Psychopharmacology

, Volume 170, Issue 1, pp 73–79

The 5-HT1A receptor agonist MKC-242 reverses isolation rearing-induced deficits of prepulse inhibition in mice

Authors

  • Masaki Sakaue
    • Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical SciencesOsaka University
  • Yukio Ago
    • Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical SciencesOsaka University
  • Akemichi Baba
    • Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical SciencesOsaka University
    • Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical SciencesOsaka University
Original Investigation

DOI: 10.1007/s00213-003-1515-x

Cite this article as:
Sakaue, M., Ago, Y., Baba, A. et al. Psychopharmacology (2003) 170: 73. doi:10.1007/s00213-003-1515-x

Abstract

Rationale

Prepulse inhibition (PPI) of startle provides an operational measure of sensorimotor gating in which a weak stimulus presented prior to a startling stimulus reduces the startle response. PPI deficits observed in schizophrenia patients can be modeled in rats by individual housing from weaning until adulthood. The deficits in PPI produced by isolation rearing can be reversed by antipsychotics. We previously found that (S)-5-[3-[(1,4-benzodioxan-2-ylmethyl)amino]propoxy]-1,3-benzodioxole HCl (MKC-242), a highly potent 5-HT1A receptor agonist, reduced aggressive behavior selectively in isolation-reared mice.

Objective

This study examines whether isolation rearing of mice produces PPI deficits and whether PPI deficits are attenuated by 5-HT1A receptor activation.

Methods

Male ddY mice, 4 weeks old, were housed for more than 6 weeks singly or in groups of five or six. The PPI of the acoustic startle response was measured using SR-LAB systems.

Results

The PPI was less in isolation-reared mice than in group-reared mice. Oral administration of MKC-242 at 0.1–0.3 mg/kg reversed PPI deficits in isolation-reared mice, although it did not affect PPI in group-reared mice. MKC-242 did not affect MK-801-induced and apomorphine-induced PPI deficits in group-reared mice. The reversal by MKC-242 of isolation-induced PPI deficits was antagonized by the 5-HT1A receptor antagonist WAY100635 at low doses.

Conclusion

These results suggest that isolation rearing produces deficits in sensorimotor gating in mice that are reversible by activation of 5-HT1A receptors, probably somatodendritic 5-HT1A autoreceptors.

Keywords

Prepulse inhibition5-HT1A receptor agonistMKC-242Isolation rearingMiceSchizophrenia

Introduction

Prepulse inhibition (PPI) refers to the normal inhibition of a startle response when a weak stimulus (the prepulse) immediately precedes the intense startling stimulus (the pulse) (Graham 1975). PPI levels indicate the current integrity of the sensorimotor gating mechanism by measuring the extent to which current information processing routines elicited by the prepulse are interrupted by the subsequent startling stimulus. Reduced levels of PPI have been reported in patients with several neuropsychiatric disorders, including schizophrenia (Braff et al. 1978, 1992), obsessive-compulsive disorder (Swerdlow et al. 1993), and Huntington's disease (Swerdlow et al. 1995). A number of studies show that PPI is disrupted in isolation-reared rats compared to socially reared controls (Geyer et al. 1993; Wilkinson et al. 1994; Bristow et al. 1995; Varty and Higgins 1995; Bakshi et al. 1998; Domeney and Feldon 1998; Bakshi and Geyer 1999; Heidbreder et al. 2001), although the effect of isolation rearing on PPI has not been studied in mice. Social isolation also induces aggressive behavior in several strains of rats and mice (Valzelli 1969; Valzelli and Garattini 1972), and the isolation-induced aggression is attenuated by serotonin (5-HT)1A receptor agonists (Olivier et al. 1989; White et al. 1991; Sánchez et al. 1993; De Boer et al. 1999; Mendoza et al. 1999). However, it is not known whether the isolation-induced deficits in PPI are also reversed by 5-HT1A receptor activation. We have reported that (S)-5-[3-[(1,4-benzodioxan-2-ylmethyl)amino]propoxy]-1,3-benzodioxole HCl (MKC-242), a highly potent 5-HT1A receptor agonist (Matsuda et al. 1995b), is a strong inhibitor of isolation-induced aggressive behavior in mice (Matsuda et al. 2001; Sakaue et al. 2001). MKC-242 has anxiolytic-like and antidepressant-like effects (Matsuda et al. 1995a; Abe et al. 1996, 1998). The aim of this study is to examine whether isolation rearing of mice causes PPI deficits and whether PPI deficits are attenuated by 5-HT1A receptor activation.

