Srikumar et al

Contrasting Effects of Bromocriptine on Learning of a Partially-baited Radial Arm Maze Task in the Presence and Absence of Restraint Stress B.N. Srikumar1, T.R. Raju and B.S. Shankaranarayana Rao* Department of Neurophysiology, National Institute of Mental Health and Neuro Sciences, PB # 2900, Hosur Road, Bangalore -560 029.

1

Present address

Dept of Physiology, University of Utah School of Medicine Salt Lake City, UT 84108

*Corresponding author Dr. B.S. Shankaranarayana Rao Department of Neurophysiology National Institute of Mental Health and Neuro Sciences (NIMHANS) Hosur Road, PB # 2900 Bangalore -560 029. INDIA Phone: +91-80-2699 5175, Fax: +91-80-2656 2121/2656 4830 Email: [email protected] / [email protected]

Acknowledgements Bromocriptine was a generous gift from Serum Institute of India, Mumbai, India. The authors thank Titus, Bindu and Harsha for their technical assistance in the neurochemistry experiments, Bhagya and Veena for their help in data entry.

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Srikumar et al Abstract Rationale: Severe, traumatic stress or repeated exposure to stress can result in long-term deleterious effects, including hippocampal cell atrophy and death, which, in turn, result in memory impairments and behavioural abnormalities. The dopaminergic D2 receptor agonist, bromocriptine has been shown to modulate learning and chronic stress is associated with dopaminergic dysfunction. Objectives: In the present study, we evaluated the effects of bromocriptine in the presence or absence of restraint stress. Methods: Adult male Wistar rats were subjected to restraint stress for 21 days (6h/day) followed by bromocriptine treatment and learning was assessed in the partially baited radial arm maze task. In a separate group of animals the effects of bromocriptine per se was evaluated. Dopamine levels were estimated by high-performance liquid chromatography with electrochemical detection. Results: Stressed rats showed impairment in both acquisition and retention of the radial arm maze task, and bromocriptine treatment following stress showed a reversal of stressinduced impairment. Interestingly, in the absence of stress bromocriptine exhibited dosedependent differential effects on learning. While rats treated with bromocriptine 5 mg/kg, i.p., demonstrated impairment in learning, the bromocriptine 10 mg/kg and vehicle treated groups did not differ from normal controls. In order to understand the neurochemical basis for the effects of bromocriptine, dopamine levels were estimated. The stress-induced decrease in dopamine levels in the hippocampus and frontal cortex were restored by bromocriptine treatment. In contrast, bromocriptine alone (5 mg/kg, i.p.) decreased dopamine levels in the frontal cortex and striatum.

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Srikumar et al Conclusions: Our study shows that amelioration of stress-induced learning impairment correlates with restoration of dopamine levels by bromocriptine treatment.

Keywords: Restraint stress; dopamine; radial arm maze; spatial memory; hippocampus; reference memory; bromocriptine, dose-dependency; D2 receptor; frontal cortex.

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Srikumar et al

Introduction Severe and prolonged stress precipitates affective disorders and cause impairment in learning and memory. Earlier, we have demonstrated that 21 days of restraint stress impairs acquisition of T-maze (Sunanda et al. 2000a) and radial arm maze tasks (Srikumar et al. 2006). Furthermore, stress-induced impairment in learning is shown in other paradigms like the Y-maze (Conrad et al. 1996), Barnes maze (McLay et al. 1998) and Morris water maze (Bodnoff et al. 1995). In addition to stress, enhanced glucocorticoids (either cortisol or corticosterone), a hallmark of stress has been shown to produce learning deficits (Herbert et al. 2006). Recent and delayed declarative memories are impaired in both young and ageing humans, by excess corticosteroids (Lupien et al. 2002a; 2002b). Similarly, it has been demonstrated in animals, that excessive corticosterone can impair spatial learning (de Quervain et al. 1998; McLay et al. 1998). Further, the cognitive impairment and the hippocampal degeneration associated with chronic stress are thought to be at least in part due to the elevated levels of glucocorticoids since blockade of glucocorticoid receptors or glucocorticoid synthesis prevents stress-induced dendritic atrophy and cognitive deficits (de Quervain et al. 1998; Krugers et al. 2006; Luine et al. 1993; Magarinos and McEwen 1995). Several neurotransmitter systems like the dopaminergic (Finlay and Zigmond 1997), cholinergic (Srikumar et al. 2006), glutamatergic (Sunanda et al. 1997; 2000b), noradrenergic (Srikumar et al. 2006; Sunanda et al. 2000b) and serotonergic (McEwen et al. 1997; Sunanda et al. 2000b) systems are involved in the stress-induced deficits. Among these, alteration in the dopaminergic system is one of the main neurochemical changes following stress. Previous studies indicate that chronic restraint stress

