Embryonic chicken gizzard: smooth muscle and non-muscle myosin isoforms

Antibodies to smooth muscle and non-muscle myosin allow the development of smooth muscle and its capillary system in the embryonic chicken gizzard to be followed by immunofluorescent techniques. Although smooth muscle development proceeds in a serosal to luminal direction, angiogenetic cell clusters develop independently at the luminal side close to the epithelial layer, and the presumptive capillaries invade the developing muscle in a luminal to serosal direction. The smooth muscle and non-muscle myosin heavy chains in this avian system cannot be separated by SDS polyacrylamide gel electrophoresis and do not show isoform specificity in immunoblotting, unlike the system found in mammals. Only two myosin heavy chains with M_r of 200 and 196 kDa were separable and considerable immunological cross-reactivity was found between the denatured myosin isoform heavy chains.     

Embryonic chicken gizzard: expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase

The patterns of expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase were investigated in the developing chicken gizzard. Immunofluorescent studies revealed that both proteins were expressed as early as E5 throughout the mesodermal gizzard anlage, together with actin, -actinin and a small amount of nonmuscle myosin. These proteins appear to form the scaffold for smooth muscle development, defined by the onset of smooth muscle myosin expression. During E6, a period of extensive cell division, smooth muscle myosin begins to appear in the musculi laterales close to the serosal border and, later, also in the musculi intermedii. Until about E10, myosin reactivity expands into the pre-existing thin filament scaffold. Later in development, the contractile and regulatory proteins co-localize and show a regular uniform staining pattern comparable to that seen in adult tissue. By using immunoblotting techniques, the low-molecular mass form of caldesmon and myosin light chain kinase were detected as early as E5. During further development, the expression of caldesmon switched from the low-molecular mass to the high-molecular mass form; in neonatal and adult tissue, high-molecular mass caldesmon was the only isoform expressed. The level of expression of myosin light chain kinase increased continously during embryonic development, but no embryospecific isoform with a different molecular mass was detected.     

Cytochemical localization of alkaline phosphatase in the ependyma of the rat medulla oblongata

Summary The ependyma of the IVth ventricle and the central canal of the rat medulla oblongata was investigated using the cytochemical technique for alkaline phosphatase (AlPase) which revealed two types of ependymal cells in the medulla. The central canal type of the ependymal cell occupying the dorsal part of the central canal in the lower medulla exhibited intense AlPase activity with light microscopy. These cells had reaction products in all plasma membranes, including the microvilli and the cilia at the luminal cell surface. Some cells appeared to be tanycytes, since the process reached the basement membrane of the parenchymal blood vessel. The ventricular type of ependymal cells, which form the floor of the IVth ventricle and the central canal, contained no reaction products in any structure of the luminal cell surface.The possible relationship between the cerebrospinal fluid and the nervous tissues through the ependymal linings is discussed.     

Immunocytochemical localization of alkaline phosphatase in absorptive cells of rat small intestine after colchicine treatment

Summary The purpose of this study was to investigate the effect of colchicine and vinblastine on the localization of alkaline phosphatase (AlPase) in rat duodenum in relation to structural changes. AlPase was localized on the membranes of rough endoplasmic reticulum, Golgi stacks, cytoplasmic vesicles, microvilli, on lateral plasma membranes, and in some lysosomes of the duodenal epithelial cells of rats treated with either lumicolchicine or 0.9% NaCl alone. Microvilli were most intensely stained, and AlPase-positive Golgi stacks were regularly distributed in the supranuclear regions. After colchicine treatment, microvilli were shortened and the staining intensity became weaker, whereas basal as well as lateral plasma membranes showed stronger staining. The AlPase-positive microvilli appeared not only on the luminal surfaces, but also on the baso-lateral plasma membranes and even on the surfaces of characteristic intracytoplasmic cysts. Golgi stacks became smaller and their distribution became less localized, and the staining intensity of the Golgi stacks became weaker. AlPase localization in rats treated with vinblastine was almost identical with that of rats treated with colchicine. Thus, colchicine and vinblastine appeared to have elicited a disorientation of intracellular transport of intestinal AlPase by inhibiting microtubule organization.     

