1. Introduction

Apo morphine is a dopaminergic D1/D2 receptors agonist [1] with slightly higher affinity for D2-like dopamine receptors [2]. Sensitization to Apo morphine (1.0 mg/kg) develops upon repeated administration, as assessed in an open field [3]. This hyperactivity induced by Apo morphine is suggested to be mediated by the stimulation of dopamine auto-receptors [4,5]. Apo morphine (2.0mg/kg) induced locomotor sensitization varies with peak concentration of drug as well as the habituation. Repeated Apo morphine (1.0 mg/kg) administration increases behavioral sensitivity, which could be attenuated upon repeated co-administration with 7-hydroxy 7-hydroxy-N, N-dipropyl-2-aminotetralin (7-OH-DPAT; dopamine receptor agonist) [4]. This Apo morphine-induced sensitization could be monitored after single injection of the drug as well [6].

Researchers have reported that serotonin and dopamine modulate the neurotransmission of each other [7-10]. Serotonin has an inhibitory effect dopaminergic neurotransmission while an increase in dopaminergic activity may also modulate serotonergic functions [11]. Evidences suggest that DR-induced hyperactivity in rat’s results in an increased in the inhibitory influence of 5-HT on the activity of dopaminergic neurons possibly due to increase super sensitivity of 5-HT_1Areceptors [12,13]. The present experiment is designed to monitor the effects of Apo morphine on locomotor activity in diet-restricted rats.

2.  Materials and Methods

2.1.  Animals and Treatment 

Locally bred female Sprague-Dawley rats weighing 165.4±15.2g were housed individually under 12-hour light dark cycle and controlled room temperature (22±2ºC) with the access of cubes of standard rodent diet and water, a week. Animals were cared according to a protocol approved locally which is consistent with the NIH guidelines for the care and use of laboratory animals.

 2.2.  Experimental Protocol

 One week after arrival 24 female Sprague-Dawley rats were randomly divided into Freely Feeding (FF) and Diet Restriction (DR) group of 12 each. Food was available for 24 h to FF group while animals of DR group were given access to food daily for 2 h. Food intake and body weights of the two groups were monitored weekly. After 4 weeks of treatment FF group and DR group animals were divided into 4 groups containing six animals each: 

·         FF-saline (1.0ml/kg),

·         FF-Apo morphine (1.0mg/kg)

·         DR-saline (1.0ml/kg) and

·         DR-Apo morphine (1.0mg/kg).

Animals were placed in the activity box (one in each box) 15 min before injection. Animals were then injected with the respective dose of Apo morphine or saline for seven days in week 5. Activity in familiar environment was monitored for a period of 15 minutes’ post injection every day.

2.3.  Drugs and Doses

Apo morphine-HCl (Sigma, St. Louis, USA) was dissolved in saline and injected intraperitoneally. Drug solutions were freshly prepared before each experiment. Control animals were injected with saline (0.9% NaCl) at a dose of 1.0 ml/kg.

2.4.  Behavioral Assessment

Activity Monitoring Activity Cage

To monitor the behavioral response of Apo morphine on locomotor activity an animal was transferred to transparent, Perspex activity cages (26× 26× 26 cm) with a saw-dust covered floor were used to monitor activity.  Experiment was conducted in a separate quite room. Locomotor activity was observed in activity boxes for 15 minutes in terms of numbers of cage crossings in all groups in a balanced design as described earlier [14-16].

2.5.  Statistical Analysis

Results are represented as means±S.D. Data were analyzed by two-way ANOVA (Repeated Measure Design). Post hoc comparisons done by Tukey’s test. p values < 0.05 were taken significant.

3.  Results

 Effect of Apo morphine administration on food intake of rats fed on DR schedule of 2 h/day is shown in (Figure 1) Two -way ANOVA (d.f. 1, 20) showed significant effect of DR (F = 21.5; p < 0.01) interaction (d.f.1, 20; F=126.8, p<0.01) significant; Apo morphine (d.f.1,20; F=1.8, p>.0.05) was insignificant. Post hoc test showed that food intakes were significantly (p < 0.01) decreased in saline injected and Apo morphine injected DR groups than respective FF group. 

Body weight changes in FF and DR groups are shown in (Figure 2) Two -way ANOVA (d.f. 1, 20) showed significant effect of DR (F = 31.68; p < 0.01), interaction (d.f.1, 20; F=131.5, p<0.01) significant; Apo morphine (d.f.1, 20; F=2.86, p>.0.05) was insignificant.  Post hoc test showed that body weights were significantly (p < 0.01) decreased (16.4%) in Apo morphine and 18.8 % in saline injected DR group when compared to respective controls.

