Introduction

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Neurotensin (NT) is a tridecapeptide that was first structurally characterized from extracts of bovine hypothalamus by Carraway and Leeman (1973). It has since been found to be heterogeneously distributed throughout the central nervous system of many mammals, including humans (Uhl, 1982; Mai et al., 1987). The hypothesis that NT might be an endogenous neuroleptic was first posited 20 years ago (Nemeroff, 1980). Since that time, a large database has been accrued in an attempt to determine whether the NT system plays a seminal role in mediating the effects of antipsychotic drugs. Centrally administered NT has been found to possess many of the same properties of peripherally administered antipsychotic drugs (for review, see Bissette and Nemeroff, 1995). For example, both typical antipsychotic drugs and centrally administered NT are known to increase dopamine (DA) turnover in the terminal fields of the mesolimbicocortical DA pathway. Similarly, both i.c.v. NT and NT microinjected directly into the nucleus accumbens block the hyperactivity produced by d-amphetamine and other psychostimulants known to increase the synaptic availability of DA. Centrally administered NT also inhibits avoidance (but not escape) behavior in a discrete-trial, conditioned avoidance paradigm, potentiates barbiturate- and ethanol-induced sedation, blocks intracisternal electrical self-stimulation from the ventral tegmental area (VTA) after injection into the nucleus accumbens, and produces hypothermia, all effects of classical antipsychotic drugs. In contrast to typical antipsychotic drugs, however, NT does not induce catalepsy in rats and fails to decrease stereotyped sniffing elicited by DA-stimulating drugs, leading to the hypothesis that NT may have a pharmacological profile closer to that of atypical antipsychotic drugs such as clozapine (Jolicoeur et al., 1993).

Alterations in the NT system also have been observed in schizophrenia. Clinical studies examining drug-free schizophrenic patients demonstrated that there is a subset of schizophrenic patients with decreased cerebrospinal fluid (CSF) NT levels (Bissette et al., 1985; Garver et al., 1991; Sharma et al., 1997). After antipsychotic drug treatment, NT concentrations in the subset of schizophrenic patients with decreased NT concentrations increased to control levels. There were no consistent changes in CSF NT concentrations of schizophrenic patients that originally had CSF NT concentrations indistinguishable from controls. There also appears to be a correlation between NT concentrations in the CSF and the magnitude of psychopathology (Garver et al., 1991; Breslin et al., 1994; Sharma et al., 1997). Schizophrenic patients with low CSF concentrations of NT are lithium nonresponders, and have a greater degree of thought disorder, delusions-hallucinations, behavioral disorganization, and impaired functioning. Postmortem studies in schizophrenia also indicate alterations in the NT system. Wolf et al. (1995) reported that NT receptor binding is decreased in the entorhinal cortex of schizophrenics compared with controls.

Govoni et al. (1980) first reported that the clinically efficacious antipsychotic drugs haloperidol, chlorpromazine, trifluoroperazine, and pimozide specifically increased NT concentrations in the nucleus accumbens and caudate nucleus of rats, whereas clinically ineffective phenothiazines (e.g., promazine and promethazine), as well as other classes of psychoactive compounds such as tricyclic antidepressants, anxiolytics, and antihistamines failed to alter NT concentrations in any brain region (Govoni et al., 1980; Frey et al., 1986; Eggerman and Zahm, 1988; Myers et al., 1992).

The effects of antipsychotic drug administration on almost every aspect of the NT system (NT metabolism, NT/Neuromedin N (NT/NN) mRNA expression, NT tissue concentrations, NT extracellular release, NT receptor mRNA expression, and NT receptor binding) have now been examined. Of particular interest has been the finding that typical and atypical antipsychotic drugs differentially regulate the NT system in that typical antipsychotic drugs have actions on both the mesolimbic (VTA to nucleus accumbens) and nigroneostriatal (substantia nigra to caudate/putamen) NT systems, whereas atypical antipsychotic drugs act preferentially on the mesolimbic NT system (Kilts et al., 1988; Merchant et al., 1994). This last finding has generated the hypothesis that the nigroneostriatal NT system may play a role in the side effect profile of typical antipsychotic drugs, whereas the mesolimbic NT system may be involved in the clinical efficacy of all antipsychotic drugs.

