Elsevier

Brain Research

Volume 953, Issues 1–2, 25 October 2002, Pages 63-72
Brain Research

Research report
Targeted inactivation of the neurotensin type 1 receptor reveals its role in body temperature control and feeding behavior but not in analgesia

https://doi.org/10.1016/S0006-8993(02)03271-7Get rights and content

Abstract

Three subtypes of neurotensin receptor have been described, two members of the heptahelical transmembrane domain G protein-coupled receptor superfamily NT-1R and NT-2R, and NT-3R unrelated to this family. We have generated NT-1R deficient (NT-1R−/−) mice. NT-1R−/− mice were born at the expected Mendelian frequency without obvious abnormalities and they were fertile. The NT-induced analgesia on the writhing induced by phenyl-p-benzoquinone administration remained at wild-type levels in the NT-1R−/− mice demonstrating that the NT-1R is not implicated in the analgesic effect of NT in this test. The NT-1R−/− mice were hyperthermic; their body temperature was not affected by intracerebroventricular (i.c.v.) administration of NT, contrasting with the hypothermia induced in NT-1R+/+ mice. NT-1R−/− mice showed a small significant increase in body weight compared to the NT-1R+/+ congeners as early as 10 weeks after birth, correlated with a higher food intake. NT-1R−/− mice showed similar spontaneous locomotion to the control littermates, but did not respond to i.c.v. NT-induced hypolocomotion. I.c.v. injection of NT inhibited feeding in fasted wild-type mice, but had no effect on feeding of the NT-1R−/− mice. I.c.v. administration of the orexigenic neuropeptide Y (NPY) stimulated feeding to the same extent in both wild-type and NT-1R−/− mice. This analysis of NT-1R-deficient mice shows that the NT-1R does not play a role in NT-induced analgesia, but that it is clearly implicated in thermal and feeding regulation, weight control, and NT-induced hypolocomotion.

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.

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