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Srikumar et al significantly decreases dopamine (DA) levels in the hippocampus (Sunanda et al. 2000b; Torres et al. 2002). It has been reported that chronic variate stress or isolated housing of rats increases the 3,4 dihydroxy phenyl acetic acid (DOPAC) levels in the frontal cortex and hippocampus (Gamaro et al. 2003; Miura et al. 2005) and in pigs, acute immobilization stress also affects the hippocampal dopamine turnover (Piekarzewska et al. 1999). Repeated maternal separation stress and exposure to novel environment upregulated D1 receptor density in the stratum radiatum and moleculare of the hippocampal CA1 region (Ziabreva et al. 2003), and affected D2 receptor binding in the periaqueductal grey and ventral tegmental area (Ploj et al. 2003). Thus, there are multiple lines of evidence that dopamine levels, turnover and receptor status are affected in stress. Dopamine is known to play an important role in learning and memory. For example, dopamine plays a central role in working memory (Goldman-Rakic 1995) and several others show that dopamine can modulate learning in the Morris water maze (Miyoshi et al. 2002; Da Cunha et al. 2003) and radial arm maze tasks (Packard and White 1989; 1991). Thus, dopamine is involved in learning and memory and stress is associated with learning deficits and dopaminergic dysfunction. In this study, we hypothesized that this stress-induced dopaminergic dysfunction produces learning deficits and administration of a dopaminergic agonist could ameliorate the stress-induced deficits. Bromocriptine, a dopaminergic D2 receptor agonist has been shown to affect a variety of neuronal processes. Bromocriptine demonstrated protection of the hippocampal CA1 neurons against cerebral ischemia induced cell death in gerbils (Liu et al. 1995; O'Neill et al. 1998). Further, bromocriptine is reported to have antidepressant activity in the chronic mild stress (CMS) model of depression (Muscat et al. 1992) wherein, the

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Srikumar et al CMS induced decrease in sucrose consumption was reversed by chronic intermittent bromocriptine treatment. In another study, the water immersion stress-induced gastric lesion has been shown to be prevented by pre-treatment with bromocriptine (Samini et al. 2002). Furthermore, in a restraint stress model, bromocriptine attenuated the stressinduced gastric mucosal lesions and prevented the elevation of plasma corticosterone levels (Puri et al. 1994). Thus, although earlier studies have evaluated the effect of bromocriptine on stress-induced gastric lesions and depression, none of them have examined its effects on stress-induced deficits in learning and memory. Earlier studies in an animal model of brain trauma demonstrate that bromocriptine attenuates the controlled cortical impact induced deficits in performance of the water maze task (Kline et al. 2002; 2004). Accordingly, in this study, we have evaluated the effect of bromocriptine treatment following chronic restraint stress and in the absence of stress on the radial arm maze performance. Further, the dopamine metabolism was assessed in the hippocampus, frontal cortex and striatum.

Materials and methods Experimental animals Adult male Wistar rats (200-250g; 2 to 2.5 months old) obtained from Central Animal Research Facility (CARF), NIMHANS, Bangalore were used in the study. Rats were housed three per cage in polypropylene cages (22.5×35.5×15cm) in a temperature (25±2°C), humidity (50-55%) and light-controlled (12h-light-dark cycle) environment, with food and water ad libitum except during the periods of stress. An Institutional

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Srikumar et al animals ethics committee approved the experimental protocols. All efforts were made to minimize both the suffering and the number of animals used.

Stress and drug treatment Animals were randomly assigned to the different treatment groups. Normal control rats did not undergo stress or drug treatment. In the stress group, rats were encaged in a rodent restrainer, 6h/day for 21 days as described earlier (Shankaranarayana Rao et al. 2001; Srikumar et al. 2006). This form of restraint stress produces gastric ulcers, increases the adrenal weights (Sunanda et al. 1997; Sunanda et al. 2000b) and plasma corticosterone levels (Luine et al. 1996). Solution of bromocriptine mesylate (a gift from Serum Institute of India, Mumbai, India) was freshly prepared in 50 % DMSOsaline. After the completion of stress protocol, stress + bromocriptine group of rats received 10 daily injections of bromocriptine (5 mg /kg or 10 mg /kg, i.p.). The stress + vehicle group received 10 daily injections of the vehicle alone (DMSO-saline; 2ml/kg, i.p.). In another set of experiments, rats that were not subjected to stress were given 10 daily injections of either vehicle alone or bromocriptine at two different doses. The number of animals in each group was 9-12 in case of behavioural experiments and was 6 for neurochemical experiments.