To test this hypothesis, the effects of various doses of the atypical antipsychotic drugs risperidone, sertindole, quetiapine, and olanzapine on NT concentrations and NT receptor binding were compared with the effects of the typical antipsychotic drug haloperidol and the prototype atypical antipsychotic drug clozapine in the rat brain. Acute dose-response curves for the effects of these antipsychotic drugs on NT tissue concentrations were first performed, and then the subchronic effects of antipsychotic drug administration on both NT tissue concentrations and NT receptor binding were examined.

	Materials and Methods

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Animals and Housing. Adult male Sprague-Dawley rats (200-250 g; Harlan Sprague-Dawley, Inc., Indianapolis, IN) were housed under 24-h light/dark cycle (lights on 7:00 AM; lights off 7:00 PM) in an environmentally controlled animal facility with food and water available ad libitum. All rats were handled daily for 1 week before treatment and housed four per cage. The Emory University Institutional Animal Care and Use Committee approved all animal protocols.

Acute Administration of Antipsychotic Drugs. Each rat received a single s.c. injection of one of the following: 1) Haloperidol (Sigma, St. Louis, MO) was administered s.c. (0.1, 0.33, 1.0, or 3.3 mg/kg) in a vehicle of 0.3% tartaric acid (pH = 6.0). 2) Clozapine (Novartis Pharmaceuticals, Basle, Switzerland) was administered s.c. (1.0, 3.3, 10.0, 17.6, or 33.3 mg/kg). Clozapine and olanzapine were dissolved in a minimal volume of glacial acetic acid and brought up to volume with 0.3% tartaric acid (pH = 6.0). 3) Olanzapine (Eli Lilly Co, Indianapolis, IN) was administered s.c (0.1, 0.33, 1.0, 3.3, 10.0, or 33.3 mg/kg). 4) Risperidone (Janssen Research Foundation, Beerse, Belgium) was administered s.c. (0.1, 0.33, 1.0, 3.3, or 10.0 mg/kg) in a vehicle of 0.3% tartaric acid (pH = 6.0). 5) Sertindole (Abbott Laboratories, North Chicago, IL) was administered s.c. (0.1, 0.33, 1.0, 3.3, or 10.0 mg/kg) in a vehicle of polyethylene glycol-400 and 0.3% tartaric acid at a ratio of 3:1 (pH = 6.0). 6) Quetiapine (Zeneca Pharmaceuticals, Wilmington, DE) was administered s.c. (0.33, 1.0, 3.3, 10.0, or 33.3 mg/kg) in a vehicle of 0.3% tartaric acid. Eighteen hours after the single injection, rats were sacrificed by decapitation, and the brains rapidly removed and frozen on dry ice. Rat brains were stored at 70°C until use.

Subchronic Administration of Antipsychotic Drugs. Doses used in these studies were chosen based on the results of the acute dose-response studies. Osmotic minipumps (model 2 ML4; Alzet, Palo Alto, CA) containing either haloperidol (2.0 mg/kg/day), clozapine (10.0 or 40.0 mg/kg/day), olanzapine (10.0 mg/kg/day), risperidone (1.0, 3.3, or 10.0 mg/kg/day), quetiapine (10.0 or 33.3 mg/kg/day), sertindole (2.0 or 10.0 mg/kg/day), or vehicle were implanted in adult male Sprague-Dawley rats (200-250 g, n = 10 for each group). Fourteen days after the minipumps were implanted, the rats were sacrificed by decapitation, and the brains were rapidly removed and frozen on dry ice. Brains were stored at 70°C until use.

