Abstract
Prepulse inhibition (PPI) of the acoustic startle reflex is a commonly used measure of preattentive sensorimotor gating. Disrupted PPI in rodents represents an animal model of the sensorimotor gating deficits characteristic of schizophrenia. The neurotensin (NT) system is implicated in the pathophysiology of schizophrenia, and NT has been hypothesized to act as an endogenous antipsychotic. In rats, NT receptor agonists restore PPI disrupted by dopamine receptor agonists and N-methyl-d-aspartate receptor antagonists, and pretreatment with an NT receptor antagonist blocks restoration of isolation rearing induced deficits in PPI by some antipsychotic drugs. The current studies further scrutinized the role of the NT system in the regulation of PPI and in antipsychotic drug-induced restoration of PPI using NT-null mutant mice (NT-/-). NT-/- mice exhibited significantly higher pulse alone startle amplitudes and disrupted PPI compared with NT+/+ mice. Haloperidol (0.1 mg/kg) and quetiapine (0.5 mg/kg) administered 30 min before PPI testing significantly increased PPI in NT+/+ mice but had no effect on PPI in NT-/- mice. In contrast, clozapine (1.0 mg/kg) significantly increased PPI in both NT-/- and NT+/+ mice, whereas olanzapine (0.5 mg/kg) had no effect on PPI in either NT-/- or NT+/+ mice. In a separate experiment, amphetamine (2.0 mg/kg i.p.) significantly disrupted PPI in NT+/+ mice but not NT-/- mice. These results provide evidence that the effects of antipsychotic drugs (APDs) may be differentially affected by the state of NT neurotransmission and, moreover, that APDs differ in their dependence on an intact NT system.
Neurotensin (NT) is a tridecapeptide (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH) that was first discovered in extracts of bovine hypothalamus (Carraway and Leeman, 1973). In the more than 30 years since elucidation of its structure, considerable information regarding the neuroanatomical distribution and physiological functions of NT has accrued. The genes encoding NT and four putative NT receptors have been successfully cloned and sequenced (for review, see Kinkead and Nemeroff, 2004), and several small molecule NT receptor antagonists, including SR48692 and SR142948A, have been developed. Of considerable interest has been the hypothesis that NT may represent an endogenous antipsychotic compound (Nemeroff, 1980). In addition to the considerable behavioral and biochemical similarities between the effects of centrally administered NT and those of systemically administered antipsychotic drugs (APDs), preclinical data has revealed markedly enhanced NT neurotransmission after APD treatment (for review, see Kinkead and Nemeroff, 2002). In addition, several clinical studies have revealed that a sizable subgroup of untreated patients with schizophrenia have abnormally low cerebrospinal fluid NT concentrations, which normalize after APD treatment (Widerlöv et al., 1982; Lindström et al., 1988; Nemeroff et al., 1989; Garver et al., 1991; Sharma et al., 1997).
There is increasing evidence that a deficit in sensorimotor gating is one of the cardinal features of the underlying pathophysiology of schizophrenia. The hypothesized deficit in gating or internal screening of sensory input in schizophrenic patients is viewed as leading to an involuntary flooding of indifferent sensory data, likely contributing to the cognitive fragmentation and thought disorder characteristic of this disease (McGhie and Chapman, 1961; Freedman et al., 1991). One test commonly used to assess these deficits in sensorimotor gating is prepulse inhibition (PPI) of the acoustic startle reflex. PPI of the acoustic startle reflex is defined by a decrease in the startle reflex induced by an acoustic stimulus when preceded by a weak prepulse. In humans, PPI has consistently been shown to be disrupted in schizophrenic patients and patients with schizotypal personality disorder (for review, see Swerdlow and Geyer, 1998). In rats, dopamine receptor agonists, N-methyl-d-aspartate receptor antagonists, hippocampal lesions, or isolation rearing disrupt PPI (Swerdlow et al., 2000; Geyer et al., 2001). These disruptions are reversed by administration of typical and atypical APDs but not by treatment with antidepressant or anxiolytic drugs (Swerdlow et al., 2000; Geyer et al., 2001).
NT has been shown to modulate PPI in a manner similar to APDs. Feifel et al. (1997) observed that low doses (0.25 and 1.0 μg) of NT administered directly into the nucleus accumbens, like APDs, blocked amphetamine-induced PPI disruption. In addition, peripheral administration of either of two NT receptor agonists (PD149163 and NT69L) reverses PPI deficits induced by amphetamine and the noncompetitive N-methyl-d-aspartate receptor antagonist dizocilpine (Feifel et al., 1999; Shilling et al., 2003). There is also evidence supporting a role for endogenous NT in the regulation of PPI (Binder et al., 2001).