Materials and methods

Animals

Male ddY mice (4 weeks old) were either housed in groups of five or six per cage (24×17×12 cm) or isolated in the same size cage for more than 6 weeks before experiments under controlled environmental conditions (22±1°C; 12-12 light-dark cycle, lights on at 0800 hours, food and water ad libitum) (Matsuda et al. 2001; Ago et al. 2002). Different mice were used in each experiment. Procedures involving animals and their care were conducted according to Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society.

Apparatus

All PPI testing occurred within startle chambers acquired from San Diego Instruments (San Diego, Calif., USA). Each startle chamber consists of a 5.1 cm (outside diameter) Plexiglas cylinder mounted on a platform (20.4 cm length×12.7 cm width×0.4 cm thick) with a piezoelectric accelerometer unit attached below the Plexiglas cylinder. The piezoelectric unit transduces vibrations into signals that are rectified and stored by a microcomputer interface. The Plexiglas cylinder and platform are located in a sound attenuated chamber (San Diego Instruments) with a loudspeaker (28 cm above the cylinder), and house light. Calibration procedures using a vibrating standardization unit (San Diego Instruments) were performed between experiments to ensure equivalent sensitivities across chambers. The sound levels for the background noise and various stimuli in each chamber were calibrated with a digital sound level meter.

Acoustic startle response profile

Each test session began by placing a subject in the Plexiglas cylinder where it was left undisturbed. After a background noise of 65 dB was presented for the 5-min acclimation period, each subject was exposed to five consecutive blocks with a total of 100 trials over the approximately 30-min test session. One block consisted of 20 trials including six different trial types: Pulse-alone trials in which a 40 ms broadband 120 dB burst was presented; four different Prepulse+pulse trials in which the onset of a 20 ms broadband noise preceded the onset of the 120 dB startle pulse by 100 ms (prepulse intensities that were 1, 3, 6 and 12 dB above the 65 dB background noise were used); and No stim trials in which only the background noise was presented. Trials were presented in a pseudo-random order, separated by an average of 15 s (range: 7–23 s). The startle response was recorded for 100 ms (measuring the response every 1 ms) starting at the onset of each startle stimulus. The maximum startle amplitude recorded during the 100-ms sampling window was used as the dependent variable.

Drugs

The following drugs were used: MKC-242 and N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide (WAY100635) (Mitsubishi Pharma Co., Yokohama, Japan); dizocilpine (MK-801) and risperidone (Sigma Chemical Co., St Louis, Mo., USA); apomorphine and (±)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (RBI, Natick, Mass., USA). All other chemicals used were of the highest commercially available purity. MKC-242 was freshly suspended in 0.5% w/v carboxymethylcellulose. Risperidone was dissolved in saline containing <0.1% v/v acetic acid. WAY100635, MK-801 and 8-OH-DPAT were dissolved in saline. Apomorphine was dissolved in saline containing 0.1% w/v ascorbic acid. Drugs were administered at 10 ml/kg orally (MKC-242), intraperitoneally (8-OH-DPAT, WAY100635, risperidone, MK-801) or subcutaneously (apomorphine).

Data analyses

The amount of PPI was calculated as a percentage score for each prepulse trial type. The following formula was used: % PPI=100−{[(startle response to Prepulse+pulse trial)/(startle response to Pulse-alone trial)]×100}. Startle amplitude was calculated as the average response to all of the Pulse-alone trials. Data were analyzed using Student's t-test, two-way analysis of variance (ANOVA) followed by the Tukey-Kramer test and one-way ANOVA followed by the Dunnett test. Statistical analyses were performed using a software package (Stat View 5.0) for Apple Macintosh computer. P values of 5% or less were considered statistically significant.

The main effects of prepulse intensity were not discussed, since they were always significant. PPI was also analyzed for each prepulse trial type. Data from the No stim trials are not included in Results because the values were negligible relative to values on trials containing startle stimuli. The habituation of the startle response was investigated by averaging the startle amplitudes for each block (each block containing eight Pulse-alone trials) and measuring the decrease across five blocks.