Testosterone and FSH have independent,synergistic and stage-dependent effects upon spermatogenesis in the rat testis

Summary Adult rats were hypophysectomized and treated with ethane dimethanesulphonate (EDS) selectively to eliminate the Leydig cells in the testis. By removing the source of endogenous gonadotrophins and androgens, the subsequent effects on the seminiferous epithelium were studied after 20 days of treatment with vehicle, or FSH (2x50 g/day) or a low dose of testosterone (0.6 mg testosterone esters every 3rd day) alone or in combination. Compared to vehicle-treated hypophysectomized rats with Leydig cells, testis weight in saline-treated hypophysectomized rats treated with EDS declined by 50%, spermatogenesis was disrupted severely and only 18% of the tubules contained spermatids, these being confined to stages I–VI of the spermatogenic cycle. Treatment with either FSH or testosterone esters alone significantly (P<0.01) increased testis weight compared to vehicle-treated hypophysectomized rats treated with EDS and 40% of tubules contained spermatids either at stages I–VI after FSH, or at all stages I–XIV after testosterone treatment. Treatment with FSH and testosterone esters together maintained testis weights approximately 20% above vehicle-treated hypophysectomized controls; over 70% of the seminiferous tubules contained spermatids and there was a marked stimulation of spermatogenesis at all stages of the spermatogenic cycle. The results suggest, that in the absence of the pituitary gland and the Leydig cells, FSH alone partially supports spermatogenesis up to the development of round spermatids whereas testosterone is capable of maintaining spermatid development at all 14 stages of the cycle. When FSH and testosterone were administered in combination, the effects upon spermatogenesis were far greater than the response expected if their individual effects were simply additive. It is therefore concluded that FSH may play a role in normal spermatogenesis and that this role is essentially that of augmenting the response of the testis to testosterone. The biochemical mechanisms via which this might occur are discussed and hypophysectomized rats treated with EDS used in the present studies should provide a useful approach for their identification.     

Analytical subcellular fractionation of rat pituitary homogenates with special reference to the subcellular localization and properties of alkaline phosphatases

Alkaline phosphatase activities of the virgin rat anterior pituitary were studied with a highly sensitive fluorometric assay. Tissue whole homogenates were fractionated on sucrose density gradients in a Beaufay automatic zonal rotor and the gradient fractions assayed for alkaline phosphatase, prolactin and various organelle marker enzymes. Alkaline phosphatase was distributed between two peaks on the gradient. The low-density (1.10–1.15 g·cm^?3) alkaline phosphatase component co-sedimented with the plasma membrane marker, 5′-nucleotidase, had an apparent K_m for 4-methylumbelliferyl phosphate of approx. 59 μM, and was inhibited by levamisole. The high-density (1.20–1.25 g·cm^?3) peak was resistant to levamisole-inhibition, had an apparent K_m of approx. 30 μM and its distribution was distinct from plasma membrane, Golgi, lysosome, endoplasmic reticulum, mitochondria and prolactin granule markers on the isopycnic gradients.     