Dissection of Rat Brain. The rat brains were dissected based on the method of Glowinski and Iversen (1966). The brain regions examined consist of those previously implicated in the pathophysiology of schizophrenia and in the mechanism of action of antipsychotic drugs (coordinates according to the atlas of Paxinos and Watson, 1986): the prefrontal cortex (cortex anterior to A2.7 relative to bregma), the nucleus accumbens and anterior caudate nucleus (between A2.7 and A1.2 relative to bregma), the posterior caudate nucleus (between A1.2 and A0.2 relative to bregma), the substantia nigra and VTA (between P4.8 and P5.8 relative to bregma), and the hippocampus (taken from a wedge of tissue between P4.3 and P5.8 on the dorsal side and P4.8 and P5.8 on the ventral side). Individual brain regions were stored at 70°C in polypropylene microcentrifuge tubes until assay.

Radioimmunoassay. NT concentrations were determined by using a highly specific and sensitive NT radioimmunoassay. Brain regions were extracted in ice-cold 1.0 M HCl by ultrasonic dismembranation, and the homogenates were centrifuged at 10,000g for 15 min at 4°C. The supernatant was then transferred to a fresh microcentrifuge tube, vortexed, and duplicate 100 µl aliquots were transferred to borosilicate glass tubes and stored at 70°C. On the day of the assay the frozen aliquots were lyophilized, reconstituted in assay buffer, and then assayed by a single equilibrium radioimmunoassay according to methods previously described (Bissette et al., 1984). The assay buffer consisted of 10 mM NaH₂PO₄, 0.15 M NaCl, 0.01% NaN₃, 0.1% gelatin, 2.5 mM EDTA, and 0.05% Triton X-100 adjusted to pH 7.6 with NaOH. The antiserum used (Peninsula Laboratories, Inc., Belmont, CA) is directed toward the middle portion of the NT molecule and was used at a final dilution that provides 30% binding of the labeled NT (normally 1:13,000). Synthetic NT_1-13 (Bachem Inc., Torrance, CA) was used as a standard, and monoiodinated [Tyr³]-NT was obtained from DuPont/NEN (Wilmington, DE). Goat anti-rabbit antiserum (Arnel Products, New York, NY) was used as second antibody. The assay has a sensitivity of 1.25 pg/tube and an IC₅₀ of 80 pg/tube. The pellets from the extraction were resuspended in 1.0 M NaOH by sonication and assayed for protein concentration by the method of Lowry et al. (1951) with BSA used as standard. NT concentrations are expressed as picograms of NT per milligram of protein.

NT Receptor Binding. Previously dissected tissue stored at 70°C was weighed and then homogenized (Brinkmann polytron) in 10 volumes of buffer A (5.0 mM Tris-HCl, 1 mM EDTA, pH 7.4 at 4°C). The homogenate was centrifuged at 40,000g for 20 min at 4°C, the supernatant removed, and the pellet resuspended in buffer A (10.0 mg/ml) and recentrifuged a total of three times. The final pellet was then resuspended in buffer B (50 mM Tris-HCl containing 0.2% BSA, 0.1 nM phenanthroline, pH 7.4). The homogenate was used fresh for all assays. All incubations were performed at 25°C in buffer B. Homogenate was combined with ¹²⁵I-NT (specific activity 2200 Ci/mmol) and buffer B with or without 1.0 (M unlabeled NT_1-13 for determination of nonspecific binding. The final reaction volume was 500 µl. After a 20-min incubation the reactions were terminated by the addition of ice-cold buffer C (50 mM Tris-HCl, pH 7.4) followed by rapid filtration under reduced pressure through glass fiber filters presoaked in ice-cold buffer C containing 0.3% polyethylenimine. Filters were then rinsed three times with 5 ml of ice-cold buffer C. Radioactivity was counted on an LKB Clinigamma model 1274 with 67% counting efficiency.

Statistical Analysis. Results are presented as mean ± S.E. Differences between the means were determined by one-way ANOVA. After a significant difference, the Student-Newman-Keuls multiple comparison test was applied to identify groups differing significantly from control values. Significant differences were determined by t test in instances where there was only one treatment group. Differences were considered significant if the probability that they were zero was less than 5%. Sample size was determined to be adequate when the power of the performed test was greater than 0.8.

