The effects of inositol treatment in animal models of psychiatric disorders

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Abstract

Clinical trials indicate that inositol may be effective in the treatment of patients with depression, panic disorder and obsessive compulsive disorder (OCD), but not in the treatment of patients with schizophrenia, Alzheimer’s disease, ADHD or autism. This spectrum of clinical action parallels that of serotonin selective reuptake inhibitors (SSRIs), but inositol is a precursor in the phosphatidylinositol cycle, a second messenger system distal to the receptor for 5HT-2. To study its mechanism of therapeutic action there is a need to test inositol’s activity in animal models of psychopathology. In rats, chronic inositol was demonstrated to increase activity levels, reduce immobility time in the forced swim test and in the reserpine-induced hypoactivity models of depression, and reduce anxiety-like behaviors in the elevated plus-maze. The reduction in anxiety-like behaviors appears to be related to baseline levels of activity. Inositol treatment was not observed to have any effect on amphetamine-induced hyperactivity, apomorphine-induced stereotypy, or on the performance of memory tasks by monkeys. Clinical controlled trials of inositol in patients with depression, panic disorder, and OCD were small, and positive psychoactive effects in animals clearly strengthen the case for further clinical trials and potential for general therapeutic use in humans.

Introduction

The development of novel drugs for the treatment of psychiatric disorder and the understanding of their mechanism of action is dependent on our ability to test pharmaceutical compounds in appropriate animal models of these diseases (Abramson and Seligman, 1977, Willner, 1991a). Beyond the thoroughly discussed problematics of animal models for psychiatric disorders (Rodgers, 1997, Willner, 1991b), it is clear that their use is still the best option to screen potential novel drug treatments in psychiatry.

Inositol is an endogenous polyol that has emerged during the last few years as a new possible treatment in psychiatry (Levine, 1997). Inositol is the precursor of the PI cycle in the cell and as such is necessary for the production of two second messengers: Inositol triphosphate3 (IP3) and DAG. Unlike most psychotropic drugs, inositol is probably not acting directly in the synapse but in second messenger systems distal to receptors.

Inositol does not readily enter the brain and only about 3% of plasma inositol crosses the blood brain barrier (Spector, 1998). Yet, exogenously administered oral inositol was demonstrated to increase CSF inositol levels in chronic schizophrenic patients (Levine et al., 1993c). In rats, acute intraperitoneal injection of 5 g/kg resulted in increased inositol levels in cortex, hypothalamus and hippocampus (Patishi et al., 1996) and chronic dietary inositol (5% in food for 3 weeks) elevated inositol levels in the cortex and hippocampus (Kofman et al., 1998).

A possible relevence of inositol to psychiatric disorders was first suggested by Barkai et al. (1978) who reported that patients with affective disorders had reduced CSF inositol levels. Although these findings were not replicated (Levine et al., 1996), recent studies reported reduced inositol level in frontal cortex of post mortem brains of patients with bipolar disorder and victims of suicide (Shimon et al., 1997). Furthermore, clinical studies with inositol demonstrated that it may have therapeutic value in the treatment of depression (Levine, 1997, Levine et al., 1993a, Levine et al., 1995a), depression accompanying post-traumatic stress disorder (Kaplan et al., 1996), panic disorder (Benjamin et al., 1995), and obsessive-compulsive disorder (OCD; Fux et al., 1996). In contrast, no therapeutic effects were found in schizophrenic patients (Levine et al., 1993b, Levine et al., 1994), Alzheimer patients (Barak et al., 1996), individuals with Attention Deficit Hyperactivity Disorder (Levine et al., 1995b), or autistic patients (Levine et al., 1997). This clinical spectrum parallels that of the anti-depressant serotonin selective uptake inhibitors (SSRI’s).

