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Results for “Colocalization”

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PubMed2003

Two neuropeptides colocalized in a command-like neuron use distinct mechanisms to enhance its fast synaptic connection.

In many neurons more than one peptide is colocalized with a classical neurotransmitter. The functional consequence of such an arrangement has been rarely investigated. Here, within the feeding circuit of Aplysia, we investigate at a single synapse the actions of two modulatory neuropeptides that are present in a cholinergic interneuron. In combination with previous work, our study shows that the command-like neuron for feeding, CBI-2, contains two neuropeptides, feeding circuit activating peptide (FCAP) and cerebral peptide 2 (CP2). Previous studies showed that high-frequency prestimulation or repeated stimulation of CBI-2 increases the size of CBI-2 to B61/62 excitatory postsynaptic potentials (EPSPs) and shortens the latency of firing of neuron B61/62 in response to CBI-2 stimulation. We find that both FCAP and CP2 mimic these two effects. The variance method of quantal analysis indicates that FCAP increases the calculated quantal size (q) and CP2 increases the calculated quantal content (m) of EPSPs. Since the PSP amplitude represents the product of q and m, the joint action of the two peptides is expected to be cooperative. This observation suggests a possible functional implication for multiple neuropeptides colocalized with a classical neurotransmitter in one neuron.

Action Potentials↗

PubMedDate not supplied

Urokinase plasminogen activator colocalizes with CD25+ cells in atherosclerotic vessels.

Proteolytic activity in vascular tissue is necessary for cellular migration, remodelling of extracellular matrix and the development of atherosclerotic lesions. Inflammatory cells, mainly macrophages, are numerous in atherosclerotic plaques and may synthesize and secrete proteolytic enzymes. The principal activator of plasminogen in tissues is urokinase plasminogen activator (u-PA). To determine if an activated phenotype of inflammatory cells colocalizes with local expression of u-PA in atherosclerotic vessels, vascular biopsies from 15 patients with peripheral atherosclerotic disease were analyzed by immunohistochemistry on consecutive sections. Anti-CD68 antibodies were used as markers for macrophages and were positive in 14/15 specimens. Anti-CD25 (interleukin-2 receptor-alpha) antibodies were used to identify inflammatory cells with an activated phenotype and were positive in 9/14 CD68+ specimens. The same 9 specimens were positive for u-PA. A positive reaction for u-PA was found only in specimens with CD25+ cells. Specimens with positive reactions for all three antibodies were further analyzed with computer-assisted image analysis. The colocalization with u-PA was higher for CD25 compared to CD68 in all specimens. Mean percentage of the u-PA-positive area in regions positive for cellular markers was 52% (SEM 6%) for CD25 and 19% (SEM 5%) for CD68 (p < 0.01). The results indicated that the activation of macrophages in atherosclerotic vessels may modulate local proteolysis and be of importance in plaque development and stability.

Aged↗

PubMed1991

Colocalization of delta sleep inducing peptide and luteinizing hormone releasing hormone in neurosecretory vesicles in rat median eminence.

The colocalization of immunoreactivities similar to delta sleep inducing peptide (DSIP) and luteinizing hormone releasing hormone (LH-RH) was investigated by light and electron microscopic immunocytochemistry in the rat median eminence. At the light microscopic level, DSIP and LH-RH immunostained fibers, and varicosities exhibited a similar distribution pattern throughout the median eminence. Immunoreactive axons were mainly found in the lateral part of the external layer. Using an elution-restaining technique, the coexistence of LH-RH and DSIP immunoreactivities was observed in most labelled axons. To determine the intracellular localization of DSIP and LH-RH, we used double immunocytochemical labelling with species-specific antibodies and secondary antibodies conjugated to different sizes of gold particles. The two peptides were found colocalized in single axons. Immunoreactive terminals frequently showed direct membrane apposition with tanycyte processes but rare contacts with portal capillaries. No staining was observed in tanycytes and ependymal or glial elements. Moreover, we could demonstrate that LH-RH and DSIP (or a closely related molecular form) are contained not only in the same axons, but also in the same approximately 100-nm dense-core vesicles, suggesting cosecretion of these peptides.