The current study was designed to first evaluate the role of endogenous NT in the regulation of PPI using NT-null mutant mice (NT-/-), testing the hypothesis that mice lacking NT would exhibit deficits in PPI. Second, because NT-/- mice were shown to exhibit a blunted activation of striatal neurons in response to amphetamine (Dobner et al., 2003), the effect of this psychostimulant on PPI was examined. Last, because pretreatment with a NT receptor antagonist has been shown to prevent APD-induced restoration of PPI in isolation-reared rats (Binder et al., 2001), the effects of APD administration on PPI in NT-/- mice were examined.
Materials and Methods
Animals and Housing. NT knockout mice were generated as described previously (Dobner et al., 2001) and backcrossed five times with C57BL/6J mice, alternating the sex of the C57BL/6J parent for each generation. Mice for experiments were generated from heterozygous mating pairs. All animals were housed in an environmentally controlled animal facility under reverse dark/light conditions (lights off 10:00 AM; lights on 10:00 PM) with food and water available ad libitum, except during PPI testing. At weaning (postnatal day 23), mice were housed in same sex groups of three to five per cage. All animals were ear punched for identification, and the tissue was used for genotyping. All animal protocols were approved by the Emory University Institutional Animal Care and Use Committee (IACUC) in compliance with National Institutes of Health (http://grants.nih.gov/grants/olaw/olaw.htm) recommendations based on National Research council guidelines (Institute for Laboratory Animal Research, 2003).
Drugs.d-Amphetamine sulfate (Sigma-Aldrich, St. Louis, MO) was dissolved in 0.9% saline. Haloperidol (Sigma-Aldrich) was dissolved in 0.3% tartaric acid. Clozapine (Novartis, Basel, Switzerland), olanzapine (Eli Lilly & Co., Indianapolis, IN), and quetiapine (Zeneca Pharmaceuticals, Wilmington, DE) were dissolved in a minimal volume of glacial acetic acid and brought up to volume with 0.3% tartaric acid (final pH adjusted to 6.0). All drugs were administered in a volume of 1.0 ml/kg.
Startle Paradigm. PPI testing was completed in the dark phase between 11:00 AM and 4:00 PM. PPI testing was performed in a San Diego Instruments (San Diego, CA) startle chamber. The testing session began with a 5-min acclimatization to the startle chamber in the presence of a 65-dB background white noise. Testing consisted of nine 120-dB pulses alone and 18 pulses preceded (100 ms) by a prepulse of 4, 8, or 12 dB above background. Pulses were presented in a pseudorandom order with an average of 15 s between pulses. The onset latency (latency from stimulus to commencement of startle) occurring 20 to 120 ms after the pulse alone stimulus was reported in milliseconds. The peak latency (latency from stimulus to maximum startle amplitude) was defined as the point of maximal amplitude occurring within 150 ms of the pulse alone stimulus. PPI for each animal at each prepulse intensity was calculated using the following formula: %PPI = 100 - (startle amplitude with prepulse × 100/startle amplitude with pulse alone).
Characterization of Baseline Pulse Alone Startle Amplitude and Prepulse Inhibition of the Acoustic Startle Reflex. Mice (NT-/-, n = 48; NT+/-, n = 72; NT+/+, n = 52) were tested for PPI of the acoustic startle response beginning on postnatal day 42 and were tested a maximum of 12 times with 7 days between each testing.
Effect of Amphetamine on Prepulse Inhibition of Acoustic Startle Response. Adult male mice (NT-/-, n = 42; NT+/+, n = 16) were tested for PPI of the acoustic startle response three times, 7 days apart, as described above. All testing occurred between postnatal days 90 and 140. All animals were tested for baseline PPI (test week 1) and then 7 days later, all animals received a single i.p. injection of amphetamine (2.0 mg/kg, 1.0 ml/kg in 0.9% saline) 10 min before PPI testing (test week 2). Finally, 7 days after the second PPI, all animals were again tested in the PPI paradigm to verify baseline PPI (test week 3).