The present paper reviews a variety of studies of the effects of inositol treatment on animal behavior. Two studies tested the effects on normal behavior and the others tested effects on animal models of psychopathology. The goal of these studies was to confirm psychoactive effects for this compound which had been studied in only a small number of patients, and to define at least one robust effect of inositol in an animal model that could be used for detailed neurochemical studies of mechanism and dose–response relationships.

The effects of inositol treatment were tested in three different conditions related to depression: activity levels in normal rats; reserpine induced hypoactivity; the Porsolt forced swimming test.

Two studies examined the effects of inositol on activity level in rats, the first one tested the effects of acute intraperitoneal inositol treatment (Kofman et al., 1993) and the second examined the effects of chronic oral inositol administration (Kofman et al., 1998).

For the first study, Sprague Dawley rats were housed 4 per cage in a temperature controlled (23°C) and 12 h light dark cycle colony room, with food and water provided ad lib. All tests were done during the dark phase of the light dark cycle. Rats were divided to five groups which received saline (n=8); glucose 1 g/kg (n=10); glucose 5 g/kg (n=10; inositol 1 g/kg (n=9); and inositol 5 g/kg (n=10). All drugs were diluted in distilled water and administered via intra-peritoneal injection in a volume of 2 ml/100 g weight. Two hours after injection, rats were placed in automated activity monitors (Optivarimax, Columbus Instruments, USA) and their behavior monitored for horizontal and vertical activity for 20 min.

The vertical activity (number of rearings) of the inositol 1 g/kg group was significantly higher than of the other groups (ANOVA: F(4,42)=3.18, P=0.022; followed by Neuman–Keuls post-hoc test). No other significant effects were found but a similar non-significant trend was demonstrated for horizontal activity induced by the same inositol dose (1 g/kg).

For the second study, 20 male rats were housed 5 per cage in similar conditions to the previous experiments. Rats received ad lib food containing 5% inositol or 5% glucose plus mannitol at 1:2 ratio for 3 weeks. On day 22 of treatment, rats were placed in activity monitors for 30 min. Total ambulatory activity as well as horizontal activity (rearing) levels were measured and served for later analysis.

Results show that inositol treatment induced increased ambulation (approximately 30% increase, F=11.64, P=0.003) and increased rearing (approximately 60% increase, F=16.27, P<0.0008) compared with control animals.

Two experiments were conducted to test the effects of inositol in the reserpine induced hypoactivity model of depression. The difference between the two experiments was only in the dose of reserpine used to induce hypoactivity.

Rats (Sprague Dawley males weighing 200–250 g at the beginning of experiment) were housed 4 per cage, with free access to food and water, in a temperature controlled (22°C) animal colony with 12 h light dark cycle. All treatments and experimental procedures were conducted during the light phase of the cycle.

A treatment group (n=10 for each experiment) received 14 daily inositol injections (1.2 g/kg diluted in deionized water to injection volume of 12 ml/kg). A control group (n=10 for each experiment) received equivalent injections of glucose: mannitol in a 1:2 ratio. During the last 3 days of each experiment all rats were also given daily subcutaneous (SC) reserpine injections. The dose of reserpine was 0.5 mg/kg for the first experiment and 0.25 mg/kg for the second experiment. Reserpine was diluted in deionized water, with a minimal amount of citric acid added, to injection volume of 2 ml/kg.

Thirty minutes after the last reserpine injection, rats were placed in automated activity monitors (Elvicom, Israel) and their behavior monitored for ambulatory and vertical activity for 30 min. Since the activity monitors are not sensitive enough to detect locally oriented activity (and therefore differentiate between complete immobility and small movements), manual scoring of behavior was also performed and an experimenter scored complete immobility periods of rats for 1 min for each 10 min of the test session (totalling 3 min per rat). Data for all measures (ambulatory behavior, vertical activity, and immobility score) were analyzed utilizing a student’s t-test.

As shown in Table 1, inositol treatment was effective in reducing complete immobility time in both experiments. A similar trend was also demonstrated in the second experiment for increased ambulatory behavior but did not reach statistical significance.

