The blood-brain barrier in a reptile, Anolis carolinensis

An electron microscopic study was made of the ultrastructure and permeability of the capillaries in the cerebral hemispheres of the lizard, Anolis carolinensis. The brain of Anolis is vascularized by a loop-type pattern consisting exclusively of arteriovenous capillary loops. The ultrastructure of the endothelium and the arrangement of the various layers from the capillary lumen to the central nervous tissue is similar to that of mammals. The endothelial cells form a continuous layer around the lumen and are joined by tight interendothelial junctions. The basal lamina of the endothelium is also continuous and encloses pericyte processes. The cells of the nervous tissue rest directly on the basal lamina of the capillary and are separated from each other by a 200 Å space. Intravenously injected horseradish peroxidase (MW 40,000) and ferritin (MW 500,000) were used to study the permeability of the capillaries. The entry of horseradish peroxidase and ferritin into the intercellular spaces of the brain is restricted by the tightness of the interendothelial junctions. No vesicular transport of either tracer occurs; however, ferritin does enter the endothelial cells in vacuoles. No tracer molecules are present in the basal lamina, pericytes, or nervous tissue. The different responses of the endothelial cell to the tracers used in this study suggest that endocytotic activities of endothelial cells involve different processes. Vacuoles formed by marginal folds, vacuoles formed by endothelial surface projections or deep invaginations of the plasma membrane, 600–800 Å vesicles, and coated vesicles all seem to differ in the nature of the substances which they endocytose.     

The stimulatory effects of prostaglandins on the melanophores of the lizard, Anolis carolinensis

The melanosome dispersing activity of prostaglandins PGE1, PGE2, PGF1 alpha, PGF2 alpha, PGI2 and 6 beta PGI, was tested on the melanophores of Anolis carolinensis. Only PGE2 and PGE1 were active and while PGE2 was the most potent and acted synergistically with alpha-MSH, PGE1 was additive with alpha-MSH. Arachidonic acid also stimulated melanosome dispersion but its effect was blocked by indomethacin suggesting an action through its conversion to PGE1 or PGE2. The effect of alpha-MSH, on the other hand, was unaltered by indomethacin which suggests that alpha-MSH stimulated melanosome dispersion does not depend upon prostaglandin synthesis. Thus, while some prostaglandins may interact with alpha-MSH to stimulate melanosome dispersion they are unlikely to mediate its action.     

Distribution of arginine vasotocin in the brain of the lizard Anolis carolinensis

Summary The distribution of immunoreactive arginine vasotocin (AVT-ir) was determined in the brain of the lizard Anolis carolinensis. Cells and fibers containing AVT-ir were found in the medial septal region, lamina terminalis, lateral forebrain bundle, preoptic area, supraoptic nucleus, anterior hypothalamus, paraventricular nucleus, periventricular nucleus, arcuate nucleus, and ventromedial nucleus of the thalamus. Occasional AVT-ir cells were found in the interpeduncular nucleus. Fibers containing AVT-ir were found in the cortex, around the olfactory ventricle, in the diagonal band of Broca, amygdala area, dorsal ventricular ridge, striatum, nucleus accumbens, septum, ventromedial hypothalamus, lateral hypothalamus, medial forebrain bundle, median eminence, pars nervosa, nucleus of the solitary tract, locus coeruleus, cerebellar cortex (granular layer), dorsal part of the nucleus of the lateral lemniscus, substantia nigra, and myelencephalon. The intensity of AVT-ir staining was, in general, greater in males than in females. Comparison of AVT-ir distribution in A. carolinensis with those previously published for other reptilian species revealed species-specific differences in distribution of AVT.     