Animals↗

PubMed1997

Calretinin in the rat pituitary: colocalization with thyroid-stimulating hormone.

The purpose of this study was to examine the distribution of calretinin immunoreactivity (CR) in the male rat pituitary gland by immunofluorescence microscopy. CR was found in cells of the anterior pituitary and in granules in the posterior pituitary. In the intermediate lobe, nerve fibers in close proximity to the melanotropes were CR-immunoreactive (CR-ir). Fine CR-ir varicose fibers were also observed in the anterior and posterior pituitary. Colocalization studies revealed that the majority of the CR-containing cells of the anterior pituitary also contained thyroid-stimulating hormone (TSH). These CR/TSH cells represented about 32% of the thyrotrope population. Following thyroidectomy, a massive increase in both the number of CR-ir cells and in the expression of CR mRNA was observed in the anterior pituitary. Thyroxine treatment, however, resulted in a reduction in the number and size of the CR-ir cells in the same lobe. In the intermediate lobe, CR-ir was colocalized with tyrosine hydroxylase (TH) immunoreactive dopaminergic fibers. These intermediate lobe fibers disappeared following pituitary stalk section, as did the CR/TH fibers and the CR-ir granular material in the posterior pituitary. The findings in the anterior pituitary suggest that consideration be given to the idea that CR might function in the synthesis and/or release mechanism of TSH in thyrotropes and that its expression is modulated by the hypothalamo-pituitary-thyroid axis.

Animals↗

PubMed1998

C-reactive protein frequently colocalizes with the terminal complement complex in the intima of early atherosclerotic lesions of human coronary arteries.

There is increasing evidence that complement activation may play a role in atherogenesis. Complement proteins have been demonstrated to be present in early atherosclerotic lesions of animals and humans, and cholesterol-induced atherosclerotic lesion formation is reduced in complement-deficient animals. Potential complement activators in atherosclerotic lesions are now a subject matter of debate. C-reactive protein (CRP) is an acute-phase protein that is involved in inflammatory processes in numerous ways. It binds to lipoproteins and activates the complement system via the classic pathway. In this study we have investigated early atherosclerotic lesions of human coronary arteries by means of immunohistochemical staining. We demonstrate here that CRP deposits in the arterial wall in early atherosclerotic lesions with 2 predominant manifestations. First, there is a diffuse rather than a focal deposition in the deep fibroelastic layer and in the fibromuscular layer of the intima adjacent to the media. In this location, CRP frequently colocalizes with the terminal complement complex. Second, the majority of foam cells below the endothelium show positive staining for CRP. In this location, no colocalization with the terminal complement proteins can be observed. Our data suggest that CRP may promote atherosclerotic lesion formation by activating the complement system and being involved in foam cell formation.

Animals↗

PubMed1999

Cyclooxygenase-2 is widely expressed in atherosclerotic lesions affecting native and transplanted human coronary arteries and colocalizes with inducible nitric oxide synthase and nitrotyrosine particularly in macrophages.

Inflammation appears to have a major role in the development of atherosclerotic lesions affecting native and transplanted coronary arteries. The subsequent risk of plaque rupture and acute ischemic events correlates with the degree of inflammation and may be modified by aspirin, an anti-inflammatory cyclooxygenase inhibitor. Cyclooxygenase-2 (Cox-2) and inducible nitric oxide synthase (iNOS) are involved in the inflammatory response via the rapid and exaggerated production of prostanoids and nitric oxide, both of which may have proatherosclerotic effects. These effects may be mediated by the formation of peroxynitrite in the case of nitric oxide and involve "cross talk" between the two enzyme systems. This study aimed to investigate native and transplant atherosclerosis for the presence and distribution of Cox-2 and iNOS. Immunocytochemical studies were performed on atherosclerotic lesions from patients with native (n=12) and transplant (n=5) coronary disease by using antibodies to Cox-2, iNOS, and nitrotyrosine (an indicator of peroxynitrite production). Control tissue was obtained from unused donor hearts and at the time of autopsy. Cox-2 and iNOS colocalized predominantly in macrophages/foam cells in both types of atherosclerosis. Cox-2 expression was also detected in medial smooth muscle cells and endothelial cells, including those of the vasa vasorum. Nitrotyrosine was found in the same distribution as that of iNOS and was colocalized with Cox-2 in macrophages. Cox-2 and iNOS are coexpressed in native and transplant atherosclerosis, possibly allowing for interaction between the enzymes and suggesting an alternative mechanism for the benefits of aspirin via inhibition of Cox-2 activity.