Two experiments were conducted to test the effects of chronic treatment with intraperitoneal (experiment 1) or oral (experiment 2) inositol on rats’ performance in the Porsolt forced swim test, an established model of depression (Porsolt et al., 1978, Borsini and Meli, 1988).

Experiment 1 was designed to test a number of doses of inositol. Accordingly, 3 groups of rats received 2 weeks of daily intraperitoneal inositol injections at different doses: 0.3 g/kg (n=9); 0.6 g/kg (n=10); and 1.2 g/kg (n=8). All doses were diluted in deionized water to volume of 12 ml/kg. Control rats (n=10) received injections of 1.2 g/kg, 1:2 glucose: mannitol solution.

For experiment 2, rats were fed for 14 days with inositol (10% in powdered rat chow, n=20) or 1:2 glucose: mannitol (10% in powdered rat chow, n=20). The powdered food replaced the regular food chow in this experiment and was provided ad lib. Since normal daily food consumption of rats is approximately 10% of body weight, it can be estimated that the oral daily inositol dose in this experiment ∼2.5–3 g.

Swim exposure for both experiments was conducted during days 13 and 14.

The Porsolt swim test includes two exposures to a water tank, spaced 1 day apart (Borsini and Meli, 1988, Porsolt et al., 1978, Sanchez and Meier, 1997). For these experiments the tank sizes were 22 cm in diameter and 40 cm in height. The tank had a rounded lid and contained 20 cm high fresh water at 25°C. During the first exposure, rats were placed into the tank and left there for 10 min. During the second exposure (test session), rats were placed in the tank and left there for 5 min during which their behavior was videotaped.

Videotapes of the test session were scored by a blind observer for complete immobility time, small movements time, and vigorous struggle time. The division of activity levels to three rather than the traditional 2 (yes or no) levels is an elaboration of the method that may be more appropriate to examine the effects of different treatments (Borsini and Meli, 1988). Results were analyzed utilizing one-way analysis of variance (ANOVA) followed by LSD post-hoc tests for experiment 1, and by a student’s t-test for experiment 2. Significance level was set at P<0.05.

Table 2 shows the results of the forced swim test experiments. These results demonstrate that chronic inositol treatment with intraperitoneal 1.2 g/kg dose, but not with lower doses, significantly reduced immobility time compared with control animals (ANOVA: F(4)=3.07586, P=0.026; post-hoc tests: inositol (1.2 g/kg) different than control, and increased struggle time compared with controls (ANOVA: F(4)=6.23, P=0.0005; post-hoc test inositol (1.2 g/kg) different than control). Similar results were obtained for rats treated chronically with oral inositol (10% in powdered rat chow). Reduced immobility time was observed compared with control rats (t-test: t(38)=3.77286, P=0.0006) and struggle time in the treatment group appears increased although the difference did not reach statistical significance (t-test: t(38)=1.76297, P=0.086).

The elevated plus-maze serves to model anxiety-like behavior in rodents. It is based on a conflict situation between the exploratory drive of rodents and their aversion from open spaces (e.g. Rodgers and Cole, 1994). The model is one of the most frequently used models in psychopharmacology and had been validated in numerous studies (for review see File, 1992). The first attempt to examine the effects of inositol treatment in the plus-maze was done by Cohen et al. (1997a). In that study, the effects of a variety of acute doses injected peripherally, two doses injected acutely in intracerebroventricular injection, and one dose injected chronically and intraperitoneally were examined in the elevated plus-maze.

Results of the dose–response study of acute peripheral inositol treatment (0.03–2.5 g/kg) indicated that a single peripheral injection of inositol may have an anxiogenic effect compared with control solution (data not shown). However, a single ICV injection of 5 mg inositol resulted in reduced anxiety-like behaviors in the elevated plus-maze (data not shown). Thus, the data regarding the acute effects of inositol in this model are not clear. In human studies, inositol treatment was demonstrated to have a therapeutic effect in anxiety disorders only after chronic treatment (Benjamin et al., 1995). Therefore the evaluation of the effects in the model in a chronic treatment paradigm appears to be the most appropriate.

