Protein components of the blood–brain barrier (BBB) in the mediobasal hypothalamus

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Abstract

The blood–brain barrier (BBB) plays an important role in controlling the access of substances to the brain. Of the circumventricular organs (CVO), i.e. areas that lack a BBB, the median eminence and its close relationship with the hypothalamic arcuate nucleus plays an important role in controlling the entry of blood-borne substances to neurons of the mediobasal hypothalamus. In order to clarify the nature of the BBB in the median eminence–arcuate nucleus complex, we have used immunohistochemistry and antisera to protein components of the BBB—(1) tight junctions, claudin-5 and zona occludens-1 (ZO-1); (2) endothelial cells: (a) all endothelial cells: rat endothelial cell antigen-1 (RECA-1), (b) endothelial cells at BBB: endothelial barrier antigen (EBA), glucose transporter 1 (GLUT1) and transferrin receptor (TfR), and (c) endothelial cells at CVOs: dysferlin; (3) basal lamina: laminin; (4) vascular smooth muscle cells: smooth muscle actin (SMA); (5) pericytes: chondroitin sulfate proteoglycan (NG2); (6) glial cells: (a) astrocytes: glial fibrillary acidic protein (GFAP), (b) tanycytes: dopamine- and cAMP-regulated phosphoprotein of 32 kDA (DARPP-32), (c) microglia: CD11b. Neuronal cell bodies located in the ventromedial aspect of the arcuate nucleus were visualized by antiserum to agouti-related protein (AgRP). The study provides a detailed analysis on the cellular localization of BBB components in the mediobasal hypothalamus. Some vessels in the ventromedial aspect of the arcuate nucleus lacked the BBB markers EBA and TfR, suggesting an absence of an intact BBB. These vessels may represent a route of entry for circulating substances to a subpopulation of arcuate nucleus neurons.

Introduction

The blood–brain barrier (BBB) plays an important role in controlling the access of substances to the brain. It acts as a selective barrier and prevents toxic substances to pass into the central nervous system (CNS). Small lipophilic molecules can pass the BBB freely by diffusion, whereas hydrophilic molecules, such as peptides and proteins, may enter the brain through specific transport mechanisms (see Abbott et al., 2006). The BBB is constituted of endothelial cells that are joined together by junctional complexes and that line cerebral microvessels. The endothelial cells are surrounded by a basal membrane, pericytes, astrocytic perivascular endfeet, microglia and neuronal processes (see Abbott et al., 2006). In larger vessels, the pericytes are replaced by a smooth muscle layer (see Iadecola, 2004). Areas that lack a BBB and contain cell bodies that have access to circulating substances are referred to as circumventricular organs (CVOs) (Ehrlich, 1956). The CVOs include the pineal gland, posterior lobe of the pituitary gland, subfornical organ, organum vasculosum of the lamina terminalis, choroid plexus, area postrema, subcommissural organ and the median eminence of the mediobasal hypothalamus (see e.g. Ganong, 2000).

Intravenous injection of the tracer horseradish peroxidase (HRP) has revealed an accumulation of the blood-borne HRP protein in brain parenchyma around CVOs (Broadwell and Brightman, 1976, Broadwell et al., 1983). The region most heavily inundated with the HRP protein is the ventrobasal hypothalamus above the median eminence, where the HRP reaction product immediately after injection is enriched in the median eminence and a subdivision of the arcuate nucleus, to reach at the level dorsal to the ventromedial hypothalamic nucleus at 2 h post-injection (Broadwell and Brightman, 1976, Broadwell et al., 1983). These results indicate that circulating substances can reach neurons in the mediobasal hypothalamus. The mechanisms, routes of entry or the extent of which hormones reach the parenchyma of the mediobasal hypothalamus, however, remain unclear.

