The neurotensin-1 receptor agonist PD149163 blocks fear-potentiated startle

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Abstract

Preliminary evidence suggests that the neuropeptide, neurotensin (NT) may regulate fear/anxiety circuits. We investigated the effects of PD149163, a NT1 receptor agonist, on fear-potentiated startle (FPS). Sprague Dawley rats were trained to associate a white light with a mild foot shock. In one experiment, animals were treated with either subcutaneous vehicle or PD149163 (0.01, 0.1 or 1.0 mg/kg) 24 h after training. Twenty minutes later their acoustic startle response in the presence or absence of the white light was tested. In a second experiment, saline and 1.0 mg/kg PD149163 were tested using a separate group of rats. In the first experiment, PD149163 produced a non-significant decrease in baseline acoustic startle at all three doses. As expected, saline-treated rats exhibited significant FPS. An ANOVA of percentage FPS revealed no significant effect of treatment group overall but the high dose group did not display FPS strongly suggesting an FPS effect at this dose. This finding was confirmed in the second experiment where the high dose of PD149163 reduced percent FPS relative to saline (P < 0.05). These data suggest that systemically administered NT1 agonists modulate the neural circuitry that regulates fear and anxiety to produce dose-dependent anxiolytic-like effects on FPS.

Introduction

Neurotensin (NT) is a tridecapeptide with a wide distribution throughout the mammalian central nervous system (Emson et al., 1982). When administered directly into the brain, NT has been reported to have several antipsychotic-like behavioral and neurochemical effects including inhibition of mesolimbic dopamine function, antagonism of stimulant-induced hyperlocomotion (Kalivas et al., 1984, Robledo et al., 1993, Skoog et al., 1986) and stimulant-induced disruption of sensorimotor gating (Feifel et al., 1997). NT does not effectively reach the brain after systemic administration, nor does the C-terminal hexapeptide, NT(8–13), the smallest NT peptide fragment which contains full biological activity of the parent peptide (Kanba et al., 1988, Machida et al., 1993).

In order to produce viable drug candidates, several NT mimetics have been produced by chemically modifying the NT(8–13) peptide to make it more resistant to endopeptidase degradation in the periphery and thus better able to enter the central nervous system (CNS) (Cusack et al., 2000, Wustrow et al., 1995). PD149163 is one such NT mimetic produced by adding a reduced amide bond to NT(8–13) (Wustrow et al., 1995). PD149163 has strong and selective affinity for the neurotensin-1 (NT1) receptor (Petrie et al., 2004), the NT receptor type implicated in the antipsychotic-like effects of NT. PD149163 has been shown to produce robust antipsychotic-like effects (Feifel et al., 1999). Recently, PD149163 has been shown to produce pro-cognitive effects in the CNS after systemic administration (Azmi et al., 2006).

There is strong evidence that NT1 agonists modulate mesolimbic dopamine and forebrain acetylcholine transmission in the brain and this effect has been presumed to underlie the antipsychotic-like and pro-cognitive effects, respectively, of NT and NT mimetics such as PD149163 (Nakachi et al., 1995, Szigethy and Beaudet, 1987). Our laboratory discovered that NT mimetics have more diverse pharmacological effects, which raises the possibility that they may have important actions on other circuits relevant to neuropsychiatric disorders. For example, we reported that systemic administration of PD149163 blocks the behavioral effects of a serotonin-2 (5-HT2) agonist, DOI, suggesting that NT agonists also have inhibitory effects on serotoninergic transmission at 5-HT2 receptors (Feifel et al., 2003b). Serotoninergic mechanisms have been strongly implicated in anxiety and depression, and inhibition of 5-HT2 receptors specifically may be a mechanism for anxiolysis and anti-depression (Mora et al., 1997, Weisstaub et al., 2006). Other evidence also suggests that NT agonists may regulate anxiety-relevant neurocircuitry. NT is localized in several brain regions that have been associated with fear and anxiety, such as the amygdala and hippocampus (Campeau et al., 1992, Davis et al., 1993, Gewirtz et al., 2000, Paxinos and Watson, 1997). Saiz Ruiz et al. (1992) reported that NT levels were significantly decreased in patients with anxiety and were normalized after recovery. In a fear conditioning test, duration of freezing was significantly reduced by beta lactotensin, a natural ligand for NT receptors (Yamauchi et al., 2006). In addition, Shugalev et al. (2005) found that NT injections into the substantia nigra reduced fear produced by serotoninergic lesions of the dorsal raphe (Shugalev et al., 2005). As these studies did not use NT receptor selective agonists, the role of the NT1 receptor in these anti-fear effects is not known. In order to investigate the role of NT1 receptors in fear circuits and to further investigate the anxiolytic potential of NT1 agonists, we tested the effects of PD149163 on fear-potentiated startle (FPS), a commonly employed animal model of anticipatory anxiety.

FPS is decreased by drugs that reduce fear and anxiety in humans such as benzodiazepines, whereas, it is increased by drugs that have anxiogenic effects, such as yohimbine (Davis et al., 1993). Therefore FPS has been used extensively to test the anxiolytic potential of many compounds (Davis et al., 1993, Walker and Davis, 2002). In this paradigm, the magnitude of the acoustic startle reflex can be enhanced by a conditioned stimulus such as a light that has previously been associated with a shock. Fear-potentiated startle is exhibited when the startle reflex is significantly greater when the conditioned stimulus is present.

Section snippets

Animals

Seventy-six male Sprague Dawley rats (275–375 g at testing) were obtained from Harlan Laboratories, San Diego, California. Animals were housed in groups of two in clear plastic chambers in a climate-controlled room on a 12:12 hour light/dark cycle (lights on 7:00 am–7:00 pm). The rats were handled prior to testing. All testing occurred during the light phase of the rats’ circadian illumination schedule and they were allowed free access to food and water for the extent of the study, except

Baseline startle

In the first experiment, PD149163 produced a non-significant trend towards a reduction in startle magnitude at all three doses, F(3, 27) =1.70, NS (Fig. 1A). In the second experiment, PD149163 significantly decreased startle magnitude (t(30) = 2.04, P < 0.01) (Fig. 2A). Startle magnitude values in saline and PD149163 treated animals were comparable to those in the first experiment.

Fear-potentiated startle

Comparison of the startle response to noise alone versus light plus noise in saline-treated rats revealed that

Discussion

Light as a conditioned fear stimulus produced a significant enhancement in the acoustic startle response demonstrating that FPS was successfully induced by the training procedures in these experiments. The findings indicate that the highest dose of systemically administered PD149163 blocked FPS in Sprague Dawley rats. Although the ANOVA used to analyze the FPS data in the first experiment did not produce a significant main effect of overall drug treatment, this was likely due to the large

Acknowledgements

DF and PDS are partially funded by NIMH grant (MH62451). We thank Gilia Melendez for her excellent technical assistance.

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