To examine the effects of chronic inositol, groups of rats (n=10 per group) were treated with daily intraperitoneal injections of inositol (1.25 g/kg diluted to 10% in de-ionized water) or control solution (1:2 glucose/mannitol at equal concentration and dilution) for 14 days. Two hours after the last injection rats were tested in the elevated plus-maze. As shown in Fig. 1, rats treated chronically with inositol spent significantly more time in the open arms of the maze and demonstrated a similar trend in the measure of number of entries to these arms. These measures indicate that chronic inositol treatment has anxiolytic-like properties in the elevated plus-maze model. In line with the results of other studies (Kofman et al., 1993, Kofman et al., 1998), the inositol group also demonstrated increased activity compared with the control group as observed by increased number of entries to both the open and the closed arms of the maze (data not shown). This finding could imply that the anxiolytic-like effect is an artifact of hyperactivity but the fact that inositol significantly increased the time spent in the open arms supports a specific anxiolytic-like activity.

Support for the previous findings was found recently in a study of the anxiolytic-like effect in the elevated plus-maze of inositol and one of its isomers, epi-inositol (Einat et al., 1998a). Chronic intraperitoneal inositol treatment was demonstrated again to produce anxiolytic-like effects in the elevated plus-maze as shown by increase time spent and number of entries to the open arms of the maze (Table 3). Interestingly, epi-inositol appeared to be even more potent than myo-inositol in that model (data not shown).

In contrast to the previous intraperitoneal findings, an attempt to examine the consequences of oral inositol treatment (5% in food for 2 weeks) in the plus-maze did not indicate any anxiolytic-like effects (Einat et al., unpublished observations). The observation that intraperitoneal but not oral inositol has anxiolytic-like activity appears unusual because orally administered inositol was reported to increase brain inositol levels and to have behavioral effects in intact rats (Kofman et al., 1998). However, the fact that SSRIs are effective in the treatment of patients with anxiety disorders but do not have any anxiolytic effects in normal controls (e.g. Gelfin et al., 1998) suggested the hypothesis that inositol may be more active in stressed than in unstressed animals. If that is the case than rats chronically treated with injections and therefore chronically stressed by that paradigm, should respond more readily to inositol treatment than rats who received inositol in their food in a non-stressful manner. To test this hypothesis two studies were designed that included: (a) an examination of the effects of oral inositol treatment after chronic mild stress; and (b) a study of the effects of chronic oral inositol after acute stress (Kofman et al., 2000).

The first study examined the effects of chronic mild stress and inositol treatment in the elevated plus-maze. Rats were divided to 4 groups (n=10 per group) in a 2×2 design with oral inositol (5% in food for 3 weeks) treatment as one factor and chronic mild stress (daily saline injection 20 ml/kg for the last 14 days of the experiment) as the second factor. Results indicated that as shown before (e.g. Lister, 1987, Pellow et al., 1985, Treit et al., 1993), stress had a significant effect on the performance of rats in the elevated plus-maze. The effect of inositol on behavior in the plus-maze was not significant but a near-significant trend was demonstrated for the interaction between inositol and stress (data not shown). This offers support to the hypothesis that inositol effects on anxiety may be related to the level of anxiety.

