Widespread accumulation of [3H]testosterone in the spinal cord of
a wild bird with an elaborate courtship display

J. DOUGLAS SCHULTZ AND BARNEY A. SCHLINGER*Department of Physiological Science and Laboratory of Neuroendocrinology, Brain Research Institute, University of California, Los Angeles, CA 90095-1527 Communicated by Peter Marler, University of California, Davis, CA, June 22, 1999 (received for review January 28, 1999) ABSTRACT
Elaborate courtship displays are relatively
the performance of these behaviors by birds across many taxa, common features of the masculine reproductive behavior in
little is known about their hormonal and neural control. Given birds. However, little is known about their neural and hor-
that some of these displays (i) involve coordinate usage of monal control. One bird that performs such a display is the
several neuromuscular systems controlling posture and move- golden-collared manakin (Manacus vitellinus) of Panamanian
ments of the wings, legs, and tail; (ii) are often performed by forests. Adult males, but not females, perform a physically
males and not females; and (iii) are used in reproductive intense display requiring substantial neuromuscular control
contexts, we would predict that in displaying birds, the spinal of the wings and legs. We tested the hypothesis that steroid
motoneurons controlling behaviorally relevant muscles would sensitivity is a property of neurons in the manakin spinal
be sensitive to steroid hormones and would be anatomically cord. Males and females were captured from active courtship
and/or physiologically sexually dimorphic.
leks, treated with drugs to block steroidogenesis, injected with
To test these hypotheses, we have performed tritiated 3H-labeled testosterone, and the spinal cords were removed
testosterone (3H-T) autoradiography on the spinal cords of and processed for autoradiography. Sex steroid-accumulating
adult male and female golden-collared manakins (Manacus cells were widely distributed in the spinal cords in each of six
vitellinus), a common bird species of central Panamanian males and in one of five females. Cells, including presumptive
forests. Manakins are a family of suboscine, passerine birds motoneurons, reached their highest density in the ventral
that are common in forests of the New World tropics. Males horns of the cervical and lumbosacral enlargements, regions
of several manakin species, including the golden-collared associated with motor control of the wings and legs. These
manakins, perform elaborate courtship displays involving results suggest that neurons in the adult manakin spinal cord
short flights with midair acrobatics and intense jumping and can express sex-steroid receptors, but do so less in females
dancing movements. In addition, the wings of some species than in males. This evidence for androgen sensitivity and
(including golden-collared manakins) possess sexually dimor- sexual dimorphism in the adult avian spinal cord suggests that
phic feather structures (14) that assist in producing loud sex steroids may control diverse behaviors in male birds in
snapping sounds by the rapid flipping of their wings (13, 14).
part by acting directly on the spinal neural circuits.
We report that male golden-collared manakins show wide- spread accumulation of 3H-T or its metabolites in the spinal To reproduce, males perform behaviors to attract and stimu- cord, including in many large motoneurons, and this pattern of late females, defend territories and mates, copulate, and care sex-steroid accumulation is different in females. Androgen for young. Sex steroids can control the development and accumulation in spinal motoneurons in adult birds suggests expression of many of these behaviors by direct actions on the that steroid sensitivity may be present in those neural pathways central nervous system. Many studies focus on the actions of of the spinal cord that generate a range of avian courtship sex steroids on the hypothalamus, where there exists a rela- tively conserved population of neurons expressing androgen receptors (AR) and estrogen receptors (ER) within circuits controlling masculine copulatory behaviors (1–3). Steroids can also act directly on motoneurons (4, 5). For example, AR can Golden-collared manakins (six males; five females) were cap- be expressed in mammalian motoneurons of the lumbar spinal tured in mist nets from active courtship leks located in central cord that innervate muscles controlling the penis (6–9). By a Panama in June, 1995 and September, 1996. (All protocols for combination of actions of estrogens and androgens on the animal use have been approved by the Chancellor’s Animal brain and on the spinal cord, male mammals are stimulated to Research Committee and were collected under permit from copulate and are functionally able to do so.