	Results

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Acute Dose-Response Effects of Typical and Atypical Antipsychotic Drug Administration on NT Tissue Concentrations. The effects of acute administration of haloperidol, clozapine, olanzapine, quetiapine, risperidone, and sertindole on NT tissue concentrations were compared (Table 1). Haloperidol dose-dependently increased NT tissue concentrations in the anterior and posterior caudate nuclei, and both haloperidol and clozapine increased NT tissue concentrations in the nucleus accumbens. Although olanzapine and risperidone similarly increased NT concentrations in the nucleus accumbens, their effects were not limbic selective. The effects of olanzapine were similar to those of haloperidol (e.g., increasing NT concentrations in the nucleus accumbens and both the anterior and posterior caudate), whereas risperidone increased NT tissue concentrations in the nucleus accumbens and anterior caudate nucleus. Although sertindole had no effect on NT concentrations in the nucleus accumbens, NT concentrations in the anterior caudate were significantly increased at the highest dose examined. Quetiapine had no significant effect in any of the brain regions examined. None of the compounds examined had a significant effect on NT tissue concentrations in the prefrontal cortex, substantia nigra, VTA, or hippocampus.

Effects of Subchronic Administration of Typical and Atypical Antipsychotic Drugs on NT Tissue Concentrations. After subchronic administration of haloperidol (2.0 mg/kg/day), olanzapine (10.0 mg/kg/day), and risperidone (10.0 mg/kg) NT tissue concentrations remained elevated in the nucleus accumbens (Table 2). Although sertindole (2.0 and 10.0 mg/kg/day) had no effect on NT tissue concentrations in the nucleus accumbens after a single administration, both doses significantly increased NT concentrations in the nucleus accumbens after 14-day administration. Quetiapine (10.0 and 33.0 mg/kg) in contrast, significantly decreased NT tissue concentrations in the nucleus accumbens after 14-day administration. Haloperidol, olanzapine, risperidone, and sertindole also significantly increased NT concentrations in the anterior and posterior caudate. Subchronic administration of clozapine (10.0 mg/kg/day) significantly decreased NT concentrations in the posterior caudate.

Effect of Subchronic Administration of Typical and Atypical Antipsychotic Drugs on NT Receptor Binding. Subchronic administration of haloperidol significantly increased NT receptor binding in the substantia nigra (Fig. 1). In contrast, all of the atypical antipsychotic drugs tested significantly decreased NT receptor binding in the substantia nigra and the nucleus accumbens. In addition, quetiapine significantly decreased NT receptor binding in the VTA. There was a trend toward increased NT receptor binding in the posterior caudate after 14-day administration of the atypical antipsychotic drugs. None of the antipsychotic drugs tested had a significant effect on NT receptor binding in the prefrontal cortex or hippocampus.

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Effects of 14-day administration of typical and atypical antipsychotic drugs on NT-like immunoreactivity and NT receptor binding in the substantia nigra, nucleus accumbens, VTA, and posterior caudate. Data are mean ± S.E. of percentage of control. *P < .05 and **P < .001 compared with control.

	Discussion

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The introduction of increasing numbers of antipsychotic drugs with "atypical" clinical profiles (i.e., low incidence of extrapyramidal side effects and effectiveness in the treatment of negative symptoms) and relatively lower binding affinity at the D₂ DA receptor, has emphasized the need for identification of the biochemical effects common to all antipsychotic drugs. One potential candidate is increased NTergic neurotransmission in the mesolimbic projection system. Previous studies characterizing the effects of typical antipsychotic drugs (e.g., haloperidol and chlorpromazine) and the atypical antipsychotic drug clozapine on the NT system provided strong evidence for a role of increased limbic NT neurotransmission in the clinical efficacy of antipsychotic drugs (Govoni et al., 1980; Frey et al., 1986; Kilts et al., 1988; Radke et al., 1989, 1998; Merchant and Miller, 1994; Huang and Hanson, 1997), and for a role of increased NT neurotransmission in nigrostriatal projection regions in the extrapyramidal side effect liability of typical antipsychotic drugs (Govoni et al., 1980; Uhl and Kuhar, 1984; Frey et al., 1986; Radke et al., 1989, 1998; Giardino et al., 1990; Bolden-Watson et al., 1993; Merchant and Miller, 1994; Huang and Hanson, 1997).