The second experiment was designed to test the effects of chronic oral inositol treatment on anxiety-like behavior after acute stress. The paradigm used to induce stress was a single exposure to a cat. A single exposure to a predator was demonstrated to increase anxiety-like behaviors in rodents and was stipulated to model pathologic anxiety mechanisms (Hendrei et al., 1996) as well as behaviors related to PTSD (Adamec and Shallow, 1993, Cohen et al., 1997b). For the present experiment, rats were fed pellets containing 5% inositol (n=10) or 5% 1:2 glucose mannitol (n=10) for 19 days. On day 18 all rats were exposed for 10 min to a domestic cat (no physical contact was permitted) and 24 h later each rat was tested in the elevated plus-maze. Results indicate that inositol treatment reduced anxiety-like behaviors in this paradigm as observed by a significantly higher number of rats from the inositol group that entered the open arms (8/10 in the inositol group, 3/10 in the control group, χ2=5.05. P<0.02); by increased number of entries to the open arms (t=2.48, P=0.024) and by a strong trend of the inositol treated rats to spend more time in the open arms than the control animals (t=2.0, P=0.06).

The results of the last experiment combined with the trend presented in the previous experiment add support to the hypothesis that inositol’s anxiolytic-like effect is stronger when the level of anxiety is higher than normal.

Amphetamine-induced hyperactivity and apomorphine-induced stereotypy are both frequently used to models for some aspects of mania (Lyon, 1991a) and schizophrenia (Lyon, 1991b) respectively. To evaluate the possible effects of inositol in these models two preliminary experiments were conducted (Kofman et al., 1993).

The first experiment was designed to evaluate the effect of acute inositol treatment on amphetamine-induced hyperactivity. Accordingly, 4 treatment groups received treatment as follows: Group saline-saline (n=8), Group saline-amphetamine (n=9), Group glucose-amphetamine (n=8), and Group inositol-amphetamine (n=9). Rats were injected intraperitoneal with inositol or glucose (1 g/kg diluted to 20 ml/kg) or an equivalent volume of saline followed 2 h later by a SC injection of amphetamine (0.75 mg/kg diluted to 1 ml/kg) or an equal volume of saline. Five minutes after the SC injection, rats were placed in activity monitors (Optivarimax, Columbus Instruments, Columbus, Ohio) and their activity automatically monitored for 20 min. Results indicate no observable effects of inositol in this model (data not shown).

The second experiment tested the effects of acute inositol treatment on apomorphine-induced stereotypy. Rats received intraperitoneal pretreatment with inositol (1 g/kg, 20 ml/kg), glucose (same dose and dilution) or saline (same volume). Two hours later they were injected SC with 0.5 mg/kg apomorphine and their level of stereotypy rated by two blind observers according to a rating scale modified from Kelly and Iverson (1976). Results did not reveal any effects of inositol in this model (data not shown).

The last two experiments tested only acute inositol treatment and that may diminish the possibility for substantial conclusions regarding the effects of inositol in these models. However, the effects of classical anti-psychotic drugs in these models follow acute treatment (for review see Lyon, 1991b) and thus the results may be enough to conclude that inositol is not acting in any similar way to these drugs.

To test the effects of inositol on memory, 4 Rhesus monkeys were fed with 20 g/day inositol or 20 g 1:2 glucose:mannitol for 2 weeks in a cross over design. Spatial discrimination tests (Olton et al., 1980, Raveh et al., 1997) that permit the examination of a number of behavioral parameters with emphasis on working and reference memory were administered to the monkeys daily. Results indicate no effects of inositol on any of the behavioral parameters tested in this study (data not shown). These results are consistent with the lack of effects of inositol in Alzheimer’s patients (Barak et al., 1996).

Section snippets

Discussion

The effects of inositol treatment on animal behavior were tested in different administration schedules, doses and in a variety of situations ranging from free behavior to designed animal models of psychopathology. The collective results of the studies described in the present paper indicate that chronic inositol treatment may alleviate depression-like and anxiety-like behaviors in rats. No effects were observed in schizophrenia-related or mania-related models and on memory-related tasks in

Acknowledgements

The authors would like to thank Dr. Ora Kofman and Dr. Hagit Cohen for their help. Supported by a Stanley Foundation Center grant.

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