Instituto Nacionale de Recursos Naturales Renovables, gov- Steroids also influence neurons controlling other reproduc- ernment of Panama.) To reduce endogenous androgen pro- tive behaviors (4, 10), such as the widely studied neural duction, birds were injected immediately with an inhibitor of circuitry controlling song located in the telencephalon of the one of two steroidogenic enzymes, either trilostane (an inhib- oscine passerine songbirds (11, 12). Given that song is a itor of 3-␤-hydroxysteroid dehydrogenase/isomerase, Sterling- significant acoustic signal coordinating reproduction in these Winthrop Research Insitute, 2 males and 1 female) or keto- birds, it is not surprising that the song-control circuitry is conazole (an inhibitor of 17-␣-hydroxylase/C17–20 lyase, Jans- influenced by sex steroids via the expression of AR and ER.
sen; 4 males and 4 females). Studies on the effectiveness of But song is just one of a suite of avian reproductive behaviors.
these two inhibitors in birds are published elsewhere (15, 16).
Male birds can exhibit an impressive repertoire of visual and After 24 hr (trilostane) or 8–12 hr (ketoconazole), the birds acoustic reproductive displays (13). In some species, these were injected with 60–80 ␮Ci (1 Ci ϭ 37 GBq) of 3H-T visual displays can be dramatic, including acrobatic movements (specific activity 102.5 Ci/mmol; New England Nuclear) and that are enhanced by conspicuous physical ornaments. Despite sacrificed 90 min later by decapitation. The gonads were The publication costs of this article were defrayed in part by page charge Abbreviations: AR, androgen receptor; ER, estrogen receptor; 3H-T, payment. This article must therefore be hereby marked ‘‘advertisement’’ in *To whom reprint requests should be addressed at: Department of accordance with 18 U.S.C. §1734 solely to indicate this fact.
Physiological Science, P.O. Box 951527, Los Angeles, CA 90095-
PNAS is available online at www.pnas.org.
1527. E-mail: Schlinge@lifesci.ucla.edu.
Proc. Natl. Acad. Sci. USA 96 (1999) visually inspected and sizes noted. The vertebral columns were immediately dissected free of surrounding tissues and fixed briefly (Ϸ1 min) in formalin (3.7% formaldehyde in 0.9% Sex steroid-accumulating cells were found in the manakin PBS) to assist with removal of the spinal cord (which remained spinal cord with substantially more cells in males than in unfixed). The spinal cords were then cut lengthwise, into halves females. Accumulation of sex steroid was found in cells of the spinal cords of all six males (on average, 209 cells over the or thirds, flash-frozen with crushed dry ice onto cork with entire cord). By contrast, only one female approached this Tissue-Tek (Sakura Finetek, Torrance, CA), and stored in dry number (with 101 cells found). Only four, two, two, and zero ice at Ϫ80°C. The cords were transported in dry ice to the accumulating cells per spinal cord were found in the remaining United States and prepared for autoradiography.
Cords were sectioned longitudinally in the dark on a cryostat Cells with a visible nucleus comprised 59.5% of the total (at Ϫ17°C) and thaw-mounted onto microscope slides. For number of sex steroid-accumulating cells (Fig. 1). Of the autoradiographic analysis, two consecutive sections were cut at nucleated cells, 95% had a greater density of silver grains over 6 ␮m and thaw-mounted onto separate slides previously the nucleus alone as compared with the whole soma (which dipped in Kodak NTB-2 photographic emulsion (series 1 and includes the nucleus). Fig. 2 illustrates the total number of 2, respectively). The third consecutive section was sectioned at nucleated and nonnucleated cells with nuclear or somal accu- 20 ␮m and thaw-mounted on slides (Fisher Superfrost Plus).
mulation (respectively) greater than 3ϫ background. Many These slides were later stained with thionin and used to assist cells in males were present with accumulation between 3 and with preparation of a spinal cord map. The fourth consecutive 5ϫ background (64.0 on average) but were not accepted as section (cut at 6 ␮m) was usually discarded because of poor representing significant accumulation based on our criteria. In quality; this cycle was repeated through the entire cord.
