Neuropharmacology Poster Session
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Introduction
myo-Inositol is a polyol isomer of glucose that is a precursor in the phosphatidylinositol (PIP) cycle. Two second messengers are the products of the PIP cycle, diacylglycerol (DAG) which activates protein kinase C, and inositol triphosphate (IP3) which enhances intracellular calcium flow. Phosphoinositides play a role in cholinergic (muscarinic receptors), noradrenergic (alpha1 receptors), serotoninergic (5-HT2A and 5-HT2C receptors) and dopaminergic (D1 and possibly D2 receptors) systems.
Clinical studies demonstrated that inositol was effective in relieving symptoms of depression; however, the mechanism of action of the drug is not clear. The therapeutic effect may be the result of inositol serving as a precursor for the PIP cycle but it could also be a consequence of its activity elsewhere.
Further attempts to expand the use of inositol as therapy for depression depend in part on the understanding of its underlying mechanisms, and the study of mechanisms is usually dependent on the development of appropriate animal models of drug action. The present study was designed to test the effects of inositol in two animal models of depression, reserpine-induced hypoactivity, and the Porsolt forced swim test. These two models were chosen because they represent two different approaches to the development of animal models: the reserpine model is pharmacologically induced whereas the forced swim test is behaviorally induced.
Both models induce changes in a number of neurotransmitter systems: reserpine treatment reduces brain levels of noradrenaline, dopamine and serotonine and the forced swim test was reported to have dopaminergic, serotonergic, noradrenergic and cholinergic effects. Since the mechanism of action of inositol as a therapeutic agent is not known but the PIP cycle is involved in a number of systems, it appeared reasonable to utilize models that are also related to a number of neurotransmission systems.
Materials and Methods
Subjects - general
Sprague-Dawley male rats (Harlan, Jerusalem), weighing 200-250 gm at the beginning of experiment served as subjects. Rats were maintained in a temperature controlled colony room (22oC) with a 12 hr. light/dark cycle, and with free access to food and water. All experiments were performed during the light phase of the light/dark cycle.
Forced swim (Porsolt) test
For experiment 1, four groups of rats received 2 weeks of daily intra-peritoneal (IP) injections of inositol at one of three doses (0.0 - control group - n=10; 0.3 g/kg, n=9; 0.6 g/kg, n=10; and 1.2 g/kg, n=8), diluted to volume of 12 ml/kg. Control rats received similar treatment procedure but with 1:2 glucose/mannitol solution. A fifth group of rats (n=11) was added to the experiment as positive control and was treated throughout the experiment in a similar way to the control group but in addition, at days 13 and 14 it was treated IP with 2 injections of imipramine (30 mg/kg diluted in saline to volume of 30 mg/2 ml. The first injection was administered on day 13, immediately after the first swim exposure, and the second injection was administered on day 14, 30 minutes prior to the second swim exposure. Rats from all other groups were injected with equivalent volume of saline.For experiment 2, two groups of rats (n=20 per group) were treated for 14 days with oral inositol (10% in powdered rat chow) or 1:2 glucose/mannitol (10% in powdered rat chow). Swim exposure was conducted during days 13 and 14.