Effect of Antipsychotic Drugs on Pulse Alone Startle Amplitude and Prepulse Inhibition of Acoustic Startle Response. All animals were tested five separate times with 7 days between testing beginning on postnatal day 60. Baseline PPI was established in animals on test weeks 1, 3, and 5. In the first set of animals (NT-/-, n = 43; NT+/-, n = 70; NT+/+, n = 44), animals received haloperidol (0.1 mg/kg i.p.) 30 min before PPI in test week 2 and clozapine (1.0 mg/kg i.p.) 30 min before PPI in test week 4. In a second set of animals (NT-/-, n = 32; NT+/-, n = 75; NT+/+, n = 29), animals received olanzapine (0.5 mg/kg i.p.) 30 min before PPI in test week 2 and quetiapine (0.5 mg/kg s.c.) 30 min before PPI in test week 4.
Statistical Analysis. All data were first analyzed for sex differences. If there was a significant sex × genotype interaction, data from males and females were analyzed separately. Differences among group means in body weight and baseline measurement of pulse alone startle amplitude data were analyzed using two-way repeated measures ANOVA (genotype × age or genotype × test week, respectively) followed post hoc by Student-Newman-Keuls multiple comparisons test. For baseline measurement of PPI, differences among group means were analyzed using three-way repeated measures ANOVA (genotype × prepulse intensity × test week) followed post hoc by Student-Newman-Keuls multiple comparisons test. Spearman correlations were computed between body weight and pulse alone startle amplitude measures as well as pulse alone startle amplitude and PPI data. In studies examining the effects of amphetamine on PPI, differences between control PPI in test weeks 1 and 3 were first examined. In the absence of significant differences between baseline PPI within genotype in test weeks 1 and 3, data from these two PPIs were averaged to generate one control PPI value at each prepulse intensity for each mouse. Because averaging values across several control days may lead to a disproportionate deflation of variance in the control population compared with data obtained on any single control test day, we confirmed all statistical significance using data from control PPI in test weeks 1 and 3 individually for comparison. Similar methods were used in the haloperidol/clozapine and olanzapine/quetiapine studies. Differences among group means in the effect of amphetamine on PPI were analyzed using three-way repeated measures ANOVA (genotype × prepulse intensity × treatment) followed post hoc by Student-Newman-Keuls multiple comparisons test. In studies examining the effects of APDs on PPI, differences between control PPI in test weeks 1, 3, and 5 were first examined. In the absence of significant differences between baseline PPI within genotype in test weeks 1, 3, and 5, data from these three PPIs were averaged to generate one control PPI value at each prepulse intensity for each mouse. Differences among group means in the effect of APDs on PPI were analyzed using three-way repeated measures ANOVA (genotype × prepulse intensity × treatment) followed post hoc by Student-Newman-Keuls multiple comparisons test. Significant F values from all experiments are reported in Table 1.
Results
Weight. Mice (total n = 177) were weighed once a week for 16 weeks beginning on postnatal day 42 (Fig. 1). Because there was a significant effect of sex and a significant sex × genotype interaction, data from males and females were analyzed separately. There was a significant effect of genotype and age on weight in male and female mice but no significant genotype × age interaction. Post hoc analysis revealed that male and female NT-/- mice weighed significantly less than NT+/+ mice.
Characterization of Baseline Pulse Alone Startle Amplitude and Prepulse Inhibition. To examine the test to test stability of pulse alone startle and PPI, mice were tested beginning on postnatal day 42 and were tested a maximum of 12 times with 7 days between each testing. Because there was a significant effect of sex, genotype, and a sex × genotype interaction (post hoc analysis revealing that males have significantly greater pulse alone startle amplitudes than females), data from males and females were analyzed separately. Two-way repeated measures ANOVA (genotype × test week) revealed a significant effect of genotype on pulse alone startle amplitude in male and female mice. Post hoc analysis revealed that male and female NT-/- mice have significantly greater pulse alone startle amplitudes than NT+/+ mice (Fig. 2). Because there was no significant effect of test week on pulse alone onset latency or peak latency, the average onset latency and peak latency from all 12 testing sessions were analyzed. Two-way repeated measures ANOVA (sex × genotype) of mean onset latency and peak latency revealed a significant effect of sex on onset latency but no significant effect of genotype on mean onset latency or peak latency in pulse alone trials. Post hoc analysis revealed that females (11.7 ± 0.5 ms) had significantly shorter pulse alone onset latencies than males (15.2 ± 0.5 ms; see Table 2 for onset latency and peak latency by genotype). There was no significant correlation between weight and pulse alone startle amplitude.