Evaluation of behaviour in the radial arm maze Learning and memory in the radial arm maze was assessed as described earlier (Devi et al. 2003; Srikumar et al. 2004; Srikumar et al. 2006). The eight arm radial maze (RAM) consisted of a computer monitored plexiform maze (Columbus Instruments,

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Srikumar et al Ohio, USA), with equally spaced arms (42×11.4×11.4cm) radiating from an octagonal central platform and the maze was kept 80cm elevated from the ground. Prior to the training, the animals were kept on a restricted diet and body weight was maintained at 85% of their free feeding weight, with water available ad libitum. Training To acclimatize the rats to the RAM prior to the acquisition, all the arms were baited and rats were allowed to explore the maze for 10 minutes and were given two such sessions on consecutive days. Acquisition Rats were given two trials a day. At the beginning of each trial, the maze was thoroughly cleaned with 70% ethanol and four of the arms (2, 3, 6 and 8) were baited with food reinforcement (Kellogg’s Planets and Stars ™, Kellogg India Ltd., Mumbai, India). The rat was placed in the center of the octagon and was allowed a free choice. An arm choice was recorded when a rat ate a bait or reached the end of an arm. The arms were not rebaited, so only the first entry into the baited arm was recorded as a correct choice. The trial continued until the rat entered all the four baited arms or 5 minutes had elapsed. At the end of the trial, the rats were returned to the home cages and were given the second trial after an inter-trial interval of 1h. Training was continued till the rats attained the criteria of 80% correct choice (at least 4 correct entries out of 5). 10 days after acquisition of the task, rats were evaluated for retention of the task. Rats were given two trials and the average of two trials was taken for analysis.

Evaluation criteria

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Srikumar et al Data from 4 trials were averaged and expressed as blocks. The data was analysed for percentage correct choice, reference, and working memory errors. An entry into an unbaited arm was considered a reference memory error (RME) and any re-entry was considered as a working memory error (WME). A re-entry into a baited arm and a reentry into an unbaited arm were considered as working memory error correct (WME correct) and working memory error incorrect (WME incorrect), respectively.

Estimation of dopamine levels After the stress and drug treatment protocols, the hippocampus, frontal cortex and striatum were quickly dissected out and homogenized in 2ml of 3mM sodium acetate buffer with 20% methanol and centrifuged at 1850g for 30 min at 4°C. The supernatant was filtered through a 0.2μm pore size cellulose acetate filter (Sartorius, Goettingen, Germany) and was stored at -80°C till further analysis. Stock solutions of standard DA was made at a concentration of 1 mg/ml and diluted to obtain a concentration of 10pg/µl of injection volume. The analysis was done using isocratic ion-pair HPLC (Shimadzu Co., Kyoto, Japan) with electrochemical detection (Biorad, CA, USA). The mobile phase consisted of sodium acetate (20 mM), heptane sulfonic acid (5 mM), EDTA (0.1 mM), and dibutylamine (0.04%) mixed with methanol (5.4%). The electrochemical conditions maintained included an applied potential of 650 mV and sensitivity of 2 nA/V. DA and its metabolites were separated on a reverse phase Nucleosil C-18 analytical column (15 cm X 0.46 cm; 3μm particle size; Supelco, USA) with a flow rate of 0.9 ml/min and injection volume of 20μl. Chromatography data were processed and chromatograms were analyzed with Winchrom data station (Indtech Instruments, Mumbai, India) (Deepti et al.

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Srikumar et al 2004). The peaks in the samples were identified by comparing their retention time with that of the standard solution and quantified by comparing its peak area with that of the standards and the concentration of the biogenic amines was determined and expressed as ng/g tissue wet weight. Statistical analyses Data is expressed as Mean ± SEM. Either two-way or one-way ANOVA followed by Tukey’s post-hoc test was used to compare the means. The correlation of behavioural data and dopamine levels was performed by Pearson’s correlation analysis. p<0.05 was considered statistically significant.

Results Stress impairs learning in a partially baited RAM task Normal control rats reached the criterion of 80% correct choice after 24 trials (block 6). At 16 days of training, they reached a value of 89.16 ±1.78 percentage correct choice (Fig. 1a). Rats subjected to chronic restraint stress failed to attain the criterion of learning. The learning curve started to plateau after 20 trials. Even at 16 days of training the percentage correct choice was 67.73±3.48 in stressed rats (Fig. 1a).