Animals↗

PubMed1996

Endogenous retinoic acid signaling colocalizes with advanced expression of the adult smooth muscle myosin heavy chain isoform during development of the ductus arteriosus.

During fetal development, a specialized vessel the ductus arteriosus, shunts blood from the pulmonary artery to the aorta, thus bypassing the lungs. The ductus differs primarily from the great vessels in that it is a muscular rather than an elastic artery, and the etiology of this differential development remains controversial. We present evidence that retinoic acid (RA) may contribute to the unique muscle phenotype of the ductus arteriosus. Using a transgenic mouse carrying an RA response element-lacZ transgene that expresses beta-galactosidase (beta-gal) in response to endogenous RA signals during embryonic and fetal development, we observe a strong beta-gal signal in the ductus arteriosus. By immunofluorescence, this signal colocalizes with the expression of the adult-specific smooth muscle myosin heavy chain isoform, SM2. The beta-gal signal is present throughout fetal development and persists in the neonate until the ductus arteriosus is completely closed. beta-Gal-positive cells are first detected by immunofluorescence at 13.5 days postcoitum (dpc) in the mesenchyme surrounding the ductus. By 15.5 dpc, very intense beta-gal staining localizes to the ductus arteriosus but is absent or minimal in the pulmonary trunk and aortic arch; by 17.5 dpc, the smooth muscle layers of the tunica media in the ductus arteriosus exhibit positive beta-gal staining. Immunostaining with antibodies against smooth muscle myosins shows that, while SM1 is expressed in all embryonic vessels, SM2 is precociously expressed in the ductus arteriosus. Furthermore, SM2 expression can be detected in the ductus as early as 15.5 dpc. In the neonate, the beta-gal signal persists in the smooth muscle layer of the ductus and immunostaining colocalizes with SM2 expression. These data suggest that RA may play a role in inducing and maintaining smooth muscle differentiation in the developing ductus arteriosus and may promote precocious expression of the adult vascular phenotype.

Animals↗

PubMed2003

Carboxyfluorescein diacetate succinimidyl ester facilitates cell tracing and colocalization studies in bioartificial organ engineering.

BACKGROUND: We demonstrate a method that includes colocalization studies to analyze cell suspensions after isolation and to characterize 3-dimensional grafts consisting of cells and matrix in vitro and in vivo. MATERIALS AND METHODS: Neonatal rat cardiomyocytes were labelled by CFDA-SE after harvest. Cells in the isolated cell suspension, the embodied cells in the seeded scaffolds were characterized measuring features such as viability and distribution of the cell types. RESULTS: Selective cell count revealed high yields of viable cardiomyocytes. After seeding cells in collagen matrix, viability of the cells decreased gradually in the time process in vitro. Histology of implanted bioartificial myocardial tissue detected viable cardiomyocytes within the graft. CONCLUSION: Using colocalization histology we could label and track cells within the bioartificial myocardial tissue graft in vitro and post implant and assess viability and distribution.

Animals↗

PubMed2002

Fractional attachment of CD47 (IAP) to the erythrocyte cytoskeleton and visual colocalization with Rh protein complexes.