Hyperplasia of jejunal smooth muscle in the myenterically denervated rat

Summary Application of a cationic surfactant, benzalkonium chloride, to the serosa of rat jejunum results in an increase in thickness of both longitudinal and circular smooth muscle layers. The increase in thickness is due primarily to an increase in the number of smooth muscle cells (hyperplasia). Little cellular hypertrophy was observed. The time sequence of surfactant-induced effects on the muscle layers was determined. Within 24 h, total destruction of the longitudinal muscle and partial destruction of the circular muscle was evident. The myenteric plexus was also necrotic; however, the submucosal plexus remained intact. By 48 h after surfactant treatment, the smooth muscle cells remaining in the circular muscle layer had begun to divide, as indicated by the presence of mitotic figures and incorporation of ³H-thymidine. A repopulation of the longitudinal muscle layer began at this time, apparently the result of migration of cells arising in the circular muscle layer. By 5 days post-treatment, both muscle layers had regenerated to their original states. The myenteric plexus was totally absent. The denervated smooth muscle cells proceeded to divide until approximately day 15, resulting in hyperplasia of both muscle layers. Between 15 and 105 days, the number of muscle cells in the circular layer progressively declined, eventually returned to the value seen in control tissue. In contrast, the number of smooth muscle cells in the longitudinal layer remained elevated through the period of study (165 days). We hypothesize that the smooth muscle hyperplasia observed after serosal benzalkonium chloride application results from loss of the myenteric nerves.     

Electron-cytochemical localization of alkaline phosphatase to G cells of Necturus maculosus antrum

Summary Electron-cytochemical localization of alkaline phosphatase activity was performed on G cells of Necturus maculosus antral mucosa. Alkaline phosphatase activity was localized to the nuclear membrane, the Golgi/endoplasmic reticulum, and the limiting membranes of G cell peptide-secretion vesicles. There was no specific localization of alkaline phosphatase activity to the plasma membrane. Treatment of the tissues with levamisole (an alkaline phosphatase inhibitor) did not markedly reduce the specific alkaline phosphatase activity. Specific lead deposition was reduced by removal of the substrate from the reaction mixture. The results from this study on N. maculosus G cells demonstrate that alkaline phosphatase activity can be found in a non-mammalian gastric endocrine cell and that specific activity was localized primarily to those intracellular structures involved with protein biosynthesis.     

Actin,myosin, and laminin localization in retinal vessels of the rat

Summary Actin, myosin, and laminin have been localized in retinal vessels of normal rats by fluorescence microscopy. Actin was localized with the fluorescent F-actin binding toxin nitrobenzoxadiazole phallacidin (NBD-Ph). Indirect immunofluorescence was used to localize myosin and laminin. In addition, laminin localization was also performed with the Protein A-horseradish peroxidase (PA-HRP) method. NBD-Ph staining gave strong fluorescence in both retinal capillaries and larger vessels. Anti-myosin fluorescence could also be observed in trypsin digests of the retinal vasculature. Strong fluorescence of PA-HRP reaction product could be detected in the walls of vessels exposed to antilaminin antibody. Actin distribution in vessels of the RCS rat with inherited retinal degeneration (retinal dystrophic RCS rat) was also studied. After exposure to NBD-Ph, all capillaries showed fluorescence. However, it was more intense in many of the capillaries in the outer retina, which also appeared morphologically abnormal. Electron microscopy of retinal capillaries fixed in 2.5% glutaraldehyde containing 8% tannic acid revealed numerous micro filaments in the pericyte cytoplasm amd some in the basal portion of endothelial cells. In pericytes, these microfilaments are in close association with the endothelial side of the cell. Tangential sections through this region indicate that these filaments may be anchored to the membrane at this site.Supported by grants EY04831, Research to Prevent Blindness, Inc. and the Michigan Eye Bank     

Immunocytochemical localization of aromatase-containing neurons in the rat brain during pre- and postnatal development

