Neurotensin-deficient mice show altered responses to antipsychotic drugs

Abstract

The peptide transmitter neurotensin (NT) exerts diverse neurochemical effects that resemble those seen after acute administration of antipsychotic drugs (APDs). These drugs also induce NT expression in the striatum; this and other convergent findings have led to the suggestion that NT may mediate some APD effects. Here, we demonstrate that the ability of the typical APD haloperidol to induce Fos expression in the dorsolateral striatum is markedly attenuated in NT-null mutant mice. The induction of Fos and NT in the dorsolateral striatum in response to typical, but not atypical, APDs has led to the hypothesis that the increased expression of these proteins is mechanistically related to the production of extrapyramidal side effects (EPS). However, we found that catalepsy, which is thought to reflect the EPS of typical APDs, is unaffected in NT-null mutant mice, suggesting that NT does not contribute to the generation of EPS. We conclude that NT is required for haloperidol-elicited activation of a specific population of striatal neurons but not haloperidol-induced catalepsy. These results are consistent with the hypothesis that endogenous NT mediates a specific subset of APD actions.

Figure 1

Generation of NT mutant mice. (A) Strategy for NT/N gene disruption. The NT/N precursor protein is depicted; the signal sequence (Pre, dotted), neuromedin N (N, hatched), and NT (NT, black) coding domains, and the corresponding exons of the mouse NT/N gene are indicated. Mouse NT/N gene fragments were inserted on either side of β-gal and PGK-Neo genes in the targeting construct, and homologous recombination resulted in the targeted NT gene structure shown at the bottom. pUC18, cloning vector (black); RI, EcoRI; Ps, PstI; Ms, MscI; Nc, NcoI; HI, BamHI; Nt, NotI. (B) Southern blot analysis of EcoRI-digested tail DNA derived from the progeny of two NT^+/− mice. The 15- and 6.5-kb EcoRI fragments identified by the ³²P-labeled 3′ probe depicted in A represent the wild-type and targeted alleles, respectively. The positions of HindIII-digested λ DNA size markers are indicated.

Figure 2

NT^−/− mice do not express NT. (A) Northern analysis of poly(A)⁺ RNA (10 μg) isolated from the proximal small intestine (P), distal small intestine (D), and brain (B) from NT^−/− and NT^+/+ mice. The blots were hybridized with ³²P-labeled probes for NT, enkephalin (ENK), substance P (SP), dynorphin (DYN), dopamine D₂ receptor (D₂R), and tyrosine hydroxylase (TH). (B) NT was quantitated by RIA with an N-terminally directed NT antiserum after HPLC-fractionation of brain extracts from NT^−/− (●, fmol/300 μl) and NT^+/+ (○, fmol/50 μl) mice. (C) Immunohistochemical analysis of NT expression in coronal brain sections from NT^−/− (Left) and NT^+/+ (Right) mice with a C-terminally directed NT antibody.

Figure 3

Haloperidol induction of Fos is markedly attenuated in the dorsolateral and central caudate-putamen of NT^−/− mice. (A) The number of Fos-positive neurons was quantitated after immunohistochemical detection of Fos in brain sections from NT^+/+ (n = 10, Veh; n = 8, Hal) or NT^−/− (n = 9, Veh; n = 7, Hal) mice that had been treated with either haloperidol (1 mg/kg) or pH-matched vehicle 2 h before perfusion. The mean (±SEM) number of Fos-positive neurons per mm² is presented for the intermediate caudate-putamen (Bregma + 0.2), the nucleus accumbens (Bregma + 1.1 mm), and the bed nucleus (at the level of Bregma). dlCP, dorsolateral caudate-putamen; cenCP, central CP; vlCP, ventrolateral CP; medCP, medial CP; NASsh, nucleus accumbens shell; NASco, NAS core; BNST, bed nucleus of the stria terminalis. *, P < 0.05; **, P < 0.01. (B) Fos-positive neurons in the rostral striatum (Bregma + 0.9 mm) in the same experiment as described in A. **, P < 0.01; ***, P < 0.001. (C) Immunofluorescent detection of Fos expression in the dorsolateral striatum of NT^+/+ (Upper) and NT^−/− (Lower) mice after the administration of either vehicle (Left) or haloperidol (Right). The position of the corpus collosum (cc) is indicated. (Bar = 100 μm.)

Figure 4

Clozapine induction of Fos is unaltered in NT^−/− mice. NT^+/+ (n = 9, Veh; n = 6, Cloz) and NT^−/− (n = 6, Veh; n = 6, Cloz) mice were injected with either clozapine (20 mg/kg) or pH-matched vehicle 2 h before perfusion. Fos-positive neurons were quantitated as described in the legend of Fig. 3. Abbreviations are as described for Fig. 3A plus PFC, prefrontal cortex.

Figure 5

Haloperidol-induced catalepsy is unaffected in NT^−/− mice. The latency to move all four paws after placement on a vertical wire grid was measured immediately after haloperidol administration (1 mg/kg) and at 45 and 90 min after injection. A maximal score of 90 sec was allowed. The mean latency to move was plotted (±SEM) for NT^+/+ (black bars, n = 14) and NT^−/− (white bars, n = 23) mice.