Involvement of the neurotensin receptor 1 in the behavioral effects of two neurotensin agonists, NT-2 and NT69L: Lack of hypothermic, antinociceptive and antipsychotic actions in receptor knockout mice

doi:10.1016/j.euroneuro.2009.01.004

European Neuropsychopharmacology

Volume 19, Issue 7, July 2009, Pages 466-475

https://doi.org/10.1016/j.euroneuro.2009.01.004 Get rights and content

Abstract

Neurotensin (NT) is a neuropeptide implicated in the pathophysiology of schizophrenia and in mediating the efficacy of antipsychotic drugs. NT is also involved in the regulation of body temperature and pain sensitivity. Using neurotensin receptor 1 (NTR1) knockout (KO) and wild-type (WT) mice, these studies evaluated the involvement of NTR1 in the behavioral responses produced by peripheral administration of NT agonists (NT-2 and NT69L). Animals were characterized in paradigms designed to assess hypothermia, antinociception, and antipsychotic-like effects. Under basal conditions, there were no phenotypic differences between NTR1 KO and WT mice. In WT mice, both NTR1 agonists decreased core body temperature (active doses in mg/kg, i.p., for NT-2 and NT69L, respectively: 1 and 3), increased tail withdrawal latencies (1 and 3), produced decreased spontaneous climbing (0.1, 0.3, 1 and 1, 3, 10) and reversed apomorphine-induced climbing (0.3, 1 and 1, 3). In contrast, none of the effects of either agonist were present in KO mice. These results suggest that NTR1: (1) does not play a major role in the control of basal thermoregulation, nociception or psychomotor stimulation in mice (barring possible developmental plasticity), (2) does mediate these behavioral responses to NT agonists, and (3) may play a role in the potential antipsychotic effects of these agonists.

Introduction

A role for neurotensin (NT) in the pathophysiology of schizophrenia is supported by decreased brain and cerebrospinal fluid levels of NT in schizophrenic patients (Breslin et al., 1994, Garver et al., 1991, Lahti et al., 1998, Wolf et al., 1995). The magnitude of the deficit in cerebrospinal fluid levels was highly correlated with symptom severity, with NT levels showing normalization in drug-responsive patients (Widerlov et al., 1982, Garver et al., 1991). Consistent with this effect, antipsychotic administration (acute and chronic) increases NT concentration (Govoni et al., 1980), NT mRNA expression (Merchant et al., 1992), and NT release (Radke et al., 1998) in limbic regions of the brain. Further, haloperidol administration increases NT binding in the substantia nigra (Uhl and Kuhar 1984), and increases mRNA expression of neurotensin1 receptors (NTR1) in the substantia nigra and ventral tegmental area (Bolden-Watson et al., 1993). Atypical antipsychotics like clozapine increase NT release only in the nucleus accumbens (Radke et al., 1998). Such differential release (limbic over striatal) may contribute to the lowered extrapyramidal side effect (EPS) liability of atypical antipsychotics (ibid).

Given the diversity of NT receptor subtypes, several studies have attempted to determine the individual roles of these receptors in both the physiological and antipsychotic-like effects of NT. These efforts have been hampered by the lack of subtype selective antagonists and many issues remain unresolved. For example, antisense studies using rats implicate NTR1 in both the hypothermic and antinociceptive (hot plate) effects of NT (Tyler et al., 1999b), but NTR2 antisense studies using mice suggest a role for NTR2 in the antinociceptive (writhing) effects of NT (Dubuc et al., 1999). Recently, a NTR1 knockout mouse was produced that lacked the hypothermic, but not the antinociceptive (writhing), response to centrally administered NT (Remaury et al., 2002). However, another group reported a loss of both the hypothermic and the antinociceptive (hot plate) effects of centrally administered NT (and peripherally administered NT-2) in such knockouts, implicating NTR1 in both of these effects (Pettibone et al., 2002). Therefore, the identity of the receptor subtype involved in the antinociceptive effects of NT is still unclear. Further, the involvement of the NTR1 in the potential antipsychotic effects of NT agonists has yet to be investigated in these KO mice.