the present immunohistochemical study demonstrates the ontogenetic appearance of aromatase-immunoreactive neurons in several discrete regions of the hypothalamus and limbic system in the rat brain, using a purified antibody against human placental aromatase cytochrome P450. Immunoreactive cells were first detected in the preoptic area on the 13th day of embryonic life (E 13), and additionally in the bed nucleus of the stria terminalis on E 15. Labeled cells were also found in the medial amygdaloid nucleus and the ventromedial nucleus on E 16, and some were detected in the arcuate nucleus on E 19. As gestation progressed, the number and the immunoreactivity of these cells gradually increased and peaked within definite periods of perinatal life and there-after declined or disappeared. The immunoreactive cells were also found in the central amygdaloid nucleus and the lateral septal nucleus, and in the ventral pallidum, after the 14th day of postnatal life (P 14) and 30th day (P 30), respectively. The distribution of aromatase-immunoreactive neurons was similar between the sexes, while the immunoreactivity was higher in males than in females after late gestational days. No immunoreaction was detectable in other regions of the telencephalon or midbrain at any time periods studied. The aromatase-immunoreactive neurons in the specific regions may be involved in the sexual differentiation of the brain.     

A possible mechanism for the changes in hepatic and intestinal alkaline phosphatase activities in bile-duct-ligated rats or guinea pigs

The effects of bile-duct ligation on hepatic and intestinal (jejunum) alkaline phosphatase activities were studied using rats and guinea pigs. In ligated rats, the enzyme activity was increased 4.1-fold in the liver after 24 h and 2.8-fold in the intestine after 12 h. In guinea pigs, the hepatic and intestinal enzyme activities were increased 2.3-fold and 1.5-fold after 100 and 24 h, respectively. The intestinal activity was induced sooner after ligation than hepatic activity. The induction of alkaline phosphatase was inhibited by prior treatment of animals with amanitin, an inhibitor of RNA polymerase activity. This result indicates that the induction is associated with de novo enzyme synthesis. The content of cyclic AMP in liver and intestine increased immediately after ligation. The increase in alkaline phosphatase activities was also inhibited by pretreatment with chlorpromazine, an inhibitor of adenylate cyclase activity. Hence, cellular cyclic AMP may be implicated in playing a role in the induction of alkaline phosphatase by bile-duct ligation.     

Appearance of B and H blood group antigens in the developing cochlear hair cells

Summary The presence of human blood-group antigens was analyzed in the rat cochlea during its postnatal development, using anti-A, anti-B and anti-H antibodies. At no stage was reactivity with anti-A antibody observed. With the anti-H antibody, a strong reactivity was observed from 1 to 9 days after birth within hair cells and some other surface epithelial cells of the cochlear duct. After postnatal day 9, only a faint reactivity persisted in a few non-sensory cells. With the anti-B antibody, only hair cells were selectively labeled. At early stages (postnatal day 1 and 3), the reactivity was intense and observed both around the cell surface and within the supranuclear region of cytoplasm. Later on, the reactivity decreased; it was limited at postnatal day 9 to a reactive spot below the cuticular plate. Results are compared with a preliminary finding describing the first appearance of B and H antigens in the organ of Corti at a prenatal stage, and with data concerning other sensory and neural structures. The appearance and progressive disappearance of B and H antigens on sensory and non-sensory cells can be correlated with significant events in the development of the cochlea. The transient expression of B and H antigens in cochlear sensory cells may correspond to developmental changes in their surface glycoconjugates.     

Immunohistochemical localization of tissue-type plasminogen activator in ovaries before and after induced and spontaneous ovulation in the rat

Summary The observation that tissue-type plasminogen activator (tPA) activity increased dramatically in preovulatory follicles has led to the hypothesis that plasminogen activation is causally related to follicle rupture. With immunohistochemistry, we have studied the appearance of tPA in ovaries of immature rats induced to ovulate and in adult cycling rats. Treatment of immature female rats with a single dose of pregnant mare serum gonadotropin (PMSG) induced follicular maturation. A subsequent human chorionic gonadotropin (hCG) injection resulted in follicle rupture 12–14 h later. PMSG treatment alone did not induce appearance of tPA-immunoreactive cells in any ovarian compartment. After hCG stimulation, however, theca cells, granulosa cells, and oocytes of pre- and postovulatory follicles displayed distinct tPA immunoreactivity. Fibroblastlike cells in the theca layers and tunica albuginea of the follicle apex also demonstrated localized cytoplasmic tPA reactivity. In addition to tPA synthesis in preovulatory follicles, hCG also induced tPA staining in the theca (but not granulosa) layers of non-ovulatory follicles. At 24 h after hCG treatment, there was a marked tPA staining in developing corpora lutea, ovulated ova, and oviductal epithelium. Ovaries from regularly cycling adult rats displayed a similar ovulation-related pattern of tPA immunostaining. The appearance of tPA in different cell types of the preovulatory follicle and in the fibroblast-like cells at the follicle apex, strengthens the hypothesis of a direct involvement of tPA in follicle rupture. Presence of tPA in postovulatory oocytes, cumulus cells, and surrounding oviductal epithelium may also indicate a role for tPA in the transfer of eggs in the oviduct.This work was supported by NIH Research Grants HD-14084; 12303     