In this study, we examined the effects of both acute and subchronic administration of typical and atypical antipsychotic drugs on NT tissue concentrations and NT receptor binding. We used the same doses and followed the same treatment protocol as Radke et al. (1998), allowing for the comparison of the acute and subchronic effects of administration of haloperidol, clozapine, and olanzapine on NT tissue concentrations, NT receptor binding, and extracellular NT release.

As previously described, both haloperidol and clozapine significantly increased NT tissue concentrations in the nucleus accumbens after single-dose administration of these compounds (Govoni et al., 1980; Frey et al., 1986). The increase in NT tissue concentrations in the nucleus accumbens observed 18 h after the injection of haloperidol or clozapine, has been reported to be preceded by an increase in c-fos mRNA expression (30 min after injection), an increase in NT extracellular release (1 h after injection), and an increase in NT/NN mRNA expression (4-7 h after injection), indicating that the increased NT tissue concentrations are most likely due to increased transcription in response to the acute release of NT. In this study, acute administration of olanzapine and risperidone also increased NT tissue concentrations in the nucleus accumbens. In contrast, quetiapine and sertindole had no effect in this brain region at any dose examined. It is possible that the lack of effect of quetiapine is due to the short half-life of quetiapine and its fast dissociation from D₂ receptors (Gefvert et al., 1997).

Clozapine exerted no effect on NT tissue concentrations in the caudate nucleus after acute administration, whereas olanzapine, risperidone, and sertindole all increased NT concentrations in at least one subdivision of the caudate nucleus. Although these effects do not fit in with the hypothesis that increased NT neurotransmission in the caudate nucleus may be associated with induction of extrapyramidal side effects (all three of these drugs are reported to have fairly low extrapyramidal side effect liability) the incidence of these side effects clearly increases with higher doses (Hoffman and Donovan, 1995). Whether the doses of drugs that significantly increased NT concentrations in the caudate nucleus are relevant to maximal clinically used doses remains to be determined and will require measurement of plasma and central nervous system levels of these agents and their metabolites, not a trivial task. Using a different treatment protocol (five injections of drug at 6-h intervals), Gygi et al. (1994) demonstrated that haloperidol and clozapine increase NT concentrations in different subregions of the caudate nucleus. It is therefore possible, that clozapine, olanzapine, risperidone, and sertindole increase NT tissue concentrations in subpopulations of neurons in the caudate nucleus that do not mediate the induction of extrapyramidal side effects.

There is a delay in the onset of clinical efficacy after treatment with antipsychotic drugs. Effects that are therefore seen only after subchronic or chronic administration of antipsychotic drugs are more likely to be related to the clinical efficacy of these compounds. For this reason we examined the effects of subchronic administration of these same antipsychotic drugs on NT tissue concentrations. However, because the mechanisms underlying changes in tissue levels (e.g., alterations in synthesis, release, or metabolism) are unclear, we also examined NT receptor binding in these same brain regions. NT receptor binding in these studies does not discriminate between NT binding at subtypes of the NT receptor. There are currently three cloned NT receptors, NT1 (Tanaka et al., 1990), NT2 (Chalon et al., 1996), and NT3 (Mazella et al., 1998). Although it is currently thought that the NT1 receptor is the subtype most closely associated with regulation of the DA system, the role of the NT2 and NT3 receptors has not yet been fully established.