Sperm transfer and storage in the lizard,Anolis carolinensis

The relationship of the hemipenis to the cloaca in copula and sperm storage and transport in the female oviduct were studied in Anolis carolinensis using light and scanning electron microscopy. During copulation, the hemipenis does not penetrate beyond the cloaca, but the two apical openings of the bifurcate sulcus spermaticus appose the openings of the oviducts from the cloaca. Sperm enter the sperm storage tubules between 2 and 6 hr after insemination and small amounts of sperm reach the infundibulum 6 to 24 hr following mating. Sperm storage tubules are embedded in the wall of the utero-vaginal transition, and are formed by the folding and fusion of the oviducal epithelium. The importance of the hemipenile-cloacal relationship and the role of sperm storage in the life history of A. carolinensis are discussed.     

Chromatophores and color change in the lizard,Anolis carolinensis

Summary The skin of the lizard, Anolis carolinensis, changes rapidly from bright green to a dark brown color in response to melanophore stimulating hormone (MSH). Chromatophores responsible for color changes of the skin are xanthophores which lie just beneath the basal lamina containing pterinosomes and carotenoid vesicles. Iridophores lying immediately below the xanthophores contain regularly arranged rows of reflecting platelets. Melanophores containing melanosomes are present immediately below the iridophores. The ultrastructural features of these chromatophores and their pigmentary organelles are described. The color of Anolis skin is determined by the position of the melanosomes within the melanophores which is regulated by MSH and other hormones such as norepinephrine. Skins are green when melanosomes are located in a perinuclear position within melanophores. In response to MSH, they migrate into the terminal processes of the melanophores which overlie the xanthophores above, thus effectively preventing light penetration to the iridophores below, resulting in skins becoming brown. The structural and functional characteristics of Anolis chromatophores are compared to the dermal chromatophore unit of the frog.This study was supported in part by GB-8347 from the National Science Foundation.Contribution No. 244, Department of Biology, Wayne State University.The authors are indebted to Dr. Joseph T. Bagnara for his encouragement during the study and to Dr. Wayne Ferris for his advice and the use of his electron microscope laboratory.     

Annual testicular cycle of the lizard Anolis carolinensis: effects of pinealectomy and melatonin

The effects of pinealectomy and exogenous melatonin treatment on the reproductive system of male anoles were examined at several different times of year. In September pinealectomy of anoles exposed to either a stimulatory LD 14:10 light cycle or a nonstimulatory LD 10:14 light cycle induced significant testicular growth and development over that observed in sham-operated anoles. At a nonphotosensitive time of year (December) pinealectomy also had a significant progonadal effect but no effect of pinealectomy was seen in February-March. Daily melatonin injections given either in the morning or afternoon (or both) failed to block gonadal growth either (1) in sham-operated or pinealectomized anoles exposed to LD 14:10 in the fall or (2) in pinealectomized lizards exposed to LD 10:14 in the fall. Continuous melatonin administration via subcutaneous silastic implants blocked the progonadal effects of pinealectomy in the winter (December). The results show that pinealectomy can have significant progonadal effects; these effects are seasonal but can encompass phases of the annual testicular cycle which are either photoperiod-dependent or temperature-dependent; and melatonin may be a reproductively active factor involved.     

Latent effects of egg incubation temperature on growth in the lizard Anolis carolinensis

Varied egg incubation temperatures can result in immediate effects on the phenotype of reptiles, and also latent effects that can augment or contradict effects evident at egg hatching. I examined the effects of incubation temperature on embryonic development, hatching morphology, and subsequent growth in multiple populations of the lizard Anolis carolinensis. Eggs from wild-caught females in four populations were incubated at up to three temperatures, 23.5, 27, and 30 degrees C. Measures of body size were collected immediately after hatching and weekly thereafter, while juveniles were maintained in a common laboratory environment for 8 weeks. Cooler incubation temperatures resulted in longer incubation periods but did not affect conversion of egg mass to hatchling mass. Incubation temperature did not affect hatchling mass or snout vent length (SVL), but did affect subsequent growth in both mass and SVL, which varied by population. Cooler incubation temperatures generally resulted in greater overall growth over 8 weeks of housing all juveniles in a common environment. In A. carolinensis, egg incubation temperature had latent effects on juvenile growth despite the absence of any detected immediate effects on hatchling phenotype. Therefore, the total impact and evolutionary importance of developmental environment should not be assessed or assumed based solely on the phenotype of reptiles at birth or hatching.     