Interactions of CD47 and RhAG and the Rh proteins are visualized between one another and with the cytoskeleton of intact erythrocytes. In a first study, CD47 is labeled with a phycoerythrin (PhE)- tagged antibody, which generates discrete spots that reflect induced clusters of CD47. Rh and RhAG colocalize with each other and to these induced clusters, whereas Band 3 and glycophorin C remain more homogeneously dispersed on the cell periphery. In a second study, red cells are aspirated into a micropipette, and immunofluorescent maps of the surface gradients that develop for CD47 and RhAG determine cytoskeletal connectivity. CD47 and RhAG gradients on normal red cells prove to be nearly identical and also appear intermediate to those found for the fluid bilayer and network-linked glycophorin C. Similar gradients are obtained for CD47 on Rh(null) cells, suggesting that linkage of CD47 to the spectrin-actin skeleton is independent of Rh or RhAG and is not affected by CD47's reduced surface expression on these cells. The results show that CD47 colocalizes with Rh and RhAG but is fractionally attached to the red cell membrane skeleton independent of these and other major integral membrane proteins involved in cytoskeletal attachment. The results imply a homogeneous base distribution of CD47, restrained by cytoskeleton linkages, plus a smaller fraction of CD47, which is able to diffuse in the membrane.

Anion Exchange Protein 1, Erythrocyte↗

PubMed2001

Expression and colocalization of cytokeratin 1 and urokinase plasminogen activator receptor on endothelial cells.

The cellular localization of human cytokeratin 1 (CK1), urokinase plasminogen activator receptor (uPAR), and gC1qR, high-molecular-weight kininogen (HK)-binding proteins on endothelial cells, was determined. CK1 was found on the external membrane of nonpermeabilized endothelial cells by immunoperoxidase staining, immunofluorescence, and transmission electron microscopy using immunogold. Human umbilical vein endothelial cells (HUVECs) had 7.2 +/- 0.2 x 10(4) specific CK1 membrane sites/cell by (125)I-F(ab')(2) anti-CK1 antibody binding. Flow cytometry studies confirmed the presence of CK1, uPAR, and gC1qR on HUVECs. On laser scanning confocal microscopy and transmission electron microscopy, CK1 and uPAR, but not gC1qR, colocalized on the cell surface of HUVECs. The HUVEC surface distribution of these proteins was distinctly different from that for von Willebrand factor. In competitive inhibition experiments, anti-CK1, anti-uPAR, or anti-gC1qR blocked both biotin-HK binding and prekallikrein (PK) activation on HUVECs with an inhibitory concentration of 50% (IC(50)) of 300 to 350 nM, 50 to 60 nM, or 35 to 100 nM, respectively. Also, antibodies to uPAR and gC1qR each inhibited 86% of kallikrein-mediated, 2-chain urokinase plasminogen activation, whereas antibodies to CK1 only inhibited 24% of plasminogen activation. On HUVECs, CK1 and uPAR, but not gC1qR, colocalized to be a multiprotein receptor complex for HK binding, PK activation, and 2-chain urokinase plasminogen activation.

Amino Acid Sequence↗

PubMed2002

Colocalization of progesterone receptors in parvicellular dynorphin neurons of the ovine preoptic area and hypothalamus.

Recent evidence suggests that the dynorphin-kappa receptor opioid system acts to mediate the inhibitory effect of progesterone (P) on GnRH pulse frequency during the luteal phase of the ovine estrous cycle. It is known that progesterone receptors (PRs) are required for the actions of P on GnRH secretion. Therefore, if P acts directly on dynorphin (DYN) neurons, then these neurons should contain PRs. To test this hypothesis, we used a dual-label immunoperoxidase procedure to visualize PRs and DYN in the preoptic area (POA) and hypothalamus of ovary-intact ewes killed during the luteal phase of the estrous cycle. The PR was colocalized in more than 90% of parvicellular DYN neurons in the POA, anterior hypothalamus (AHA), and arcuate nucleus (ARC). By contrast, none of magnocellular DYN cells of the paraventricular and supraoptic nuclei coexpressed immunoreactive PRs. The high percentage of colocalization of PRs in parvicellular DYN cells of the POA, AHA, and ARC suggests that these cells are prime targets of P. In addition, DYN cells in the ARC, but not the POA or AHA, were found to receive synaptic inputs from DYN-positive axon terminals. This observation raises the possibility that an ultrashort feedback loop controls the release of DYN from ARC neurons.

Animals↗

PubMed1991

Immunocytochemical localization of protein kinase-C subtypes in anterior pituitary cells: colocalization in hormone-containing cells reveals heterogeneity.