The Porsolt swim test is composed of two exposures, spaced one day apart, to a water tank that does not permit escape. The water tank used was a transparent plastic tank, measuring 22 cm in diameter and 40 cm in height, with a rounded lid, containing 20 cm of fresh water at 25oC. During the first exposure, rats were placed into the tank and left there for 10 minutes. During the second exposure (test session), rats were placed into the tank and left there for 5 minutes during which their behavior was videotaped. Videotapes of the test session were scored by an experimenter blind to the treatment for complete immobility time, small movements time, and vigorous struggle time. Struggle was defined as: 1) attempts to jump out of the tank; 2) attempts to climb the walls; 3) attempts to dive into the tank. All other activities were defined as small movements. The results obtained for each of the 3 measures 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.Reserpine induced immobility
Groups of rats received 2 weeks daily IP treatment of either inositol (1.2 g/kg, diluted to volume of 12 ml/kg; n=10) or 1:2 glucose/mannitol solution at the same end concentration and volume (n=10). During the last three days of treatment, rats were also treated with daily IP reserpine (Sigma) at 0.25 mg/kg dose. Reserpine was diluted in water with an addition of minimal amount of citric acid to injection volume of 2 ml/kg. Tests were conducted 30 minutes after the last reserpine injection. Rats were tested for activity levels in automated activity monitors for a duration of 30 minutes. Since the automated monitors are sensitive to ambulatory activity but less sensitive to locally oriented activity, rats were also manually scored during the session for complete immobility time. Activity monitors measures reflected the total ambulatory activity of each rat for the 30 minutes session while for manual scoring each rats was scored for 1 minute for each 10 minutes of the session (total of 3 minutes per rat). Data obtained from activity monitors and from manual score were analyzed using a student's t-test for differences between inositol and control groups. Significance level was set at p<0.05.Results
Forced swim test experiments
Experiment 1
Sub-acute treatment with imipramine was effective in reducing immobility time (t(19)=2.821, p=0.011) and increasing struggle time (t(19)=4.169, p<0.001) compared with the control group (Figure 1). The replication of the known effects of imipramine may indicate the validity of the model used in the experiment. As shown in Figure 1, compared with control rats, animals treated with chronic inositol at the 1.2 g/kg dose demonstrated significantly reduced immobility time (ANOVA: F(4)=3.07586, p=0.026; post-hoc tests: inositol (1.2 g/kg) different than control, and increased struggle time (ANOVA: F(4)=6.23, p=0.0005; post-hoc test inositol (1.2 g/kg) different than control). No significant effect was demonstrated for the lower inositol doses although a trend for increased struggle time was observed for the inositol 0.6 g/kg dose.
Fig. 1: Immobility and struggle time in the Porsolt test following IP inositol treatment.
Experiment 2
Compared with control rats, animals treated with chronic oral inositol (10% in powdered rat chow) demonstrated reduced immobility time (Figure 2; t-test: t(38)=3.77286, p=0.0006). A similar trend was demonstrated for struggle time although it did not reach statistical significance (Figure 2; t-test: t(38)=1.76297, p=0.086).
Fig. 2: Immobility and struggle time in the Porsolt test following oral inositol treatment.
Reserpine induced immobility
Chronic inositol treatment (1.2 g/kg/day for 14 days) significantly reduced complete immobility time scored manually after 3 days treatment with 0.25 mg/kg reserpine (Figure 3; t-test t(18)=4.455, p=0.0003). A similar trend that did not reach statistical significance was also observed for the automated measures of ambulatory activity (Figure 3).
Fig. 3: Immobility time and ambulatory counts in the reserpine-induced hypoactivity test.
Discussion and Conclusion
The results of the present study show that chronic inositol treatment given either orally or via IP peripheral injections was effective in two different models of depression, the Porsolt forced swim and the reserpine induced immobility test. The fact that these models are essentially different from each other, conceptually and technically, supports the notion that inositol is effective in relieving depressive-like behaviors in rats.
The present study does not elucidate the mechanism of action of inositol in depression. However, effects such as those observed in the present have been related to the dopaminergic system and the reports of decreased mesolimbic dopamine during the forced swim test combined with the reports of the involvement of the PI cycle in dopaminergic may raise the hypothesis that the effects of inositol treatment are mediated through dopaminergic mechanisms. Alternatively, the involvment of the PI cycle in serononergic neurotransmission, the serotonergic functions in motor activity and the effects of interactions between the models and the serotonergic system may imply that inositol's behavioral effects are related to the serotonergic system. Since the present study established the activity of inositol in two models of depression, these models can now be utilized to test these possibilities.
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