Detection of a novel sequence change in the major form of alpha-MSH isolated from the intermediate pituitary of the reptile, Anolis carolinensis.

Intermediate pituitaries of the reptile, Anolis carolinensis, were separately pulse labeled with [3H]Trp and [3H]Tyr. The major form of alpha-MSH was purified by immunoprecipitation and isolated by reverse phase HPLC. Tryptic peptide analysis indicated that the [3H]Trp-labeled C-terminal fragment of Anolis alpha-MSH had the same retention time as mammalian ACTH(9-13) amide; however, the [3H]Tyr-labeled N-terminal fragment did not coelute with either mammalian ACTH(1-8) or N-acetyl-ACTH(1-8). Purification of alpha-MSH from 76 Anolis intermediate pituitaries confirmed that a sequence change had occurred in the N-terminal region of Anolis alpha-MSH. The tissues were acid extracted and purified by Sephadex G-25 chromatography and reverse phase HPLC to yield 4.5 micrograms of purified Anolis alpha-MSH for amino acid composition analysis and automated Edman degradation sequence analysis. The major form of Anolis alpha-MSH is nonacetylated and has the following novel primary sequence: Ser-Tyr-Ala-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro(Val-amide). The presence of Val-amide was verified by immunological analysis, tryptic peptide analysis and amino acid composition analysis.     

Phosphodiesterase of Cone Photoreceptors from the Lizard, Anolis carolinensis

Cone and rod photoreceptors utilize cyclic guanosine monophosphate (cGMP) in the light regulation of membrane polarization. The prototype for visual transduction is established for rod photoreceptors, which utilize a cascade of reactions to regulate a cyclic nucleotide phosphodiesterase (PDE) (EC 3.1.4.17) and thereby control the intracellular concentration of cGMP. Although cones appear to utilize a comparable cGMP cascade for their phototransduction, evidence exists that the PDE from cone photoreceptors may be different from that of rods. Dissociated cone photoreceptors, isolated retinas, and cone outer segments from the lizard, Anolis carolinensis, have been used to identify and characterize a PDE enzyme complex that shares several features in common with the rod outer segment (ROS) PDE complex. Immunoadsorption and sodium dodecyl sulfate-polyacrylamide gel electrophoresis have identified a subunit of lizard cone PDE that has an apparent electrophoretic mobility of 84 kDa and a subunit of lizard rod PDE that migrates at approximately 90 kDa. The lizard cone PDE complex is similar in size, extraction, activation, and immunological characteristics to the PDE complex of rod photoreceptors from lizard, bovine, and human retinas. The lizard cone PDE complex, and perhaps that from cone photoreceptors in general, differs from that of ROS in its chromatographic properties on anion-exchange resins. The sharing of physical and activation properties of the rod and cone PDE complex is compatible with the phototransduction process occurring by a similar mechanism in both cell types. The differences in light sensitivity and speed of response may be attributable to features of the individual proteins that form the PDE complexes of rods and cones or to other undisclosed features of the respective cascades.     

Arginine vasotocin concentrations in the supraoptic nucleus of the lizard Anolis carolinensis are associated with reproductive state but not oviposition.

Arginine vasotocin (AVT) is a neuropeptide involved in reproductive function in many nonmammalian vertebrates. We determined brain and plasma AVT concentrations during the estrous cycle and oviposition in the lizard Anolis carolinensis. There were no differences in AVT concentrations in the plasma or any brain region during the ovipositional sequence. However, we found that females with an egg in each oviduct and a large pre-ovulatory follicle (diameter > 4.5 mm) in one-ovary had significantly higher AVT concentrations in the supraoptic nucleus (SON) of the hypothalamus than did females with small pre-ovulatory follicles in both ovaries. In a second study, females with an egg in each oviduct and a large pre-ovulatory follicle had significantly greater AVT concentrations in the SON than females with only one oviductal egg and a large pre-ovulatory follicle or females with an egg in each oviduct and a small pre-ovulatory follicle in each ovary. Concentrations of AVT in other brain regions and in the plasma did not differ among these groups. Changes in steroid profiles during estrous and/or direct neural communication between the uterus, ovary, and brain may account for the changes in AVT concentrations seen in the supraoptic nucleus during the estrous cycle of Anolis carolinensis.     