We have examined the distribution and colocalization of protein kinase-C (PKC) in cultured rat anterior pituitary cells by light microscopic immunocytochemistry using monoclonal antibodies to group A rat brain PKC subspecies. Type I (PKC gamma) was not detected in the cells, in line with the assertion that the gamma-enzyme is expressed specifically in central nervous tissues. The other subspecies recognized by the antibodies (PKC beta and PKC alpha) were present throughout the cytoplasm in a diffused pattern, while the nuclei were apparently unstained. The number of cells stained with the antibodies in juvenile animals (12 days old) increased rapidly with age and reached a plateau between adult (5-month-old) and older (1-yr-old) rats. Type II (PKC beta) was the predominant subspecies detected in anterior pituitary cells. Double immunofluorescence staining techniques enabled the colocalization of PKC with various anterior pituitary cell types. Surprisingly, not all of the hormone-producing cells were stained with the PKC antibodies. Moreover, within the different pituitary cell types, the percentage of PKC-stained cells varied, revealing heterogeneity among the various cell populations. Thus, among somatrophs, mammotrophs, thyrotrophs, ACTH-containing cells, and gonadotrophs, only 9%, 22%, 13%, 44%, and 26%, respectively, reacted with the PKC antibodies. We suggest that activation of pituitary PKC might mobilize only a fraction of the hormone-containing cells.

Animals↗

PubMed1992

Immunocytochemical colocalization of hypothalamic progestin receptors and tyrosine hydroxylase in steroid-treated monkeys.

Progesterone (P)-induced PRL secretion in estradiol (E)-primed monkeys is not due to direct pituitary stimulation, because lactotropes do not express progestin receptors (PR). However, the hypothalamus, particularly the tuberoinfundibular dopaminergic system (TIDA), plays a major role in the regulation of PRL secretion. To determine whether hypothalamic dopamine neurons are progestin target cells, the colocalization of PR and tyrosine hydroxylase (TH), a phenotypic marker of dopaminergic neurons, was examined with double immunocytochemistry. Two methods for visualizing the antigens were applied; the first was a dual peroxidase method, and the second was a peroxidase-alkaline phosphatase method. In addition, the question of whether E induces PR in dopamine neurons was explored. Spayed female monkeys were treated with empty Silastic capsules, E-filled capsules for a period of 28 days, or E capsules supplemented with P capsules for the last 14 days of E treatment. Only the E- plus P-treated monkeys exhibited an increase in serum PRL during the P treatment period. Frontal sections at the level of the optic chiasm and arcuate nucleus were examined for the colocalization of TH and PR. After E treatment, hypothalamic PR-positive cells increased in both intensity and number. Neurons expressing both TH and PR were detected in the rostral hypothalamus, lateral to the third ventricle (A11-rostral) and in a discrete subventricular population (A11-subvent). The lateral population continued caudally (A11-caudal). The A11-subvent population exhibited little steroid regulation. Of the remaining A11 TH neurons, approximately 20% exhibited PR in the spayed and E-treated groups. Addition of P doubled the percentage of PR-containing TH neurons in this group. Although very few TH-positive neurons in the ventral arcuate nucleus contained PR (A12-ventral), many double labeled neurons were observed in the dorsal arcuate region (A12-dorsal). Ventral arcuate TIDA neurons were not regulated by steroids, but E plus P increased PR expression in A12-dorsal. Double labeled cells were rarely seen in the zona incerta (A13) or the emerging ventral tegmental area (A10). In summary, P probably does not act directly on ventral arcuate TIDA neurons to stimulate PRL secretion. However, the frequency of PR-positive dopamine neurons in the A11-rostral, A11-caudal, and A12-dorsal groups increased with E and P treatment. Therefore, the contribution of the PR-positive periventricular dopamine neurons to progestin-stimulated PRL secretion may be important.

Animals↗

PubMed1993

Colocalization of immunoreactive endothelin-1 and neurohypophysial hormones in the axons of the neural lobe of the rat pituitary.