Distribution of activity of alkaline phosphatase and Mg-dependent adenosine triphosphatase in the cranial dura mater-arachnoid interface zone of the rat

Summary The distribution of the activity of alkaline phosphatase and Mg-dependent adenosine triphosphatase was studied in the encephalic dura mater-arachnoid borderline (interface) zone of albino Wistar rats. Intense clustering of electron-dense granules that indicated alkaline phosphatase activity was observed in the inner dural cells, the neurothelial cells, the outermost row of the outer arachnoidal cells and in the intercellular cleft between the latter two (the so-called electron-dense band). The remainder of the outer arachnoidal cells contained almost no reaction product. Mg-adenosine triphosphatase activity was distributed differently; a lack of reaction product was observed not only in the outer arachnoidal cells, but also in the zone occupied by the electron-dense band. The data confirm histochemically the barrier properties of the dura mater-arachnoid interface zone.     

Chloroquine and monensin inhibit induction of DNA synthesis in rat arterial smooth muscle cells stimulated with platelet-derived growth factor

Summary The weak base chloroquine and the Na⁺/H⁺ ionophore monensin were used to study the role of lysosomes in the induction of DNA synthesis by platelet-derived growth factor (PDGF) in rat arterial smooth muscle cells cultivated in vitro. The results show that PDGF initiates DNA synthesis in a defined, serum-free medium. This indicates that a single factor may control, directly or indirectly, the transition from the G0 to the G1 phase, the progress through the G1 phase, and the entrance into the S phase of the cell cycle. It is further demonstrated that PDGF has to be present throughout most of the prereplicative period (12–16 h) to induce DNA synthesis in the maximum number of cells, suggesting that one or more processes need to be stimulated continually or successively to push the cell into the S phase. Chloroquine and monensin inhibit induction of DNA replication by PDGF, with maximum effect at 50 M and 5 M, respectively. To be fully active, the drugs have to be added within 4–8 h after the growth factor, but a partial inhibition persists if they are added at any time during the prereplicative period. Both drugs reduce PDGF-stimulated RNA and protein synthesis, and suppress degradation of [³H]leucine-labeled cellular protein and [¹²⁵I]-labeled PDGF. Fine-structurally, they give rise to an accumulation of lysosomes or prelysosomal vacuoles with inclusions of incompletely degraded material. These findings suggest that the mitogenic effect of PDGF is dependent on a normal function of lysosomes during the prereplicative phase, especially its first half (0–8 h).     

The immunocytochemical localization of angiotensinogen in the rat ovary

Summary The present study examined the presence and cellular distribution of angiotensinogen, the precursor to the angiotensin peptides, in the ovary of the normal cycling rat by immunocytochemistry. Angiotensinogen staining was present in the granulosa cells of maturing follicles and to a lesser extent in those undergoing atresia. Staining was not seen in the granulosa cells of primordial or early primary follicles. In maturing follicles intense staining for angiotensinogen was confined to the antral cell layers, cells of the cumulus oophorus and in the follicular fluid. Strong immunostaining was also seen in the germinal epithelium covering the ovary. Lighter angiotensinogen staining was observed in some parts of the cortical and medullary stroma and occasionally in corpora lutea. No variation in the intensity or pattern of angiotensinogen staining was observed throughout the estrous cycle. Comparison of the distribution of angiotensinogen with the previously described localization of renin, AII, angiotensin converting enzyme and AII receptors, suggests that there are a number of intra-ovarian sites at which AII could be produced.     