Results

We first examined the effects of 5-HT1A receptor ligands on PPI of the acoustic startle response in group-reared mice. The 5-HT1A receptor agonists MKC-242 [F(3,65)=1.663, P=0.1842] and 8-OH-DPAT [F(2,38)=2.546, P=0.0924] and the 5-HT1A receptor antagonist WAY100635 [F(2,41)=0.876, P=0.4244] did not affect PPI in group-reared mice (Fig. 1). Apomorphine (1.0 mg/kg, SC) caused a marked reduction of PPI of the acoustic startle response in group-reared mice (P=0.0005, analyzed by Student's t-test), and this disruption was reversed by risperidone [F(1,57)=13.398, P=0.0006], but not by MKC-242 at doses from 0.1 to 1.0 mg/kg [F(3,58)=0.763, P=0.5196] (Fig. 2). MKC-242 [F(3,58)=1.155, P=0.3354] or risperidone [F(1,57)=2.362, P=0.1302] with and without apomorphine did not affect startle response in group-reared mice (data not shown). We also observed that MK-801 (0.5 mg/kg, IP) caused a marked reduction of PPI of the acoustic startle response in group-reared mice (P<0.0001, analyzed by Student's t-test), but the disruption was not reversed by MKC-242 at doses from 0.1 to 1.0 mg/kg [F(3,66)=0.681, P=0.5667] (data not shown).
Fig. 1.

Effects of 5-HT1A receptor ligands on PPI of the acoustic startle response in group-reared mice. MKC-242 (0.1, 0.3, 1.0 mg/kg) was adminstered orally 60 min, and 8-OH-DPAT (0.3, 1.0 mg/kg) and WAY100635 (0.1, 1.0 mg/kg) were injected IP 30 min before the experiments. The percentage of PPI in group-reared mice is shown for 77 dB of prepulse intensity. Results were means±SEM of 13–17 mice

Fig. 2.

Effects of MKC-242 and risperidone on apomorphine-induced disruption of PPI of the acoustic startle response in group-reared mice. MKC-242 (0.1, 0.3, 1.0 mg/kg, PO) and risperidone (0.3 mg/kg, IP) were administered 60 and 30 min, respectively, before saline or apomorphine (1.0 mg/kg, SC), which was injected 10 min before the experiment. The percentage of PPI in group-reared mice is shown for 77 dB of prepulse intensity. Results were means±SEM of 14–15 mice. **P<0.01, compared with the vehicle/saline-treated group, and ††P<0.01, compared with the vehicle/apomorphine-treated group

Figure 3 shows the effect of isolation rearing on PPI of the acoustic startle response in mice. PPI of the acoustic startle response was less in isolation-reared mice than in group-reared mice (analyzed by Student's t-test). Isolation-reared mice exhibited more pronounced startle response than group-reared mice (P<0.0001, analyzed by Student's t-test), although there was no difference in startle habituation between isolation- and group-reared mice (Fig. 4). Repeated two-way ANOVA revealed significant main effects of block [F(4,120)=20.746, P<0.0001], indicating normal habituation of the startle response. However, there were no significant interactions between block and rearing [F(4,149)=1.981, P=0.1023], suggesting that isolation-reared mice were not impaired in startle habituation.
Fig. 3.

Effect of isolation rearing on PPI of the acoustic startle response in mice. The percentage of PPI in group- (open symbols) and isolation-reared (closed symbols) mice is shown for each prepulse intensity. Results were means±SEM of 13–17 mice. *P<0.05, **P<0.01, ***P<0.001, compared with group-reared mice

Fig. 4.

Effect of isolation rearing on habituation of the acoustic startle response in mice. The averages of startle amplitude for each block in group- (open symbols) and isolation-reared (closed symbols) mice are shown. Results were means±SEM of 13–17 mice

Isolation-induced disruption of PPI of the acoustic startle response was reversed by MKC-242 [F(3,51)=2.973, P=0.0409] and risperidone [F(3,51)=12.353, P<0.0001] in a dose-dependent manner (Fig. 5). The significant effect of MKC-242 was observed at 0.1 and 0.3 mg/kg, but not at 1.0 mg/kg. MKC-242 showed a tendency to decrease the startle response, although it was not significant [F(3,51)=1.550, P=0.2138] (Table 1). In contrast, risperidone decreased the startle response in a dose-dependent manner [F(3,51)=10.811, P<0.0001] (Table 1). The effect of MKC-242 was antagonized by WAY100635 at low doses of 0.03 and 0.1 mg/kg [F(2,89)=6.437, P=0.0025] (Fig. 6). No experiment showed a statistically significant effect of any drug on startle habituation.
Fig. 5.