Control of the pars intermedia of the lizard,Anolis carolinensis

Summary The capacity of colour change in intact lizards and in animals with a transected hypophysial stalk was studied during extended periods. It was concluded that, with certain exceptions, the skin colour of the lizards gives information on the circulating levels of MSH, and thereby on the function of the pars intermedia.After transection of the hypophysial stalk, three phases of chromatic behaviour were recognised. During Phase I, which lasted for about 6 days (average), dark skin was retained irrespective of the colour of the background (= high MSH levels), whereas Phase II (19 days) was characterised by the inability of the animals to become brown (= low MSH levels). A gradual recovery toward normal adaptive capacity was seen during Phase III.In the disconnected neuro-intermediate lobe, aldehyde fuchsin (AF)-positive material in the rostral region rapidly clumped and disappeared within a few days. In the caudal portion, the stainability and the varicose arrangement of fibres were retained longer, but disappeared within approximately 14 days after the operation. Proximal to the lesion, the preoptic system exhibited a marked increase in stainability with AF, starting in the median eminence and progressing in a retrograde direction toward the peptidergic nuclei.Revascularisation of the disconnected neuro-intermediate lobe occurred during the first few days. A reinnervation of AF-fibres across the transected area into the neural lobe was observed during Phase III in most animals, but AF-fibres did not reoccupy the entire lobe. In association with an outgrowth of capillaries, the regenerating fibres formed new neural lobules. This regenerative process was accompanied by an increase in blood supply from the primary plexus of the median eminence to the neuro-intermediate lobe.Supported by grants from the Swedish Natural Science Research Council and the Royal Physiographic Society of LundThe authors are indebted to Mrs. Ingrid Hallberg and Mrs. Kirsten Thörneby for valuable technical assistance and skillful attention to the animals and to Miss Inger Norling for photographic aid     

Development and keratinization of the epidermis in the common lizard, Anolis carolinensis

Epithelial-mesenchymal interactions play important roles in the development of the vertebrate integument with its diverse appendages. As a result of these interactions, specific morphogenetic events occur which result in the formation of distinct epidermal appendages. Following the early morphogenetic events involving cell proliferation and movement, other developmental events such as stratification, histotypic differentiation, and terminal cytodifferentiation occur in the epidermis. Using the common lizard Anolis carolinensis, we are seeking to obtain a better understanding of the relationship between the various developmental events and the expression of alpha and beta keratins, with the aim of eventually understanding the mechanisms by which tissue-specific keratinization patterns are established in the integument. As a first step, we have used immunoblot analyses and indirect immunofluorescence procedures with antisera specific for either alpha or beta keratins to determine the temporal and spatial appearance of these keratins at specific developmental stages. We have found that: 1) There are relatively low molecular weight alpha keratin polypeptides present in the epidermis early in development as morphogenesis is taking place. 2) After morphogenesis occurs and histogenesis is well under way, the alpha keratins which characterize the adult epidermis appear. 3) Only alpha keratins are found in the basal cells of all regions of the epidermis. 4) beta keratins are found only in the suprabasal layers of well-developed scales and show region-specific distribution in overlapping scales.     