the female in which we found a large number of cells meeting Although only one series was analyzed from a given bird, there the 5ϫ criteria, 39 additional cells met the 3ϫ criteria. If these was usually 38 ␮m of tissue between each section in a given cells do indeed express AR or ER, but at low levels, then our series. Slides from series 1 and 2 were sealed in light-tight, results may underestimate the total number of sex steroid- desiccated containers and stored at 4°C. After 3, 6, 9, or 12 accumulating cells in the manakin spinal cord (21). No addi- months, autoradiographic slides were immersed consecutively tional cells that met the 3ϫ criteria were found in the in Kodak D-19 developer, distilled H2O, and Kodak fixer, thionin stained, dehydrated in a graded series of alcohols, and Sex steroid-accumulating cells with an obvious nucleus fell into two general size categories, small and large cells with
For each bird, the slide series demonstrating optimal cellu- mean areas of Ϸ1,000 ␮m2 and 4,000 ␮m2, respectively (Fig. 3).
lar-to-background silver grain density was chosen for analysis.
Most large cells were located ventrally, whereas small cells Cells that appeared to have high levels of accumulation with were distributed widely, especially in middle and dorsal levels a distinct nucleus were analyzed over both the nucleus and soma (which included the nucleus). Cells that appeared to have Even though sex steroid-accumulating cells were found in all high levels of accumulation but lacked an obvious nucleus were six males, only four males provided histology that was quali- analyzed for grain accumulation over the whole soma. These tatively sufficient to allow for precise localization and map- were measured by capturing the cellular image (at ϫ1,000) on ping. Sex steroid-accumulating cells of the remaining two a Macintosh computer by using a Zeiss Axioskop microscope males were present with their size and position consistent with linked to a Sony charge-coupled device video camera. By using the large motoneurons of the ventral cervical and lumbosacral enlargements. For purposes of description, the spinal cord was ference of the nucleus, soma, or selected region of neuropil was subdivided rostrocaudally into the high cervical, cervical en- traced, and silver grain size was defined. We computed both largement, midthoracic, and lumbosacral enlargement regions and dorsoventrally into the ventral, middle, and dorsal regions the area and the number of silver grains contained within the (Fig. 4). Cells were included on the map without distinguishing tracing. Silver-grain density was determined by dividing the between those with and without nuclei. The relative abun- area measurement into the number of silver grains counted. A dance and distribution of sex steroid-accumulating cells in the similar measurement was then made over the remaining spinal regions of the four males and five females is summarized neuropil adjacent to the cell in the captured microscope image (background). The average background area measured was six times larger than the average motoneuron somal area. We then calculated the ratio of silver-grain density over the soma or nucleus to background density. Data were recorded for all cells in which this cellular or nuclear-grain density was Ն3 times the background; accumulation was considered significant when cellular or nuclear-grain densities were Ն5 times background (17, 18), and these cells are hereafter referred to as sex A map of the position of all sex steroid-accumulating cells in the spinal cord was created for each individual bird; afterward, a single composite map was created summarizing the general distribution of all birds. Cellular position in the rostrocaudal plane was established by observing landmarks such as visible dorsal-root ganglion, the beginning of the cervical enlargement that starts at C10 in the pigeon (19), and the lumbosacral enlargement that starts at L1 in the pigeon (20). Cells in the dorsoventral axis were located by estimating the proximity of the cell to Lamina IX (20), a morphologically FIG. 1. Representative sex steroid-accumulating, thionin-stained prominent motoneuronal cell column throughout the ventral neurons from the ventral cervical enlargement of an adult male Proc. Natl. Acad. Sci. USA 96 (1999) females in the middle third of the cord, and a group of cells from one male was found in the dorsal third of the cord.