The intracellular localization of immunoreactive endothelin (ET)-1, and its colocalization with vasopressin (VP) and oxytocin (OT) in the rat neural lobe were investigated by immunogold techniques using specific antisera raised against ET-1, VP, and OT. There were two types of axons: the first contained VP-immunolabeled neurosecretory granules, and the second contained OT-immunolabeled neurosecretory granules. A considerable number of the neurosecretory granules in both types of axon were immunolabeled with antibodies against ET-1, although the VP-immunolabeled granules were more heavily labeled with anti-ET-1 antiserum than OT-immunolabeled ones. Double immunogold labeling clearly demonstrated the intragranular colocalization of immunoreactions for ET-1 and VP and that of immunoreactions for ET-1 and OT. These results suggest that ET in the neural lobe may be released concomitantly with neurohypophysial hormones. Its biological significance remains to be elucidated.

Animals↗

PubMed1993

Ultrastructural colocalization of growth hormone binding protein and pituitary hormones in adenohypophyseal cells of the rat.

GH receptor immunoreactivity is widely distributed within the rat pituitary gland, although apart from somatotrophs the cell types with GH receptor immunoreactivity have yet to be identified. It is also unknown whether this immunoreactivity reflects the presence of GH binding proteins (GHBPs) or authentic receptors. The possible colocalization of GHBPs and pituitary hormones in somatotrophs, lactotrophs, gonadotrophs, thyrotrophs, and corticotrophs was therefore examined using immunogold electron microscopy. Pituitary sections were indirectly immunostained with antibodies for GH, PRL, LH, FSH, TSH, or ACTH using gold-labeled immunoglobulin G. The same grids were also immunostained with a polyclonal antibody raised against rat GHBP using protein A conjugated to gold particles of a different size. Some sections were gold-labeled using a monoclonal antibody (MAb 4.3) raised against the unique hydrophobic tail of the GHBP, using gold-labeled immunoglobulin G. The cellular and ultrastructural distribution of immunoreactivity within the pituitary gland was similar after labeling with either GHBP antiserum. In all immunoreactive cells the labeling was most intense in secretory granules. Specific staining was not, however, demonstrated in the nucleus, in contrast with earlier findings using MAb 263. Moreover, while staining with MAb 263 appeared to be ubiquitous, some pituitary cells were not labeled by the polyclonal GHBP antiserum or by MAb 4.3. GHBP immunoreactivity was, however, colocalized with hormones in all pituitary cell-types, although in some cells GHBP immunoreactivity was not present in all secretory granules. These results clearly demonstrate the presence of GHBPs in adenohypophyseal cells, in which they appear to be stored or secreted with GH, PRL, LH, FSH, TSH, and ACTH. The function of GHBPs in the pituitary gland is, however, uncertain.

Adrenocorticotropic Hormone↗

PubMed1997

Thyroid hormone receptor (alpha) distribution in hamster and sheep brain: colocalization in gonadotropin-releasing hormone and other identified neurons.

Thyroid hormones appear to play an important role in the seasonal reproductive transitions of a number of mammalian and avian species. These seasonal transitions as well as the effects of thyroid hormones on the reproductive neuroendocrine axis are mediated by the GnRH system. How thyroid hormones affect the GnRH system is unclear. Double label immunocytochemistry was used to examine GnRH- and other neurotransmitter/neuropeptide-containing neurons for thyroid hormone receptor (alphaTHR) colocalization in two seasonal breeders, the golden hamster and the sheep. AlphaTHR was identified in hamster and sheep brain by Western blot analysis. Furthermore, alphaTHR immunoreactivity was widely distributed in brain and was colocalized in identified populations: GnRH neurons (hamster, 28%; sheep, 46%); dopaminergic neurons of the A14 (hypothalamic) and A16 (olfactory bulb) cell groups, but not in the hypothalamic A13 cell group; and neurophysin-immunoreactive neurons of the supraoptic and paraventricular nuclei. The finding of alphaTHR in GnRH and A14 dopamine neurons provides an anatomical substrate for direct thyroid hormone action on the reproductive neuroendocrine system of these two seasonally breeding species. It remains to be determined whether the GnRH gene itself or the gene of another constituent within the same GnRH neuron is responsive to thyroid hormones.