To examine differences in habituation to startle, the nine pulse alone startle amplitudes spaced throughout the testing session were analyzed. Because there was no significant effect of test week on pulse alone startle amplitude in male or female mice, the average pulse alone startle amplitude from all 12 testing sessions was analyzed. As described, there was a significant effect of genotype on pulse alone startle amplitude in male and female mice (Fig. 2, insets). In addition, there was a significant effect of trial number in male and female mice but no genotype × trial number interaction. All groups habituated to pulse alone startle.
Although there was a significant effect of sex on PPI, there were no significant interactions between sex (males have slightly higher PPI than females) and genotype, prepulse intensity, or test week. Therefore, PPI data from male and female mice were analyzed together. There was a significant effect of test week, prepulse intensity, and genotype on PPI. There was no significant genotype × PPI test week interaction. Because there was no significant interaction between prepulse intensity and either genotype or test week, all data are shown together across prepulse intensity (Fig. 3). Post hoc analyses revealed that NT-/- mice have significantly reduced PPI compared with NT+/+ mice. PPI during test weeks 1 and 2 was significantly less than PPI after test week 3 (approximately 60 days of age) in all genotypes. To reduce the potential confound of age related effects on PPI, mice in the antipsychotic drug and amphetamine experiments were not tested until after 60 days of age. The potential confound of multiple PPI testing was addressed individually within the antipsychotic drug and amphetamine experiments [two-way repeated measures ANOVA (genotype × test week)].
Because there was no significant effect of test week on onset latency, peak latency, or peak latency facilitation (peak pulse alone latency - peak prepulse/pulse latency) in prepulse/pulse trials, the average onset latency, peak latency, and peak latency facilitation from all 12 testing sessions were analyzed. Two-way repeated measures ANOVA (sex × genotype) of onset latency, peak latency, and peak latency facilitation revealed a significant effect of sex on onset latency but no significant effects of genotype on onset latency, peak latency, or peak latency facilitation in prepulse/pulse trials. Post hoc analysis revealed that females had significantly shorter onset latencies than males (Table 2).
Effect of Amphetamine on Pulse Alone Startle Amplitude and PPI. Amphetamine (2.0 mg/kg i.p.) had no significant effect on pulse alone startle amplitude (Table 3). Basal PPI values (test weeks 1 and 3) recorded before and after PPI testing with amphetamine (test week 2) were not significantly different from each other; therefore, the average basal PPI value for each mouse was used for further analysis. There was a significant effect of genotype and prepulse intensity on PPI and a significant genotype × treatment interaction (Fig. 4). Post hoc analyses revealed that basal PPI was significantly lower in NT-/- mice compared with NT+/+ mice and that amphetamine reduced PPI only in NT+/+ mice.
Effect of Haloperidol and Clozapine on Pulse Alone Startle Amplitude and PPI. There was a significant effect of sex on pulse alone startle amplitude but no sex × genotype or treatment interactions. Therefore, pulse alone startle data from male and female mice were analyzed together. Two-way repeated measures ANOVA (genotype × test week) of baseline pulse alone startle amplitude in test weeks 1, 3, and 5 of mice tested with haloperidol and clozapine (weeks 2 and 4) demonstrated a significant effect of genotype. Post hoc analysis revealed that NT-/- mice had significantly higher pulse alone amplitudes than NT+/+ mice (Table 3). There was no significant difference in pulse alone startle amplitude between test weeks within genotype (data not shown). Therefore, for further analysis, pulse alone startle values in test weeks 1, 3, and 5 were averaged for each mouse to generate one control pulse alone startle amplitude value for each animal. Clozapine significantly increased pulse alone startle amplitude in NT+/+ mice (Table 3).