Composition in situ and in vitro of vascular smooth muscle laminin in the rat

Vascular smooth muscle cells are surrounded by a basal lamina containing an array of macromolecules: included among these are the laminins, a family of oligomeric glycoproteins composed of subunits encoded by different genes. In this study, we have used monoclonal antibodies to several of these subunits, including S-laminin, laminin B2, and laminin B1, to study these proteins in tail artery, superior mesenteric artery, and aorta of rats. In situ, immunostaining for the B2 and S chains was present in the basal lamina, between the smooth muscle cells, throughout the tunica media. In contrast, B1 chain immunostaining was concentrated around cells in the inner media. To investigate whether smooth muscle cells can produce S-laminin, laminin B2, and laminin B1, smooth muscle cells from the superior mesenteric artery were grown in culture and laminin subunit expression determined. In early culture (4 days), immunostaining showed abundant laminin B2 and less B1 synthesis and incorporation into the matrix. Staining for S-laminin was even less intense than for B1 and was localized to areas where cells were densely packed. The same pattern of S-laminin immunostaining was seen during early culture in cells grown on fibronectin, type IV collagen, or gelatin. Immunoblotting detected S-laminin in the conditioned medium from early cultured cells. In later culture (12 days), S-laminin incorporation into the matrix increased markedly compared to incorporation at 4 days. At this time, cells are much more densely packed and multilayered with extensive matrix accumulation. Cyclical stretching of cells in vitro did not increase immunostaining for S-laminin. Together these data show that S-laminin is a component of the arterial media in situ and that in vitro S-laminin is synthesized by smooth muscle cells. Increased incorporation of S-laminin into the matrix in later culture correlates with the presence of a more extensive matrix, suggesting that matrix organization may be critical to S-laminin incorporation.     

Cyclic AMPand cytochalasin B-induced arborization in cultured aortic smooth muscle cells: its cytopharmacological characterization

Summary The present study analyzed effects of dibutyryl cyclic AMP (DB-cAMP) and cytochalasin B (CB) on the morphology of cultured aortic smooth muscle cells (SMC) from rat using phase-contrast microscopy, scanning electron microscopy, and fluorescence staining of actin filaments by the NBD-phallacidin method. The exposure of SMC to each of these agents led to rapid, extensive, and reversible (within 1–2 h of drug withdrawal) changes in their morphology including cytoplasmic arborization (stellation). The latter was preceded by (i) marginal membrane ruffles (DBcAMP) and (ii) increased zeiotic activity (CB), which were visible within 20 min of the exposure, followed (30–90 min incubation) by a centripetal retraction of the cytoplasm and progressive development of complete or partial arborization. Further, the effects of substances interfering with the assembly-disassembly of microtubules (colchicine, taxol, lidocaine) on DB-cAMPand CB-induced arborization were studied. None of these agents antagonized CB-induced morphological changes. Colchicine, but not lumicolchicine, taxol, or lidocaine (in a short-term study) prevented DBcAMP-induced arborization. Taxol added to cell cultures for 24 h promoted DB-cAMP-induced arborization. Both DB-cAMP and CB resulted in the disintegration of actin filaments. The present data suggest that the arborization of cultured aortic SMC is a cytoskeleton-based process involving stabilization of microtubules and disintegration of actin filaments. Our study also suggests that the SMC arborization may represent an in vitro case of SMC stellation found in situ.     