Sex steroid-accumulating cells were widespread in the lum- bosacral enlargement (L1–L6) throughout its full rostrocaudal extent. A majority of these cells were found in the ventral third of the enlargement and were found in one male and in one female. Numerous labeled cells were also found in medial portions of the middle third of the lumbosacral enlargement, especially in two males. One of these males also had a significant number of cells in the dorsal third of the enlarge- ment. We were unable to reliably distinguish the glycogen body DISCUSSION
These results suggest that some neurons in the manakin spinal cord express AR as described in other vertebrates (6, 9, 22). It is also possible that 3H-T was aromatized into [3H]estradiol FIG. 2. Motoneuronal silver grain density (cellular silver grain number as a multiple of background silver grain number) over nuclei and that ER are expressed in the manakin spinal cord, as they (nucleated cells) and somas (nonnucleated cells) after injection of are in rats (23). The presence of widespread sex-steroid 3H-labeled testosterone into adult male and female golden-collared accumulation in cells in the spinal cords of males compared with females suggests that there is a sex difference in the magnitude of AR and/or ER expression or in the number of Within the high cervical cord (rostral to the cervical en- AR- and/or ER-expressing cells. It is also possible that sex largement), accumulating cells were found in all four males steroid availability in the manakin spinal cord may differ and in none of the females. Here, the number of cells were between males and females because of sex differences in roughly evenly distributed dorsoventrally, with the middle steroid metabolism (24, 25). Presumably, sex steroids act on third of the high cervical region having the most cells relative spinal neural circuits in manakins to control expression of to the ventral and dorsal regions. Localization was mostly confined to an area midway between the hindbrain and the Many sex steroid-accumulating cells in the manakin spinal cervical enlargement (see Fig. 4), approximately C5.
cord are located in lamina IX (20) and are of a large size, In the cervical enlargement (C10–C13), sex steroid- consistent with that of motoneurons (Fig. 2). Similar AR- accumulating cells were abundant in the ventral third for three expressing motoneurons have been found in rat (23, 26) and in of the four males and for one female. Relative to the ventral Xenopus (27), leading us to suspect that these ventral mo- third, there were comparatively fewer cells in the middle third, toneurons in manakins are also androgen-, and not estrogen-, and accumulation was present in all four males and none of the sensitive. Some smaller sex steroid-accumulating cells found in females. In the ventral and middle third of the cervical dorsal regions of the manakin spinal cord may be sensory enlargement, cells were found along the entire rostrocaudal neurons binding estrogen as reported in rats (23) and ring length. In the dorsal third, we found one male with two distinct doves (28). Subpopulations of AR-expressing motoneurons clusters of cells, one each in the rostral and caudal portions of the cervical enlargements. We found no sex steroid- Table 1. Relative abundance of sex steroid-accumulating cells in accumulating cells in this region of any other bird.
A small number of sex steroid-accumulating cells were present in the midthoracic region, approximately T3, midway between the cervical and lumbosacral enlargements. Only one male showed a few accumulating cells in the ventral third of the cord. Small cell clusters were found in two males and two Areas of nucleated (hatched bars) and nonnucleated (open bars) sex steroid-accumulating cells in the spinal cords of adult male M, male; F, female; Ϫ, no cells; ϩ, 1–5 cells; ϩϩ, 16–50 cells; ϩϩϩ, and female golden-collared manakins.
51–100 cells; ϩϩϩϩ, 101–200 cells; ϩϩϩϩϩ, 201–224 cells.
Proc. Natl. Acad. Sci. USA 96 (1999) steroids act directly on spinal neural circuits to control the expression of these courtship behaviors. Males, but not fe- males, court actively, presumably because testosterone circu- lates at higher levels in males than in females. We suspect that androgens, or their metabolites, act centrally to increase the motivation to court and peripherally to increase the neuro- muscular capacity to perform the displays. Androgens also act on neurons centrally and peripherally in frogs and rats to activate masculine reproductive behaviors (5, 30, 31). In the spinal cords of these species, sex differences in AR expression in part underlie the observed sex differences in behavior (5).