Effects of MKC-242 and risperidone on the disruption of PPI of the acoustic startle response in isolation-reared mice. MKC-242 (0.1, 0.3, 1.0 mg/kg, PO) and risperidone (0.1, 0.3, 1.0 mg/kg, IP) were injected 60 min and 30 min before the experiments, respectively. The percentage of PPI in isolation-reared mice is shown for 77 dB of prepulse intensity. Results were means±SEM of 13 mice. *P<0.05, **P<0.01, compared with the vehicle-treated mice

Table 1.

Effects of MKC-242 and risperidone on startle amplitude in isolation-reared mice. In experiment 1, vehicle and MKC-242 at the indicated doses were administered orally 60 min before the experiment. In experiment 2, vehicle and risperidone at the indicated doses were injected IP 30 min before the experiment. Results are means±SEM of 13 mice

Treatment

Average startle amplitude

Experiment 1

Vehicle

1047±82

MKC-242

0.1 mg/kg

779±170

0.3 mg/kg

674±134

1.0 mg/kg

861±103

Experiment 2

Vehicle

1006±78

Risperidone

0.1 mg/kg

826±77

0.3 mg/kg

480±82**

1.0 mg/kg

452±91**

**P<0.01, compared with vehicle-treated group

Fig. 6.

Effect of WAY100635 on MKC-242-induced increase in PPI of the acoustic startle response in isolation-reared mice. WAY100635 (0.03, 0.1 mg/kg, IP) and MKC-242 (0.3 mg/kg, PO) were injected 90 min and 60 min before the experiments, respectively. The percentage of PPI in isolation-reared mice is shown for 77 dB of prepulse intensity. Results were means±SEM of 15 mice. **P<0.01, compared with the saline/vehicle-treated group, and ††P<0.01, compared with the saline/MKC-242-treated group

Discussion

Since psychotomimetic compounds such as amphetamine, apomorphine, MK-801 and phencyclidine have been reported to decrease PPI in animals (Geyer et al. 2001), these drugs have been used in animal models with PPI deficits. However, these methods are not concerned with environmental or developmental factors in the ontogeny of PPI deficits. Isolation rearing has received considerable attention as an animal model of sensorimotor gating deficits in schizophrenia, since it has a developmental perspective. In this study, we examined whether isolation rearing of mice causes PPI deficits of the acoustic startle response and then whether PPI deficits are reversed by 5-HT1A receptor activation.

We observed that isolation rearing caused PPI deficits of the acoustic startle response in mice, in agreement with previous studies in rats (Geyer et al. 1993; Wilkinson et al. 1994; Bristow et al. 1995; Varty and Higgins 1995; Bakshi et al. 1998; Domeney and Feldon 1998; Bakshi and Geyer 1999; Heidbreder et al. 2001). The finding that the mouse is also a useful animal to study isolation rearing-induced PPI deficits appears to be important, in view of recent development of gene-targeting strategy in mice. The present study also showed that isolation rearing resulted in an increase in startle reactivity, as evidenced by the elevated startle magnitude in isolation-reared mice compared to group-reared mice. This effect on startle magnitude is consistent with previous studies in rats (Geyer et al. 1993; Wilkinson et al. 1994; Varty and Higgins 1995; Bakshi et al. 1998). However, other studies have indicated that PPI deficits can be observed in isolation-reared rats in the absence of alterations in startle reactivity to the Pulse-alone trials during the PPI test session (Wilkinson et al. 1994; Bristow et al. 1995; Bakshi and Geyer 1999). It is likely that startle amplitude and PPI are independent variables (Bakshi et al. 1998; Cilia et al. 2001).

The most important finding of this study is that MKC-242 reverses isolation rearing-induced PPI deficits of the acoustic startle response without any effect on the startle amplitude. In contrast to MKC-242, risperidone at higher doses not only reversed the PPI deficits of the acoustic startle response but also attenuated the startle amplitude. This supports the idea as discribed above that startle amplitude and PPI are independent variables. The effect of MKC-242 was antagonized by the selective 5-HT1A receptor antagonist WAY100635, suggesting the involvement of 5-HT1A receptors. The present study shows that neither the 5-HT1A receptor agonists 8-OH-DPAT and MKC-242, nor the 5-HT1A receptor antagonist WAY100635, affect PPI of the acoustic startle response in group-reared mice of ddY strain. In contrast, previous studies showed that 8-OH-DPAT increased PPI in mice (Dulawa and Geyer 2000; Dulawa et al. 1997, 2000), and that the 5-HT1A receptor agonist decreased PPI in rats (Rigdon and Weatherspoon 1992; Sipes and Geyer 1995). The apparent discrepancy of the effects of 8-OH-DPAT might be due to a strain difference, since Dulawa and Geyer (2000) reported that the significant effect of 8-OH-DAPT on PPI was observed in 129 Sv and ICR strains, but not in C57BL/6.