Organotypic culture, a powerful model for studying rat and mouse fetal testis development

The key role of the fetal testis in the masculinization of genital organs has been known for a long time. More recently, the observed increases in male reproductive disorders has been postulated to be the result of changes in fetal and neonatal testis development in response to increasing environmental pollution. However, few tools are available for studying fetal testis development and the effects of physiological or toxic substances. We have developed an organ culture system in which rat fetal testis is grown on a filter floating on a synthetic medium containing no serum, hormones or biological factors. In this study, we have compared the long-term morpho-functional development of the various testicular cell types in this system with that observed in vivo and have extended this system to the mouse. Rat Leydig, Sertoli and germ cells and macrophages develop normally over a period of 1–2 weeks in this system. Fewer cells are produced than in vivo but the level of differentiated function is similar. Germ cells, which are difficult to culture in vitro, resume mitosis after a quiescent period, at the same time as in vivo. Similar results have been obtained with mouse fetuses, except that Leydig cells dedifferentiate in vitro if the testis is explanted after 13.5 days post conception. Testicular architecture and intercellular communication are sufficiently preserved for the development of the main fetal and neonatal testicular cell types in vitro with no added factors. Our floating-filter organotypic culture system in synthetic medium therefore allows the morpho-functional development of somatic and germ cells in fetal testis explants taken at all developmental stages in rat and at early stages in mouse. This method is potentially useful for studies of the effects of various factors, and of xenobiotics, in particular.This work was supported by the Université Paris 7, Commissariat à l’Energie Atomique (CEA) and the Institut National de la Santé et de la Recherche Médicale (INSERM) and by grant Agence Francaise de Sécurité Sanitaire et Environnementale (AFSSE). G. Delbes is the recipient of a fellowship from the Ministère de la Recherche et de la Technologie and from the Association pour la Recherche contre le Cancer.This work is dedicated to the memory of José Maria Saez in recognition of his helpful and constant encouragement and discussions over the last decade.     

Postnatal development of autonomic and sensory innervation of thoracic hairy skin in the rat

Summary Histochemical, immunocytochemical, and radioenzymatic techniques were used to examine the neurotransmitter-related properties of the innervation of thoracic hairy skin in rats during adulthood and postnatal development. In the adult, catecholamine-containing fibers were associated with blood vessels and piloerector muscles, and ran in nerve bundles throughout the dermis. The distribution of tyrosine hydroxylase (TH)-immunoreactive (IR) fibers was identical. Neuronal fibers displaying neuropeptide Y (NPY) immunoreactivity were seen in association with blood vessels. Double-labeling studies suggested that most, if not all, NPY-IR fibers were also TH-IR and likewise most, if not all, vessel-associated TH-IR fibers were also NPY-IR. Calcitonin gene-related peptide (CGRP)-IR fibers were observed near and penetrating into the epidermis, in close association with hair follicles and blood vessels, and in nerve bundles. A similar distribution of substance P (SP)-IR fibers was evident. In adult animals treated as neonates with the sympathetic neurotoxin 6-hydroxydopamine, a virtual absence of TH-IR and NPY-IR fibers was observed, whereas the distribution of CGRP-IR and SP-IR fibers appeared unaltered. During postnatal development, a generalized increase in the number, fluorescence intensity, and varicose morphology of neuronal fibers displaying catecholamine fluorescence, NPY-IR, CGRP-IR, and SP-IR was observed. By postnatal day 21, the distribution of the above fibers had reached essentially adult levels, although the density of epidermal-associated CGRP-IR and SP-IR fibers was significantly greater than in the adult. The following were not evident in thoracic hairy skin at any timepoint examined: choline acetyltransferase activity, acetylcholinesterase histochemical staining or immunoreactivity, fibers displaying immunoreactivity to vasoactive intestinal peptide, cholecystokinin, or leucine-enkephalin. The present study demonstrates that the thoracic hairy skin in developing and adult rats receives an abundant sympathetic catecholaminergic and sensory innervation, but not a cholinergic innervation.