Control of the pars intermedia of the lizard,Anolis carolinensis

Summary The ultrastructure of the intermediate lobe of the hypophysis was studied in Anolis carolinensis with the use of a threefold aldehyde fixative. Lizards with a brown skin were selected. The possibility of two types of secretory cells is discussed; neither cell type is innervated. Type I cells are rarely found and contain dense granules approximately 0.3 m in diameter; Type II cells vary widely in secretory activity. Most of the Type II cells contain a large number of dense secretory granules (up to about 1.3 m in diameter) almost filling the cytoplasm. Rough endoplasmic reticulum (RER), Golgi apparatus and mitochondria are poorly developed. Only some of these cells show signs suggesting a high secretory activity, namely a well developed RER, Golgi apparatus and numerous mitochondria. In these cells the RER sometimes forms large intracisternal droplets (up to 7 m in diameter). Two of the animals exhibited a more uniform, high secretory activity. Large (about 2 m in diameter), pale vacuoles, probably of extracellular character, were found mostly in the vicinity of the perivascular septum. Their role in the release of MSH is discussed. The present data, which are discussed with reference to earlier findings (Forbes, 1972), form the morphological basis for an experimental study on regulation of MSH release (Larsson et al., 1979).Supported by grants from the Swedish Natural Science Research Council (to P. Meurling) and the Royal Physiographic Society of LundThe authors are indebted to Mrs. Ingrid Hallberg, Mrs. Kirsten Thörneby and Mrs. Lena Sandell for valuable technical assistance and to Miss Inger Norling for photographic aid     

Control of the pars intermedia of the lizard,Anolis carolinensis

Summary The distribution of the tracer substance horseradish peroxidase (HRP, Mw 40,000) in the neuro-intermediate lobe of the lizard, Anolis carolinensis, was studied at various time intervals (13 min to 24 h) after vascular injection. HRP rapidly entered the extracellular lumen of the neural lobe, but did not penetrate into the third ventricle. The tracer was found in micropinocytotic vesicles (MPVs) of ependymal cells within 13 min after injection. The number of cellular inclusions containing HRP increased during the period of observation (24 h). The tracer was sparsely taken up by aminergic and peptidergic nerve terminals of the external layer. After transection of the hypophysial stalk, numerous dense, labelled droplets were found in the peptidergic terminals, and the number of labelled inclusions in ependymal cells increased.MPVs were frequently found in extensions of stellate cells of the intermediate lobe, and endocytotic vacuoles (EVs) developed especially in the perikaryon. HRP was also found in large cisternae of the secretory cells, appearing predominantly towards the perivascular septum (PVS). These cisternae were found to communicate with the extracellular lumen, probably representing a system of the extracellular space extending into the secretory cell. After transection of the hypophysial stalk, there was an increase in the number of small EVs in secretory cells of the intermediate lobe.The results are discussed in terms of MSH-release regulation and possible participation of the extracellular lumen, glial and stellate cells in the transport of regulating factors and secretory material.Supported by grants from the Swedish Natural Science Research Council (to Dr. Patrick Meurling) and the Royal Physiographic Society of LundThe author is indebted to Mrs. Lena Sandell and Miss Inger Norling for excellent technical assistance and photographic aid; and to Dr. Rolf Libelius and Dr. Ingmar Lundquist for generous advice and stimulating discussions concerning the tracer technique     