(Figure 3) shows effects of administration of Apo morphine on motor activity in a familiar environment. Repeated measure two-way ANOVA revealed significant effects of Apo morphine on days (df = 6, 20; F = 259.32; p<0.01), repeated monitoring days ×Apo morphine (df = 12, 20; F = 154.915; p<0.01), interaction (df = 1, 20; F = 544.6; p<0.01) and Apo morphine (df = 1, 20; F = 113.1; p<0.01) were significant. Post hoc test showed that activity scores (number of cage crossings) were greater in DR than FF group. Administration of Apo morphine elicited hyperactivity (p < 0.01) in FF as well as DR groups and the scores were greater (p < 0.01) in DR than FF group. Post hoc analysis by Tukey’s showed that Apo morphine significantly (p<0.01) increased motor activity from day 3 to day 7 in FF as well as in DR groups. The increases were insignificant on day 1 and day 2 both in FF and DR group with respective saline injected group.

4.  Discussion

In the present study rats fed on DR schedule of 2 h/day for five weeks exhibited decreased in body weight (16.4%) in Apo morphine and (18.8 %) in saline injected DR group when compared to respective controls, decreased in food intake and increased motor activity in activity box. Similar (15-25%) reduction in body weight and behavioral deficits have also been reported in diet restriction induced AN [13,17,18].

The aim of the present study was to observe the behavioral response to Apo morphine in DR rats. Results showed that Apo morphine induced increases of motor activity were greater in DR groups compared to FF group in activity box. Number of cage crossing in activity box was greater in DR-Apo morphine treated rats than respective DR-saline treated group. Many studies have reported that there is a considerable amount of interaction between the dopaminergic and serotonergic neurotransmitter systems [19]. Some authors reported evidence for a facilitator role for 5-HT over DA. There is an abundance of evidence demonstrating that dopaminergic neurotransmission is functionally regulated by serotonin, which has important implications for 5-HT in controlling the behavior commonly exhibited in AN.  Pretreatment with 5-hydroxytryptophan or TRP decreased Apo morphine-induced hyperactivity [20]. Disruption of the 5-HT system will disrupt the DA system and affect DA mediated behaviors.5-HT neurons send projections to DA cell bodies located in the midbrain regions including the Substantial nigra and ventral tegmental area. In addition, they project to DA terminals present in the striatum, nucleus accumbens, and prefrontal cortex. The 5-HT innervations of dopaminergic cell bodies and terminals allows for the functional regulation by 5-HT of both DA neuronal firing and DA release. Results from electrophysiological and neurochemical studies on rodents have generally shown that 5-HT exerts an inhibitory influence on midbrain dopamine cell bodies. 5-HT influence over DA release in terminal regions, however, is less clear as both inhibitory and excitatory effects have been observed [19]. It has been reported that by repeated administration of Apo morphine induced sensitization increased Somatodendritic 5-HT_1Areceptors activity [17] which was attenuated by Buspirone. 

 In the present study, our results showed that daily administration of Spomorphine (1.0 mg/kg) for seven days progressively increased locomotor activity. Greater activity of DR than FF rats in the activity box in Apo morphine-induced hyperactivity in the present study might be due to an increased sensitivity of Somatodendritic 5-HT_1Areceptors. This results in release of dopamine because of the inhibitory influence of 5-HT.

 5.  Conclusion

 The present study concluded that greater increase in the hyperactivity in Apo morphine treated DR rats could be due to increased Somatodendritic 5-HT_1Areceptors activity. The study also suggests that the pharmacological agents who tend to desensitize 5-HT_1A receptors might be helpful in reducing the hyperactivity associated with diet-restriction.

Figure 1: Values are means±S.D (n=6). Significant differences by Tukey's test^*p<0.01 from respective FF group following two-way ANOVA.

Figure 2: Body weight of Freely Feeding (FF) and Diet Restriction (DR) group of rats.

Values are means±S.D (n=6). Significant differences by Tukey's test. ^*p<0.01 from

Figure 3: Activity in activity box in Freely Feeding (FF) and Diet Restriction (DR) group of rats. Values are means±S.D (n=6). Significant differences by Tukey's test^*p<0.05, ^**p<0.01 from respective FF saline injected; +p<0.05, ++p<0.01 from respective FF Apo morphine injected animals following two-way ANOVA (repeated measure design).

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