There are two distinct neuronal populations located in the ventral aspect of the arcuate nucleus that possess receptors for circulating hormones (see Morton et al., 2006, Meister, 2007). Neurons in the ventromedial aspect of the nucleus produce the orexigenic peptides neuropeptide Y (NPY) and agouti-related peptide (AgRP). Neurons in the ventrolateral division of the arcuate nucleus produce the anorexigenic peptides α-melanocyte-stimulating hormone (α-MSH), a product from the pro-opiomelanocortin (POMC) precursor, and cocaine- and amphetamine-regulated transcript (CART). Hormones produced in the periphery such as leptin and ghrelin have an influence on energy balance and operate via hypothalamic neurons (see, e.g. Morton et al., 2006, Meister, 2007). They have to reach their neuronal target receptors in the arcuate nucleus in order to convey satiety or hunger. Whether neuronal populations in the arcuate nucleus are directly accessible for blood-borne hormones is still a matter of debate. Neurons of the arcuate nucleus send projections to a large number of regions in the CNS, containing second-order neurons that affect feeding behavior and energy expenditure. Leptin, a protein of 167 amino acids that is predicted not to freely cross the BBB, influences both NPY/AgRP and POMC/CART neurons (Balthasar et al., 2004, Gong et al., 2008; see, e.g. Morton et al., 2006, Meister, 2007). Evidence of a saturable transport system responsible for leptin transport across the BBB has been presented (Banks et al., 1996). However, the existence of vessels in the mediobasal hypothalamus with a weak BBB which leptin may cross on the way to target leptin receptors in the arcuate nucleus should also be considered as an alternative route of entry for leptin into the mediobasal hypothalamus.

We have used immunohistochemistry and antisera to different proteins that represent constituents of the BBB in order to study in detail the cellular localization of markers for the BBB in the arcuate nucleus–median eminence complex and in an attempt to identify any vessels in this region that may not have an intact BBB.

The following antibodies/antisera were used to visualize components of the BBB—(1) tight junctions: claudin-5 and zona occludens-1 (ZO-1); (2) endothelial cells: (a) all endothelial cells: rat endothelial cell antigen-1 (RECA-1), (b) endothelial cells at BBB: endothelial barrier antigen (EBA), glucose transporter 1 (GLUT1) and transferrin receptor (TfR), and (c) endothelial cells at CVOs: dysferlin; (3) basal lamina: laminin (a general vascular marker); (4) vascular smooth muscle cells: smooth muscle actin (SMA); (5) pericytes: chondroitin sulfate proteoglycan (NG2); (6) glial elements: (a) astrocytes: glial fibrillary acidic protein (GFAP), (b) tanycytes: dopamine- and cyclic-adenosine-3′:5′-monophosphate (cAMP)-regulated phosphoprotein of 32 kDA (DARPP-32) and (c) microglia: CD11b.

This study provides a detailed analysis of the cellular localization of proteins that constitute the BBB in the rat mediobasal hypothalamus. The results obtained in this investigation suggest the existence of some vessels in the far ventromedial aspect of the arcuate nucleus that lack protein markers present in vessels that are known to possess a BBB. These vessels may represent a route of entry for circulating substances that regulate energy balance and which influence target neurons of the arcuate nucleus.

Section snippets

Material and methods

All studies were performed in accordance with guidelines from the Swedish National Board for Laboratory Animals and were approved by the local ethical committee. Male Sprague–Dawley rats weighing 150–200 g (Scanbur-BK, Stockholm, Sweden) were used. In order to visualize neuronal cell bodies, some rats were anesthetized with a combination of ketamin (75 mg/kg) + medetomidin (1 mg/kg) i.p. and were treated with an injection of colchicine (120 μg in 20 μl 0.9% NaCl; Sigma, St. Louis, MO, USA) into the

Tight junctions—claudin-5 and ZO-1

Mouse monoclonal antibodies to claudin-5 or ZO-1 combined with rabbit antiserum to the basal lamina marker laminin, a general marker for vessels, revealed the existence of strongly claudin-5- and ZO-1-immunoreactive vessels in the dorsal and lateral parts of the arcuate nucleus, whereas laminin-positive vessels in the ventromedial part of the nucleus showed weaker claudin-5 (Fig. 1A and B) and ZO-1 (Fig. 2A and B) immunoreactivity. There were also a few weakly claudin-5-positive vessels in the

Discussion

This study shows in detail the distribution of proteins that constitute cellular components of the BBB in the mediobasal hypothalamus. Although it is well known that the median eminence belongs to the CVOs, it has not been made clear whether neuronal subpopulations in the nearby arcuate nucleus are fully accessible for circulating substances. In an attempt to find differences and/or similarities in the cellular localization among BBB markers in the median eminence–arcuate nucleus complex, we

Acknowledgements

This research was supported by grants by the European Union Framework Programme VI Integrated Project (LSH-CT2003-503041), The Swedish Research Council (72X-10358), Novo Nordisk Foundation, Funds from Karolinska Institutet and Åhlén-stiftelsen. We thank K. Dürr, Y. Ishii, S. Unnerståle and S. Willumsen.

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