS, with Axl and Mer detected in only a limited number of sites in the adult. Tyro-3 expression was low in the embryo and increased markedly during early postnatal stages, with a time course paralleling that of synaptogenesis. Axl and Mer were expressed at low but relatively constant levels throughout development. In the cerebellum, all three receptors were found in Purkinje cells, and Tyro-3 was also detected in both granule neurons and Bergmann glia. Insofar as Gas6 has been previously shown to also be expressed by Purkinje cells, it may be engaged in both autocrine and paracrine signaling. The three receptors were also detected in cerebellar white matter, primarily during myelination. In the cortex, Tyro-3 was expressed at high levels during postnatal development and in the adult. Beginning at P6 in the hippocampus, Tyro-3 was expressed at high levels in CA1 pyramidal neurons and at lower levels in CA3 and was not detected in dentate granule neurons. Axl and Mer were found in the molecular layer of the dentate gyrus and were absent from the pyramidal and dentate granule neurons. In that Gas6 is expressed throughout the pyramidal cell layer, it may activate these cells in both an autocrine and a paracrine manner. These studies provide initial clues for elucidating the cellular functions of the Axl subfamily members and suggest potential complex Gas6/RPTK as well as RPTK/RPTK signaling interactions in the mature and developing CNS.

Regional patterning of reticulospinal and vestibulospinal neurons in the hindbrain of mouse and rat embryos.

The dispositions and axonal trajectories of bulbospinal neurons in the pons and medulla of mouse and rat embryos is described from the earliest times these projections can be labelled retrogradely from the cervical spinal cord. Reticulospinal and vestibulospinal neurons are clustered into identifiable groups, each with a characteristic combination of spatial domain and axon trajectory. The various groups can be labelled retrogradely in a specific developmental sequence. The position of some groups shifts from medial to lateral with development, apparently through cell migration. These observations show that the basic regional organization of the reticulospinal and vestibulospinal projections is similar in mouse and rat and is already established during early stages of axon outgrowth.

A calcium agonist, Bay k 8644, suppresses the embryotoxic effects induced by dihydropyridines calcium channel blockers in cultured rat embryos.

Day 9 rat embryos were exposed to 1,4-dihydropyridine calcium channel blockers; nifedipine (NIF), nicardipine (NIC) or nitrendipine (NIT), for 48 hr in the whole embryo culture system. There were dose-dependent growth retardation and abnormalities, predominantly in cardiovascular system. The three compounds exhibited very similar pattern of dysmorphogenic effects, but the potency of these compounds were quantitatively different. The incidences of embryos with the abnormalities were 100%, 100% and 85% following either exposure of NIF, NIC or NIT at concentration of 300, 8 and 15 microM, respectively. This study was to investigate whether these blocker-induced embryotoxicity was due to calcium channel blocking properties themselves in the embryos. Day 9 rat embryos were co-exposed to 1,4-dihydropyridine calcium channel agonist, Bay k 8644 (BAY) and each calcium channel blocker under the same culture condition. The retarded embryonic growth induced by 200 or 300 microM of NIF, 8 microM of NIC and 15 microM of NIT nearly of completely ameliorated when embryos were co-exposed with BAY at one-third or half concentration of each calcium channel blocker. Supplementation of BAY reduced the incidence of abnormalities by NIF-, NIC- and NIT-alone. These results suggested that one of mechanisms for embryotoxicity induced by calcium channel blocker was directly related to channel blocking property of the chemicals.

Effects of amniotic fluid on opioid activity and fetal responses to chemosensory stimuli.

The present study provides evidence that milk or amniotic fluid (AF) can promote activity in the endogenous opioid system of the E20 rat fetus. Fetal responses to a chemosensory test stimulus (lemon) were reduced after intraoral infusion of milk (Experiment 1). The effect of milk was mimicked by the kappa opioid agonist U50,488 (Experiment 2), and blocked by pretreatment with naloxone (Experiment 3), confirming opioid involvement. E20 fetuses also showed reduced responses after exposure to AF collected on E20 or E21, but not to AF collected on E19 (Experiment 4). The effects of AF on fetal responses were blocked by pretreatment with naloxone (Experiment 5), and by a selective kappa opioid antagonist, but not by a mu antagonist (Experiment 6). These findings suggest that the fetus may experience activation of the kappa opioid system for several days before birth as a consequence of its exposure to AF in utero.

Ontogeny of NADPH-diaphorase in rat forebrain and midbrain.

This study characterizes the developmental expression of NADPH-diaphorase from embryo to adulthood in the forebrain, midbrain and cerebellum of rat brain via histochemical staining. On embryonic day 12 no neurons stained. Labeling was observed in certain nuclei from E15 through the postnatal period to adulthood. Labeling in neurons increased or maintained a constant level with increased age. The embryo demonstrated substantial labeling in neurons of the caudate putamen, bed nucleus of the stria terminalis, preoptic area, lateral hypothalamic area, paraventricular thalamic nucleus, ventromedial hypothalamic nucleus, magnocellular nucleus posterior commissure, and periaqueductal central gray. Additional neuronal labeling was observed postnatally in the olfactory bulb, cerebral cortex, amygdala, various nuclei of the thalamus, interpeduncular nucleus, linear nucleus of the raphe, pretectal area and superior colliculus. In the cerebellum, labeling appeared only after P14 in cells of the molecular cell layer and granular cell layer. The sizes of labeled neurons developed significantly from P4 to P14 in several nuclei. The distinctive temporal and spatial expression pattern of NADPH-diaphorase implies that the NO/cGMP system may play an important role in physiological and developmental functions.

Injection technique and scanning electron microscopic study of the arterial pattern of the 20 gestation days (G20) rat fetus.

A technique to obtain microvascular corrosion casts of the G20 rat fetus and the normal pattern of the main arteries of the G20 rat fetus are described. The casts were studied by means of scanning electron microscopy (SEM). The arterial pattern is similar to that described in the adult; however, several variations have been found. It is concluded that the use of vascular corrosion casts studied by SEM may be particularly helpful to observe the extremely small arteries of rat fetuses. Moreover, we suggest that this technique may be useful in practical teratological studies.