Control of the pars intermedia of the lizard,Anolis carolinensis

Summary Morphological changes in the disconnected neuro-intermediate lobe were studied in the lizard, Anolis carolinensis from the 2nd to the 14th post-operative day using a threefold aldehyde fixative (Rodríguez, 1969). Two phases of colour change capacity were exhibited: Phase I started immediately after the transection, lasted for 6 days (mean) and was characterised by an excessive MSH release (brown skin). This phase proceeded gradually into Phase II, designated by an interruption of the MSH release (green skin).The degenerative processes and final elimination of neurons in the disconnected neural lobe propagate in a rostro-caudal direction from the transected area. The aminergic fibres (Type II) disappear within 2 days postoperatively, whereas the degeneration continues for more than 10 days in the peptidergic fibres (Type III, IV and V). The glia cells (ependyma and pituicytes) serve as very active macrophages, engulfing fragments of axons already affected by autolysis and transferring them into glial lysosomes. No apparent morphological changes occur in the shift from Phase I to II.The great majority of the secretory cells of the intermediate lobe are not affected by degenerative processes and appear to be markedly activated by the stalk transection. They exhibit numerous mitochondria, well-developed Golgi complexes forming numerous Golgi vesicles and extensive parallel cisternae of the rough endoplasmic reticulum, sometimes forming large intracisternal droplets (7 m in diameter). Numerous pale vacuoles are seen, especially toward the intact capillaries, suggesting their coupling to the MSH release by extension of the active membrane area toward the perivascular septum. The number of these vacuoles is very markedly reduced in Phase II (no release), whereas the formation of new granules seems to proceed in early stages. The interruption of the MSH release implies a successive refilling of gradually growing secretory granules and a concomitant reduction in the development of the synthetic apparatus. Mechanisms probably involved in the control of the synthesis and release of MSH are discussed.Supported by grants from the Swedish Natural Science Research Council (to P. Meurling) and the Royal Physiographic Society of LundThe authors are indebted to Mrs. Lena Sandell, Mrs. Ingrid Hallberg and Mrs. Kirsten Thörneby for technical assistance and skillful attention of the animals and to Miss Inger Norling for photographic aid.     

Control of the pars intermedia of the lizard,Anolis carolinensis

Summary The ultrastructure of the neural lobe of the lizard, Anolis carolinensis, was studied after fixation in a threefold aldehyde solution. The neural lobe appeared as narrow vertical diverticula separated from one another and from the pars intermedia by a continuous vascular septum. No nerves passed through this septum. The ependymal, fibrous and external layers were readily recognized. Peptidergic fibres were the main component of the fibrous layer. The peptidergic endings were in intimate contact with the ependymal cells, suggesting that the ependyma mediates the release of neural lobe peptides. The external layer contained ependymal end-feet and numerous aminergic terminals, ending directly on the perivascular basal lamina and/or on the ependymal end-feet. The functional aspects are discussed in terms of intermediate lobe control. The findings suggest that aminergic substances take part in the control of the intermedia, but do not exclude the involvement of peptide hormones.Supported by grants from the Swedish Natural Science Research Council and the Royal Physiographic Society of LundThe authors are indebted to Mrs. Lena Sandell for valuable technical assistance and to Miss Inger Norling and the late Mr. Lajos Erdös for photographic aid     

Seasonal changes in the effects of arginine vasotocin and stretch on Anolis uterine contractions in vitro

We determined the effects of acute stretch on spontaneous and arginine vasotocin (AVT)-driven contractions of the Anolis carolinensis uterus in vitro. Whole uteri from reproductively inactive females (October) were placed in a bath of oxygenated 32 degrees C Anolis "Ringer's." Two initial tensions were utilized, 1.5 g or 15 g, the latter being an estimate of the tension on the wall of a uterine compartment. Uteri were then exposed to either saline or AVT (50 ng/ml), and spontaneous or AVT-driven contractions were recorded for 20 min with the use of a strain gauge and physiograph. A similar experiment was performed on uteri from reproductively active females in the summer (June). Our results indicate that the effects of acute stretch and AVT on uterine contractility were qualitatively similar in summer and fall. That is, AVT induced a tonic contraction; stretch decreased the duration of the tonic contraction; the saline-treated uteri exhibited spontaneous rhythmic contractions; AVT increased the amplitude of the rhythmic contractions, but only at the lower tension; there were no effects of AVT on the timing (contraction interval, duration, rest interval) of the rhythmic contractions; and stretch increased the frequency of the rhythmic contractions. Season greatly influenced the magnitude of these contractile phenomena. Uteri tested during the breeding season exhibited greater distensibility, an increase in the amplitude and duration of the AVT-driven tonic contraction, and an increase in the frequency of both spontaneous and AVT-driven rhythmic contractions because of a decrease in both contraction duration and rest interval.(ABSTRACT TRUNCATED AT 250 WORDS)