Research reportTargeted inactivation of the neurotensin type 1 receptor reveals its role in body temperature control and feeding behavior but not in analgesia
Introduction
Neurotensin (NT) is a tridecapeptide originally isolated from calf hypothalamus [4]. High concentrations of neurotensin are found in numerous areas of the central nervous system (CNS) where it plays a role as neurotransmitter and neuromodulator [17] and in peripheral tissues where it can be considered as a hormone producing a diversity of pharmacological effects [14]. When NT is administered i.c.v. a variety of CNS effects are observed, including decreased locomotor activity, hypothermia, antinociception, and reduced food consumption. The hypolocomotion and hypothermia are thought to be associated with the modulatory effect of NT on the dopaminergic systems [21]. Very early anatomical and pharmacological data suggested a close connection between NT and dopaminergic systems, a connection that has increased the interest for NT-containing circuits because of their potential implications in abnormal behavior and neuropsychiatric disorders. The periaqueductal gray, which is the major brain region involved in the processing of nociceptive information, contains NT and a high density of NT receptors and is the region that most probably is involved in the antinociception of NT [22]. The regulation of feeding behavior by NT is suggested by several studies, for example, NT secretion is triggered by fat ingestion, it inhibits food intake when centrally administrated, and it mediates the feeding inhibitory activity of leptin [1]. The paraventricular nucleus and the ventromedian nucleus of the hypothalamus, where NT and NT receptor-containing neurons are present, are candidate areas for the anorexic activity of NT [42].
In peripheral tissues NT and NT receptors have been described primarily in the digestive tract where they play a role in the stimulation of pancreatobiliary secretion and increasing the contractile capacity of the colon [35]. NT and NT receptors have also been implicated in the modulation of lymphocyte function [11] and several reports recently pointed out that NT might play a growth factor-like activity facilitating the proliferation of several types of cancer cells [10]. The pharmacological effects displayed by NT are mediated by at least three subtypes of receptor, NT-1R [18], [34], [40] and NT-2R [41], both members of the heptahelical transmembrane domain G protein-coupled receptor superfamily, and NT-3R, which is identical to gp95/sortilin, with only a single transmembrane domain [19].
Several non-peptide antagonists and peptide agonists for the NT receptors have been used in an attempt to understand the relationship between the different biological activities and the different receptors. However, clear-cut information from the use of these ligands is complicated by several factors: (i) NT is an antagonist for human NT-2R [41], but is a partial agonist for rat NT-2R [43]; (ii) in vitro NT-2R is constitutively active [29], although it is unknown whether it has this property in vivo; (iii) the high affinity, potent NT-1R antagonists SR 48692 and SR 142948 are potent agonists for NT-2R [41]. To better understand the role played by each of the NT receptors we report here the construction of mice lacking the NT-1R and an analysis of some of the CNS effects resulting from the deletion.
Section snippets
Drugs
[125I]Monoiodo-Tyr3NT and [125I]peptide PYY (2000 Ci/mmol) were purchased from NEN (Boston, MA, USA). Unlabelled NT, NPY(1–36), bovine serum albumin (BSA), 1,10-ortho-phenanthroline and phenyl-p-benzoquinone (PBQ) were from Sigma (St. Louis, MO). For in vivo tests NT and NPY were dissolved in saline and PBQ in 5% ethanol in saline. The anti-histamine agent, levocabastine, was obtained from Janssen Pharmaceutica (Geel, Belgium).
Animals
Female and male wild-type C57BL/6j mice (Iffa Credo, L’Arbresle,
Construction of NT-1R−/− mice
The coding region of the NT-1R gene consists of four exons as a single copy in the H region of mouse chromosome 2 [15]. Exon 1, containing the initiation codon and 700 bp coding for the first four transmembrane regions of the receptor, was replaced in C57BL/6 embryonic stem cells by homologous recombination with a green fluorescent protein gene and a neomycin resistance gene cassette (Fig. 1a). Correctly targeted embryonic stem cell clones identified by PCR analysis were used to create
Discussion
At birth the NT-1R−/− mice exhibited a phenotype apparently identical to that of their wild-type littermates. It has been suggested that NT plays a role in cortical development in the rat [25], [32] on the grounds that NT-1R is present in the cortex in very high concentrations during late gestation. Although the apparent normal development of the NT-1R−/− mice did not provide support for such a role in the mouse, further work is needed to completely exclude the involvement of NT-1R in
Acknowledgements
We thank Bruno Verschuere for animal care and David Graham, Jean-Pierre Maffrand and Bernard Scatton for suggestions and critical reading of the manuscript.
References (44)
Neuropeptides and obesity
Nutrition
(2000)- et al.
Evidence that hypothalamic neurotensin signals leptin effects on feeding behavior in normal and fat-preferring rats
Biochem. Biophys. Res. Commun.
(1998) - et al.
The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami
J. Biol. Chem.
(1973) - et al.
Neurotensin: antinocisponsive action in rodents
Eur. J. Pharmacol.
(1979) - et al.
Differential ontogenetic patterns of levocabastine-sensitive neurotensin NT2 receptors and of NT1 receptors in the rat brain revealed by in situ hybridization
Dev. Brain Res.
(1999) - et al.
Neurotensin in the human brain
Neuroscience
(1987) - et al.
The 100-kDa neurotensin receptor is gp95/sortilin, a non-g-protein-coupled receptor
J. Biol. Chem.
(1998) - et al.
Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization
FEBS Lett.
(1996) The interaction of neurotensin with dopaminergic pathways in the central nervous system: basic neurobiology and implications for the pathogenesis and treatment of schizophrenia
Psychoneuroendocrinology
(1986)- et al.
The ontogeny of brain neurotensin receptors studied by autoradiography
Neuroscience
(1988)