Similar to the results of Frey et al. (1986), tolerance to the effects of clozapine on NT tissue concentrations in the nucleus accumbens developed after 14-day administration of this drug. However, judging by both the increased basal levels of NT in the extracellular fluid (Radke et al., 1998) and the decrease in NT receptor binding, it would appear that although tissue concentrations are not significantly different from control levels, there is an increase in NT neurotransmission in the nucleus accumbens. This is supported by the findings of Merchant et al. (1994) in which 28-day administration of haloperidol and clozapine increased NT/NN mRNA expression in the nucleus accumbens. An additional study by Kilts et al. (1988) in which rats received injections of clozapine (20.0 mg/kg i.p.) for 14 days did report an increase in NT tissue concentrations in the nucleus accumbens but used a much more sensitive micropunch dissection method.

In contrast to clozapine, subchronic administration of haloperidol increased NT tissue concentrations and basal NT release in the nucleus accumbens (Radke et al., 1998), but does not alter NT receptor binding in this same brain region. Differential effects of haloperidol on subregions of the nucleus accumbens may explain these effects. Huang and Hanson (1997) report that haloperidol increased NT tissue concentrations in the core, but not the shell, of the nucleus accumbens 24 h after a single injection. In addition, a very high dose (10.0 mg/kg) of haloperidol increased NT/NN mRNA expression in the subregion of the nucleus accumbens high in D₂ receptor binding, and decreased NT/NN mRNA expression in the ventromedial shell, a subregion high in D₃ receptor binding.

Examination of the effects of subchronic administration of olanzapine, risperidone, quetiapine, and sertindole indicate that these antipsychotic drugs may regulate the NT system by very different mechanisms. Similar to haloperidol, olanzapine increased NT concentrations in the nucleus accumbens and basal NT release (Radke et al., 1998) after 14-day administration. In contrast to haloperidol, however, olanzapine also significantly decreased NT receptor binding in the nucleus accumbens.

After subchronic administration, risperidone increased and quetiapine decreased NT tissue concentrations in the nucleus accumbens. At the same time, both drugs decreased NT receptor binding in this brain region in a manner similar to that of clozapine and olanzapine. Microdialysis studies examining the effects of subchronic administration of risperidone and quetiapine on extracellular NT release will further clarify their actions on this system.

As previously reported, subchronic administration of haloperidol increased NT receptor binding in the substantia nigra (Uhl and Kuhar, 1984; Giardino et al.,1990). All of the atypical antipsychotic drugs examined significantly decreased NT receptor binding in the substantia nigra. One plausible explanation of this finding is that drugs with high D₂ receptor affinity (e.g., haloperidol) act more on the D₂ receptor-responsive striatopallidal neurons, whereas the atypical antipsychotic drugs have greater effects on the D₁ receptor-responsive striatonigral neurons. D₂ antagonists increase NT-positive fibers in the ventral pallidum and globus pallidus (Brog and Zahm, 1996). In contrast, methamphetamine-induced increases in NT-positive terminals in the substantia nigra pars reticulata are blocked by antagonism of D₁ but not D₂ receptors (Letter et al., 1987; Castel et al., 1993, 1994).

It is unclear from these results which receptor subtypes are responsible for the effect of antipsychotic drugs on the NT system. All of the antipsychotic drugs examined in this study bind not only to the D₂ receptor but also have relatively high affinity for serotonergic receptors, -receptors, muscarinic receptors, adrenergic receptors (both the ₁-and ₂-subtypes), histamine₁ receptors, and other DA receptor subtypes (D₁, D₃, D₄) (Richelson, 1999). The ratio of binding affinities at different receptors (mainly 5-hydroxytryptamine₂/D₂ receptor-binding affinities) has been proposed to be responsible for the atypical profile of antipsychotic drugs (Seeman et al., 1997).

Although these studies do not conclusively support the hypothesis that increased NT neurotransmission is involved in the clinically relevant effects of all antipsychotic drugs, the extant evidence clearly suggests that further study is warranted. These results indicate that measurement of tissue concentrations is probably not an ideal means of detecting similarities and differences between pharmacological agents. Measurement of extracellular release of NT by using in vivo microdialysis remains an alternative, but the different subsets of NT neurons on which these drugs are having an effect must be identified.