Research reportImmunohistochemical characterization of localization of long-form leptin receptor (OB-Rb) in neurochemically defined cells in the ovine hypothalamus☆
Introduction
The central regulation of food intake, bodyweight homeostasis and neuroendocrine function involves a number of neuropeptides, which are expressed in different nuclei/areas of the hypothalamus. For example, cells that produce neuropeptide Y (NPY), pro-opiomelanocortin (POMC) (and its products), melanin-concentrating hormone (MCH), galanin (GAL), corticotropin-releasing factor (CRF), orexin (ORX) and the opioids have been implicated in the regulation of feeding behavior as well as neuroendocrine function [1], [2], [3], [5], [12], [25], [60], [61]. Alterations in nutritional status lead to a shift in the level of expression of mRNA for various neuropeptides in the hypothalamus [2], [27], [39], [63].
Leptin is secreted by adipocytes and is involved in the regulation of energy homeostasis and neuroendocrine function [1], [3], [4], [15], [26], [36], [55], [74]. The weight-reducing effects of leptin are the result of central action, effected via leptin receptors [30], [31], [48] in various neuropeptidergic neurons localized in different hypothalamic nuclear groups and regions [7], [8], [33], [34], [35]. The leptin receptor exists in several isoforms [45] and immunohistochemical and in situ studies show widespread distribution of both short (OB-Ra) and long (OB-Rb) isoforms throughout the brain [14], [23], [24], [34], [42]. OB-Rb is considered to be the major signaling form of the receptor, activating the JAK/STAT pathway [11], [30], [31], whereas OB-Ra is thought to be involved in the transport of leptin across the blood–brain barrier [72].
Using immunohistochemistry, the presence of the leptin receptor in various neuropeptidergic cells of the rat brain has been reported [34], but the antibody used did not distinguish between the long and short isoforms of the receptor. Co-localization of OB-Rb has been observed in NPY and POMC neurons in the rat [7], [8] and NPY neurons in the sheep [75], but specific distribution of this isoform is not known for other neuropeptides. Thus, the degree of co-localization of OB-Rb with various neuropeptides is not fully investigated in any species. Localization of OB-Rb protein to different neuronal populations will provide a greater insight into the regulatory actions of leptin within the brain. The present study was undertaken to determine the extent of the localization of OB-Rb to various neurochemically defined cells in the ovine hypothalamus.
Section snippets
Animals and tissue collection
All procedures and tissue collections were carried out with the prior approval of the Animal Experimentation Ethics Committee of Monash University and Victorian Institute of Animal Science.
Four adult Corriedale castrated (male) sheep of similar age (2.5 years) and weight (52–58 kg) were fed on pasture prior to use. The tissue was collected in January 2000 as previously reported [42]. Briefly, the sheep were administered 25 000 IU of heparin (i.v.) and killed 5 min later by an overdose of sodium
Results
No specific OB-Rb-like immunoreactivity (OB-Rb-ir) was observed after pre-absorption of the antisera with the immunogenic peptide (Fig. 1A–C), incubation of the tissue with pre-immune normal guinea pig serum (Fig. 1D–F) or omission of the primary or secondary antibody from the incubation medium. Specific immunoreactivity for OB-Rb was present in the membranes of the cells as well as in the cytoplasm and the cell processes (Fig. 1G–L) and the pattern of immunostaining was similar with both
General considerations
The present study provides semi-quantitative estimates of expression of OB-Rb-ir in neurochemically defined cells in the ovine hypothalamus. Both antisera, which specifically recognize the long form of the leptin receptor, demonstrated a similar pattern of immunostaining and distribution of cells contained OB-Rb-ir in the ovine hypothalamus. No OB-Rb-ir was observed after the relevant control procedures, strongly suggesting that OB-Rb-ir is specific for the long form of leptin receptor. Cells
Conclusion
We have characterized the expression of OB-Rb-ir in a variety of neuropeptidergic neurons in the ovine hypothalamus. This confirms that leptin interacts with many neurochemical systems in the hypothalamus that regulate food intake, bodyweight and energy homeostasis. Further, distribution and co-localization of OB-Rb-ir within different nuclear groupings and subsets of neuropeptides producing neurons provide evidence that leptin is directly involved in the regulation of centrally mediated
Acknowledgements
We thank Mr. Bruce Doughton and Ms. Karen Briscoe for animal care. We also appreciate the technical assistance of Sue Pankridge in preparing the figures. Supported by the National Health and Medical Research Council of Australia.
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The results of the present study were presented, in part, at the 30th Annual Meeting of the Society for Neuroscience, November 4–9, 2000, New Orleans, LA, USA, Abstract no. 440.1.