In contrast to data shown in Fig. 3, there was no significant effect of sex on PPI, but there was a significant sex × genotype interaction. Post hoc analysis revealed that female NT-/- mice did not have significantly disrupted PPI compared with female NT+/+ mice. Therefore, further analysis of the effects of haloperidol and clozapine on PPI was conducted separately in males and females. Three-way repeated measures ANOVA (genotype × test week × prepulse intensity) of baseline PPI in test weeks 1, 3, and 5 of mice tested with haloperidol and clozapine (weeks 2 and 4) demonstrated a significant effect of genotype in males and test week and prepulse intensity in males and females. Post hoc analysis revealed that there was no significant difference in PPI values between test weeks within genotype (data not shown). Therefore, for further analysis, PPI data from test weeks 1, 3, and 5 were averaged to generate control PPI values at different prepulse intensities for individual mice. Three-way repeated measures ANOVA (genotype × treatment × prepulse intensity) demonstrated a significant effect of genotype, treatment, and prepulse intensity, and treatment and prepulse intensity in females. Post hoc analysis revealed that male, but not female, NT-/- mice had significantly reduced basal PPI compared with NT+/+ mice of the same sex (Fig. 5a). Post hoc analysis in the absence of a significant genotype × treatment interaction was justified by a significant genotype × treatment interaction in the absence of the NT+/- mice (similar rationale is used in the olanzapine/quetiapine study). Haloperidol did not significantly increase PPI in either male or female NT-/- mice, but it did increase PPI in NT+/+ mice. In contrast, clozapine significantly increased PPI in both NT-/- and NT +/+ mice regardless of sex. These results indicate that NT is required for the enhancement of PPI by haloperidol but not clozapine in both male and female mice.
Effect of Olanzapine and Quetiapine on Pulse Alone Startle Amplitude and PPI. There was a significant effect of sex on pulse alone startle amplitude. However, because there was no sex × genotype or treatment interactions, pulse alone startle data from male and female mice were analyzed together. Two-way repeated measures ANOVA (genotype × test week) of baseline pulse alone startle amplitude in test weeks 1, 3, and 5 of mice tested with olanzapine and quetiapine (weeks 2 and 4) demonstrated a significant effect of genotype. Post hoc analysis revealed that NT-/- mice had significantly higher pulse alone amplitudes than NT+/+ mice (Table 3). There was no significant difference in pulse alone startle amplitude between test weeks within genotype (data not shown). Therefore, for further analysis, pulse alone startle values in test weeks 1, 3, and 5 were averaged for each mouse to generate one control pulse alone startle amplitude value for each animal. Three-way repeated measures ANOVA (genotype × treatment × prepulse) demonstrated a significant effect of genotype. There was no significant effect of olanzapine or quetiapine on pulse alone startle amplitude (Table 3).
There was a significant effect of sex on PPI but no significant interactions between sex and genotype, treatment, or prepulse intensity. Therefore, further analysis of the effects of olanzapine and quetiapine on PPI was conducted without regard to sex. Three-way repeated measures ANOVA (genotype × test week × prepulse intensity) of baseline PPI in test weeks 1, 3, and 5 of mice tested with olanzapine and quetiapine (weeks 2 and 4) demonstrated a significant effect of genotype, test week, and prepulse intensity. Post hoc analysis revealed that there was no significant difference in PPI values between test weeks within genotype (data not shown). Therefore, for further analysis, PPI data from test weeks 1, 3, and 5 were averaged to generate control PPI values for different prepulse intensities for individual mice. Three-way repeated measures ANOVA (genotype × treatment × prepulse intensity) demonstrated a significant effect of genotype, treatment, and prepulse intensity. Post hoc analysis revealed that NT-/- mice had significantly reduced PPI compared with NT+/+ mice (Fig. 6). Quetiapine did not significantly increase PPI in NT-/- mice, but it did increase PPI in NT+/+ mice. In contrast, olanzapine did not significantly increase PPI in either NT-/- or NT+/+ mice at the dose tested.
Discussion
The present results clearly indicate that 1) the NT system is involved in regulation of baseline PPI and pulse alone startle, 2) amphetamine-induced disruption of PPI is dependent on NT neurotransmission, and 3) NT neurotransmission is involved in some but not all APD effects on PPI.
Baseline Startle and PPI. NT-/- mice had significantly greater pulse alone startle amplitudes compared with NT+/+ mice. Although the mean body weight of NT-/- mice was found to be significantly lower than NT+/+, it is unlikely that differences in body weight significantly affected pulse alone amplitude or PPI because there was no correlation between body weight and pulse alone startle amplitude or PPI (data not shown).