5-HT1A receptors are expressed both presynaptically as autoreceptors by 5-HT containing neurons, and postsynaptically by a variety of other neurons (Hall et al. 1985; Azmitia et al. 1996). It appears to be important to examine which receptors are involved in the effects of 5-HT1A receptor agonists. In this respect, the presynaptic 5-HT1A receptors are considered more sensitive to agonists than the postsynaptic 5-HT1A receptors because the autoreceptors possess a large receptor reserve (Meller et al. 1990; Yocca et al. 1992). In this study, we observed the significant effect of MKC-242 at low doses but not at a high dose on isolation-induced PPI deficits. In addition, we observed that the effect of MKC-242 on isolation-induced PPI deficits was blocked by WAY100635 at lower doses. We found in a separate experiment that a low dose of WAY100635 antagonized presynaptic 5-HT1A receptor-mediated modulation of cortical 5-HT release but not postsynaptic 5-HT1A receptor-mediated modulation of cortical DA release in mice (unpublished data). Taken together, these findings suggest that the presynaptic 5-HT1A receptors play a key role in the effect of MKC-242 on isolation-induced PPI deficits of the acoustic startle response.

Several studies have reported isolation rearing-induced changes in the function of serotonergic neurons in isolation-reared rats (Gentsch et al 1982; Wright et al. 1991; Jones et al. 1992; Bickerdike et al. 1993; Fone et al. 1996; Whitaker-Azmitia et al. 2000). These include isolation rearing-induced increases in responsiveness of postsynaptic 5-HT1A, 5-HT2A and 5-HT2C receptors. In addition, we have recently found that the function of 5-HT1A receptors coupled selectively with cortical dopamine release is lower in isolation-reared mice (Ago et al. 2002). These observations suggest the dysfunction of serotonergic neurons in isolation-reared animals. The present study shows that the effect of MKC-242 on isolation rearing-induced PPI deficits is mediated by presynaptic 5-HT1A autoreceptors. Activation of the presynaptic 5-HT1A receptors results in a reduction of the firing rate of the serotonergic neurons and suppression of 5-HT synthesis, 5-HT turnover, and 5-HT release in projection areas (Barnes and Sharp 1999). This causes a reduction in signaling via all subtypes of 5-HT receptors at target cells. Then, the presynaptic 5-HT1A receptor activation may improve isolation rearing-induced changes in brain dopaminergic neurons (Hall et al. 1998; Matsuda et al. 2001) which play a role in PPI deficits (Geyer et al. 2001). The exact mechanism for the effect of MKC-242 on isolation rearing-induced PPI deficits is not known.

There is accumulating evidence suggesting the role of 5-HT1A receptors in schizophrenia. Post-mortem studies show that 5-HT1A receptors are increased in the prefrontal cortex of schizophrenic patients (Hashimoto et al. 1991; Sumiyoshi et al. 1996). Microdialysis studies show that 5-HT1A agonists increase DA release in the frontal cortex in rats (Wedzony et al. 1996; Sakaue et al. 2000) and mice (Matsuda et al. 2001; Ago et al. 2002). In addition, in vitro receptor binding studies report that atypical neuroleptics have affinity for 5-HT1A receptors (Mason and Reynolds 1992; Newman-Tancredi et al. 1998). In this regard, Rollema et al. (1997; 2000) reported that atypical antipsychotics increased dopamine release in the prefrontal cortex by 5-HT1A receptor activation. These observations suggest that 5-HT1A receptors may be implicated in the pathophysiology of schizophrenia. In this line, the present study shows that 5-HT1A receptor activation reverses the PPI deficits in isolation-reared mice. In contrast to risperidone, MKC-242 did not affect the PPI deficits induced by apomorphine or MK-801 in group-reared mice. It appears that 5-HT1A receptor activation is effective in improving a limited type of PPI deficits.

In summary, isolation rearing causes PPI deficits of the acoustic startle response in mice and the PPI deficits are reversed by 5-HT1A receptor activation. The findings implicate a role of 5-HT1A receptors in treatment of schizophrenia.

Acknowledgements

This work was supported by grants from Grant-in-Aid for Scientific Research and Mitsubishi Pharma Co. We acknowledge the donation of MKC-242 and WAY100635 from Dr. M. Egawa (Mitsubishi Pharma Co.).

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