The basis of the sex differences in sex-steroid binding in the manakin spinal cord is unknown. The differences could arise from sex differences in the numbers of some AR-expressing cells, as observed in the rat (6, 21), in which fewer AR- expressing motoneurons exist in the female lumbar spinal cord because females lack two of the target muscles (5). It is unlikely that the female manakin possesses fewer motoneurons if they are innervating essential muscles of the wings and legs. It is more likely that higher levels of circulating testosterone in male manakins transiently up-regulate spinal AR to a greater degree than in females, as is presumed to occur in Xenopus (27). Although we cannot exclude the possibility that some of the differences we observe reflect permanent sex differences in sex-steroid binding or in other cellular attributes, we assume that neuromuscular control of the wings and legs is temporally adapted for greater use by sex steroids when males are actively We found considerable variability in the numbers of sex steroid-accumulating cells across males and the extent to which androgens were bound by cells (Table 1). Cells were found most consistently in the cervical enlargement, but even here one male showed none. This variability in spinal cord steroid sensitivity may have been produced by differences in circulat- ing testosterone if males were in different reproductive con- dition. Some birds were caught in September, a time when males are beginning to cease courtship activity (14). Our data indicate that the male with the smallest testes had the lowest number of sex steroid-accumulating cells, with greater num- bers of accumulating cells found in males with somewhat larger testes. Because androgens can directly regulate AR in the spinal cord (9), males with low plasma testosterone levels before capture could have displayed less sex-steroid accumu- FIG. 4. Representative map showing the spatial distribution of sex lation. No differences in ovary size were observed that account steroid-accumulating cells in the golden-collard manakin spinal cord.
Each symbol represents three cells, with isolated cells omitted; black for differences observed across females. Variability might also dots represent cells found in males; X represents cells found in females have been produced artificially if trilostane or ketoconazole (see text for details). The cord is illustrated at three levels; all cells reduced endogenous androgen synthesis differently across found within a given level are illustrated: Ventral, the entire cord birds, creating disproportionate competition for androgen- ventral to the bifurcation of the ventral horns, including most of lamina binding sites with endogenous nonradioactive testosterone.
IX and much of lamina VIII of Leonard and Cohen (1975); dorsal, the We cannot exclude the possibility that different effects of these entire cord dorsal to the beginning of the bifurcation of the dorsal drugs on testicular or ovarian steroidogenesis might also have horns out to their tips, including all of lamina I–IV of Leonard and contributed to observed differences in spinal cord sex-steroid Cohen (1975) and part of lamina V; middle, the remaining cord between the ventral and dorsal levels. Table 1 illustrates individual differences in the pattern of accumulation.
The identification of steroid-sensitive neural circuits throughout the spinal cord of the golden-collared manakin are functionally associated with androgen-dependent muscles suggests that sex steroids may have a broader role in modu- and together control androgen-dependent penile reflexes in lating avian neuromuscular systems than previously thought.
rodents (6, 25, 9) and amplexus in Xenopus (22, 27). Therefore, Although birds have been widely studied with respect to we assume that at least some of these sex steroid-accumulating steroid actions on the brain, no avian neuromuscular system motoneurons in manakins are also part of androgen- dependent neuromuscular systems. Because in manakins the has been fully exploited to evaluate steroid control of mo- majority of sex steroid-accumulating cells are found in the toneurons and sexually dimorphic muscles that they might cervical and lumbosacral enlargements, and motoneurons in innervate. As in other species, sex steroids may regulate the these enlargements largely control muscles of the upper and interrelationship of motoneurons and their targets, possibly lower extremities (19, 29), these cells may be involved in stimulating plasticity in both (32–34). Insofar as physical multiple behavioral functions, perhaps innervating muscles displays and mechanical sounds are characteristic parts of the controlling the elaborate dancing and wing-snapping of these behavioral repertoire of a vast number of bird species, the results presented here support the view that sex steroids act on The presence of sex differences in sex-steroid accumulation the spinal cord to activate these behaviors. Further studies in the manakin spinal cord is consistent with the idea that sex defining the neuromuscular control of male courtship are Proc. Natl. Acad. Sci. USA 96 (1999) necessary to establish the role of hormones in regulating these 12. Schlinger, B. A. (1997) J. Neurobiol. 33, 619–631.
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