NT-/- mice had significantly decreased PPI compared with NT+/+ mice. Although it is possible that the decrement in PPI was due to differences in the startle response (see above), there was no correlation between pulse alone startle amplitude and PPI at any prepulse intensity. Additionally, when NT-/- and NT+/+ mice were matched for pulse alone startle amplitude, there was still a significant effect of genotype on PPI (data not shown). Although early-onset hearing loss in mice can affect both acoustic startle responding and PPI elicited by acoustic prepulses (Parham and Willott, 1988; Willott et al., 1994; Zheng et al., 1999), the C57BL/6J strain has been shown to have normal hearing before 1 year of age (Zheng et al., 1999) as well as significant cross-modal (light prepulse with an airpuff startle stimulus) PPI (Ralph et al., 2001). Although we did not directly measure hearing in the NT-/- mice, NT-/- mice had normal levels of latency facilitation, providing reasonable evidence that the prepulse is being detected, despite its reduced impact on startle magnitude.
Although NT-/- mice have significantly lower PPI than NT+/+ mice, neither central administration of NT (Feifel et al., 1997) nor peripheral administration of the NT receptor agonists PD149163 (Feifel et al., 1999) or NT69L (Shilling et al., 2003) alters baseline PPI in rats. These discrepancies may be reflective of downstream compensation in NT-/- mice or species differences between rats and mice (for review, see Geyer et al., 2001). In fact, a major species difference between rats and mice is that APDs tend to regulate baseline PPI in mice, but not rats (Geyer et al., 2001, 2002). However, isolation-reared rats are deficient in PPI compared with socially reared controls, and this deficit is associated with decreased NT mRNA expression and increased NT receptor binding in the nucleus accumbens shell (Binder et al., 2001), suggesting that decreased NT signaling in this region may underlie isolation rearing-induced deficits in PPI. Our observation that NT-/- mice display deficits in PPI lends further support to the hypothesis that NT signaling regulates basal PPI.
There were inconsistent effects of sex and sex × genotype interactions on PPI. The sex differences in PPI are most likely due to estrous cycle regulation of PPI, because PPI varies across the estrous cycle in rats (Koch, 1998). It does not seem, however, that the potential estrous cycle related effects on PPI altered the effects of APDs. In the study examining the effects of haloperidol and clozapine on PPI, despite the lack of significant differences in basal PPI between NT-/- and NT+/+ female mice, haloperidol and clozapine had the same effects on PPI as in male mice (i.e., clozapine increased PPI in both NT+/+ and NT-/- mice, and haloperidol only increased PPI in NT+/+ mice). Further studies will be necessary to examine the impact of the estrous cycle on PPI in NT-/- and NT+/+ mice.
Amphetamine Effects on PPI. Similar to other studies, amphetamine (2.0 mg/kg) significantly disrupted PPI in wildtype C57BL/6J mice (Geyer et al., 2002). In contrast, amphetamine had no effect on PPI in NT-/- mice, indicating that NT neurotransmission may be necessary for the effects of amphetamine on PPI. Although it is possible that the lack of effect of amphetamine on PPI in NT-/- mice is due to a floor effect (PPI was already significantly disrupted in the NT-/- mice), data demonstrating attenuated amphetamine-induced Fos expression in the medial striatum of NT-/- mice (Dobner et al., 2003) provide a potential biochemical correlate underlying the lack of behavioral effect. A role for NT in the behavioral effects of psychostimulants is supported by studies in which pretreatment with the NT receptor antagonist SR48692 blocked certain aspects of acute psychostimulant-induced behavioral responses and psychostimulant sensitization (Horger et al., 1994; Betancur et al., 1998; Rompré and Perron, 2000; Costa et al., 2001; Panayi et al., 2002).
APD Effects on PPI. The APDs haloperidol, clozapine, olanzapine, and quetiapine differentially affected PPI in NT-/- mice. Similar to the current results, haloperidol (McCaughran et al., 1997; Ouagazzal et al., 2001) and clozapine (Olivier et al., 2001; Ouagazzal et al., 2001) have been shown to increase PPI in C57BL/6J mice. However, whereas clozapine increased PPI in both NT-/- and NT+/+ mice, haloperidol and quetiapine had no effect on PPI in NT-/- mice. Although it is possible that higher doses of haloperidol and quetiapine would increase PPI in NT-/- mice, the behavioral effects of these two drugs are at least in part dependent on intact NT neurotransmission, because the same dose of APD that increased PPI in NT+/+ mice had no effect on PPI in NT-/- mice. These results are in agreement with our previous study in rats demonstrating that pretreatment with the NT receptor antagonist SR142948A blocked the restoration of PPI in isolation reared rats induced by haloperidol and quetiapine (Binder et al., 2001).