Animals↗

PubMed1997

Prolactin (PRL) receptors are colocalized in dopaminergic neurons in fetal hypothalamic cell cultures: effect of PRL on tyrosine hydroxylase activity.

This study examined the responsiveness of dopaminergic neurons to PRL and the expression of PRL receptors in fetal hypothalamic cells. Hypothalamic cells were cultured in medium containing 5 or 25 mM potassium (K+) with or without 5% FBS. Rat PRL (rPRL) treatment (10-1000 ng/ml) for 10 days increased tyrosine hydroxylase (TH) activity 1.6- to 1.8-fold in dopaminergic neurons cultured in serum-containing medium with 25 mM K+, but not in defined medium or any medium with 5 mM K+. The rPRL-induced increase in TH activity was observed at 10-1000 ng/ml after both 1 and 10 days of rPRL treatment, whereas 1 ng/ml was not effective. TH activity was not altered after 1-12 h of rPRL treatment (100 ng/ml), but was increased 1.4-fold after 1-3 days and 1.8-fold after 5-10 days. The colocalization of PRL receptors and TH was evaluated by double labeled immunocytochemistry. PRL receptor immunostaining was observed in most TH-immunoreactive cells cultured in either defined or serum-containing medium with or without 10 days of rPRL treatment (100 ng/ml). As assessed by reverse transcriptase-PCR, the long form, but not the short form, of the PRL receptor was expressed in the hypothalamic cells regardless of medium composition, similar to the expression pattern in adult mediobasal hypothalamus from ovariectomized rats. These data indicate that a factor present in FBS imparts PRL responsiveness to hypothalamic dopaminergic neurons in vitro. The effective PRL concentrations and the time course for PRL's action in vitro are within the physiological range in vivo. The colocalization of PRL receptor in dopaminergic neurons provides anatomical evidence for a direct effect of PRL, with the long form of the PRL receptor being the predominant form in the hypothalamic cells.

Animals↗

PubMed1999

Altered expression of agouti-related protein and its colocalization with neuropeptide Y in the arcuate nucleus of the hypothalamus during lactation.

During lactation, the levels of neuropeptide Y (NPY), which plays an important role in mediating food intake, are significantly elevated in a number of hypothalamic areas, including the arcuate nucleus (ARH). To identify additional hypothalamic systems that might be important in mediating the increase in food intake and alterations in energy homeostasis during lactation, the present studies examined the expression of agouti-related protein (AGRP), a recently described homologue of the skin agouti protein. AGRP is found in the hypothalamus and has been suggested to play an important role in the regulation of food intake. In the first experiment, animals were studied during diestrus of the estrous cycle, a stage of the cycle when estrogen levels are basal and similar to lactation, or during days 12-13 postpartum. Lactating animals had their litters adjusted to eight pups on day 2 postpartum. Brain tissue sections were used to measure AGRP messenger RNA (mRNA) levels by in situ hybridization. AGRP mRNA signal was found mostly in the ventromedial portion of the ARH, which has been shown to contain a high density of NPY neurons. A significant increase in AGRP mRNA content was observed in the mid- to caudal portion of the ARH of lactating animals compared with diestrous females. No difference was found in the rostral portion of the ARH. In the second experiment, double-label in situ hybridization for AGRP and NPY was performed in lactating animals to determine the extent of colocalization of the two peptides in the ARH, using 35S-labeled and digoxigenin-labeled antisense complementary RNA probes. It was found that almost all of the NPY-positive neurons throughout the ARH also expressed AGRP mRNA signal. Furthermore, AGRP expression was confined almost exclusively to NPY-positive neurons. Thus, the present study showed that during lactation, AGRP gene expression was significantly elevated in a subset of the AGRP neurons in the ARH. The high degree of colocalization of AGRP and NPY, coupled with previous reports from our laboratory demonstrating increased NPY expression in the ARH in response to suckling, suggests that AGRP and NPY are coordinately regulated and may be involved in the increase in food intake during lactation.

Agouti Signaling Protein↗