The purpose of this study was to determine the involvement of NTR1 in the antipsychotic-like effects of two stable, brain penetrant NT analogs (NT-2 and NT69L) using the NTR1 knockout mouse. The knock-out of NTR1 was verified by RT-PCR and [¹²⁵I]Tyr³-Neurotensin_(1–13) radioligand binding studies in brain membranes. Mice were assayed in the apomorphine-induced climbing test, a common preclinical screen for antipsychotic efficacy in rodents (Jolicoeur et al., 1991 and Jolicoeur et al., 1993, Cusack et al., 2000, Gudasheva et al., 1998). Additionally, the hypothermic and antinociceptive effects of the agonists were investigated (using a further model of thermal nociception, Tail Immersion) to confirm and /or extend previous reports (e.g. Maeno et al., 2004, Sarhan et al., 1997). As these later behaviors are also frequently affected by compounds with antipsychotic activity (Binder et al., 2001) these studies provide additional evaluation of therapeutic potential.

Section snippets

Generation of NTR1 Null Mice

A targeting vector was created to include a 1.2 kb BamHI fragment as the 5′ arm, a Lox P flanked neomycin resistance gene (neo) for positive selection, a 6 kb StuI-EcoRV fragment as the 3′ arm, and the HSV-TK gene for negative selection (Fig. 1a). This vector was linearized with NotI and introduced into 129/Ola-derived E14-1 embryonic stem cells by electroporation. The clones were selected in 310 μg/ml G418 (Gibco BRL, Gaithersburg, MD) and 2 μM gancyclovir (Roche Pharmaceuticals).

In vitro measures

Heterozygous (NTR1 +/−) F1 offspring were intercrossed to generate F2 mice, which were genotyped by PCR (Fig. 1c). All three genotypes were obtained at the expected Mendelian frequency (1:2:1). Both male and female KO mice were viable and fertile, with no significant hematologic, serologic, urologic, gross, or microscopic differences observable between the WT and the KO animals (n = 3/genotype/gender).

The absence of NTR1 mRNA in the knockouts was verified by RT-PCR (Fig. 1d). Absence of NTR1

Discussion

We found that the ability of NT agonists to attenuate apomorphine-induced climbing was lost in KO animals, suggesting that NTR1 may be a required receptor for producing antipsychotic-like effects of NT agonists. We also confirmed the roles of NTR1 in mediating hypothermic effects of NT agonists and their role in antinociception in the Tail Immersion test.

Role of the funding source

All sudies were funded by Roche Palo Alto.

Contributors

Jordan supervised the behavioral studies in the lab and was the main contributor to the manuscript; Jannette Sutton performed the in vivo studies; Amy Berson, Xiaosu Wu and Joyce Kwan performed the in vitro studies; Zhen Pang was responsible for the generation and maintenance of the mouse colony; Donald Button was the program leader for NTR1 agonists against schizophrenia and Rudy Schreiber was the senior scientist responsible for the support of CNS projects by behavioral pharmacology studies.

Conflict of interest

All authors were employed by Roche Palo Alto during the course of the studies.

Acknowledgements

The authors gratefully wish to acknowledge the contributions of the following individuals, who assisted in the various aspects of these experiments or in the preparation of this manuscript; Debra Cockayne, Ph.D. for her invaluable experience and advice in generating knockouts, Eliza Saclolo, B.A. for her tireless efforts in breeding these mice, Cory Freedland, Ph.D., Jeffrey Vivian, Ph.D. and Doug Bonhaus, Ph.D. for their critical review and editorial suggestions regarding the manuscript, and

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Involvement of the neurotensin receptor 1 in the behavioral effects of two neurotensin agonists, NT-2 and NT69L: Lack of hypothermic, antinociceptive and antipsychotic actions in receptor knockout mice

Abstract

Introduction

Section snippets

Generation of NTR1 Null Mice

In vitro measures

Discussion

Role of the funding source

Contributors

Conflict of interest

Acknowledgements

Biol. Pysch.

Schizophr. Res.

Brain Res.

J. Neurosci.

Pharmacol. Biochem. Behav.

Brain Res. Bull

J. Pharmacol. Exp. Ther.