The mechanisms underlying NT involvement in APD action remain unclear; however, previous observations that NT is required for normal haloperidol-evoked Fos expression in the dorsolateral striatum in both mice (Dobner et al., 2001) and rats (Fadel et al., 2001; Binder et al., 2004) suggest that alterations in striatal activation may be involved. The induction of NT expression in the dorsolateral striatum has been previously proposed to be involved in the production of extrapyramidal side effects (EPS). However, haloperidol-induced catalepsy is not affected in either NT-/- mice (Dobner et al., 2001) or rats pretreated with SR142948A (Binder et al., 2004), suggesting that NT is not involved in the production of EPS. Furthermore, NT receptor antagonist pretreatment potentiates haloperidol-induced catalepsy in mice at a suboptimal dose of haloperidol, suggesting that endogenous NT acts to limit catalepsy (Casti et al., 2004). However, the opposite result was obtained for haloperidol-induced hypolocomotion, indicating that NT may differentially influence diverse EPS (Casti et al., 2004). Although the rodent dorsolateral striatum is typically considered to be a motor area, lesions in the caudodorsal striatum have been demonstrated to decrease basal PPI (Kodsi and Swerdlow, 1995), suggesting that haloperidol-mediated increases in neuronal activity in this region could influence PPI. In fact, although the majority of evidence has implicated NT in the nucleus accumbens as mediating the antipsychotic-like effects of NT, NT seems to play a more important role in haloperidol-evoked Fos expression in the striatal patch compartment (Fadel et al., 2001), which has been implicated in affective and cognitive functions (Moratalla et al., 1992; White and Hiroi, 1998; Canales and Graybiel, 2000). These observations suggest that the defect in haloperidol-mediated striatal activation in NT-/- mice may explain the inability of this drug to increase PPI in these mice. In contrast, clozapine-evoked Fos expression was unaffected in both NT-/- mice (Dobner et al., 2001) and in rats pretreated with SR142948A (Binder et al., 2004), consistent with our observation that clozapine enhancement of PPI was unaffected in these mice. As discussed above, alterations in NT signaling in the ventral striatum may underlie quetiapine enhancement of PPI (Binder et al., 2001). These results suggest that certain APDs require NT for their therapeutic actions, whereas others, such as clozapine, produce similar effects through alternative mechanisms.
As stated above, only one dose of haloperidol (0.1 mg/kg) was tested in the current study. This raises the possibility that the lack of effect of haloperidol on PPI in NT-/- mice is not qualitative, but quantitative (e.g., a higher dose of haloperidol would increase PPI in NT-/- mice). One argument against this possibility is that the reduction in haloperidol-induced Fos expression in NT-/- mice and after pretreatment with NT receptor antagonists was seen at catalepsy-inducing doses of haloperidol 10- to 20-fold higher (1.0 and 2.0 mg/kg) than those used in the current study.
Our results provide additional evidence for NT system regulation of PPI and perhaps by extension the sensorimotor gating deficits characteristic of the pathophysiology of schizophrenia. Because NT-/- mice have deficits in PPI, these studies also provide further rationale for the development of NT receptor agonists as novel APDs. Studies examining the effects of APDs on PPI in NT-/- mice raise the likelihood that the state of NT neurotransmission may differentially affect the efficacy of individual APDs. In combination with the clinical research indicating that a subset of schizophrenic patients has reduced cerebrospinal fluid NT, these data suggest that the activity of NT-containing circuits may be predictive of APD efficacy.
Footnotes
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This research was supported by National Institutes of Health Grant MH-39415.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.105.087437.
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ABBREVIATIONS: NT, neurotensin; NT-/-, neurotensin-null mutant mice; SR48692, 2-{[1-(-7-chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carbonyl]amino}adamantane-2-carboxylic acid; SR142948A, 2-{[5-(2,6-dimethoxyphenyl)-1-(4-(N-(3-dimethylaminopropyl)-N-methylcarbamoyl)-2-isopropylphenyl)-1H-pyrazole-3-carbonyl]amino}adamantane-2-carboxylic acid; APD, antipsychotic drug; PPI, prepulse inhibition; PD149163, Lys(CH2NH)Lys-Pro,Trp-tert-Leu-Leu-Oet; NT69L, Nα MeArg-Lys-Pro-neo-Trp-tert-Leu-Leu; ANOVA, analysis of variance; EPS, extrapyramidal side effects.
- Received April 4, 2005.
- Accepted June 24, 2005.
- The American Society for Pharmacology and Experimental Therapeutics