Bromocriptine treatment reverses stress-induced impairment in acquisition Rats subjected to stress were treated with either vehicle or bromocriptine for 10 days. Two-way ANOVA revealed a significant interaction between the treatment and the learning of the radial arm maze [F(28,305) = 1.78; p<0.05]. Bromocriptine treatment to stressed rats resulted in amelioration of the stress-induced deficits. The effect of

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Srikumar et al bromocriptine treatment was statistically significant in block 7 [F(4,33) = 4.41; p<0.01; Fig. 1b] and block 8 [F(4,36) = 11.97; p<0.001; Fig. 1c]. However, there was no significant difference between the two doses used [p>0.05]. Reference memory impairment in stress and reversal by bromocriptine Entries into the arms 1, 4, 5 and 7 in the radial arm maze were considered as reference memory errors. Rats subjected to 21 days of restraint stress showed a significant impairment in reference memory (Fig. 2a). In block 8, the number of RMEs was 0.40 ± 0.10 and 1.70 ± 0.28 in control and stress groups, respectively. This increase in the number of RMEs in the stress group was significantly more than control rats [t(17) =

4.48; p<0.001; Fig 2a]. There was a significant effect of the groups on the number of

RMEs [F(4,308) = 18.44; p<0.001; Fig 2b]. Bromocriptine (5 mg and 10 mg/kg, i.p.) treated rats committed less number of errors compared to both stress and stress + vehicle treated rats from the sixth block onwards (Fig 2b). In the block 8, the number of RMEs in the bromocriptine treated group was significantly lesser than the stress group [F(4,36) = 5.63; p<0.01] and was comparable to the normal control (Fig 2b and c). On the contrary, the working memory was not affected by either stress or bromocriptine treatments. The working memory correct errors (WMEs correct) were not significantly different between groups in both block 7 and 8 (Table 1) and working memory incorrect errors (WME incorrect) were less than 0.1 in the block 7 and was 0 for all the groups in the 8th block.

Effect of stress and bromocriptine treatment on retention To assess whether the effect of bromocriptine was restricted to acquisition or if it had actions on retention of the RAM task also, rats were subjected to two trials of

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Srikumar et al retention test 10 days after the acquisition of the task. In the retention test, both stressed and stress + vehicle treated rats sustained the impairment seen in the acquisition [F(4,34) = 5.62; p<0.01; Fig 3a]. Interestingly, stressed rats treated with bromocriptine did not show any impairment in the retention test also. The percentage correct choice was comparable to that of the normal control group (p>0.05; Fig 3a). Similarly, the increase in the number of RMEs seen in the acquisition in the stress and stress + vehicle groups was sustained in the retention test [F(4,34) = 7.25; p<0.001] and bromocriptine treatment restored this (Fig 3b). As in the acquisition, there was no difference in the number of WMEs correct [F(4,48) = 0.57; p>0.05; Fig. 3c] and the WMEs incorrect was 0 for all the groups.

Dose-dependent effects of bromocriptine in the absence of stress on radial arm maze learning Since bromocriptine produced reversal of stress-induced deficits, we examined the effect of bromocriptine on behaviour of rats that were not subjected to stress. Twoway ANOVA showed a significant interaction between the treatment and learning [F(21,317) = 1.70; p<0.05]. Interestingly, at the low dose (5 mg/kg, i.p.), bromocriptine showed a significant impairment [F(3,317) = 23.8; p<0.001; Fig. 4b]. Contrastingly, both the vehicle and bromocriptine 10 mg/kg treated rats performed as good as the normal control (Fig. 4a). In the block 8, the percent correct choice was 65.21 ± 3.98, a 26.86% decrease compared to the normal control (Fig. 4b). Further, there was an increase in the number of RMEs in the bromocriptine 5 mg/kg treated group (Fig. 4c). This increase was statistically significant in the block 8 [F(3,32)= 11.24; p<0.001; Fig. 4d]. However,

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Srikumar et al working memory was unaffected by bromocriptine treatment and the number of WMEs correct was similar in all the groups (Table 2) and the number of WMEs incorrect was zero for all the groups in the block 8. In the retention test also, the bromocriptine 5 mg treated rats showed a sustained impairment, both in terms of percentage correct choice (Fig. 5a) and reference memory errors (Fig. 5b), where as the bromocriptine 10 mg and vehicle treated rats showed performance comparable to that of normal control rats (Fig. 5a and b). Similar to the acquisition, working memory was unaffected by drug treatment (Fig. 5c).

Effect of stress and bromocriptine treatment on dopamine levels Restraint stress decreased dopamine levels in the hippocampus, frontal cortex and striatum (Fig. 6). There was a 34%, 52% and 43% decrease in the dopamine levels in the hippocampus, frontal cortex and striatum, respectively following restraint stress. Bromocriptine treatment significantly restored the decrease in dopamine levels in the hippocampus [F(4,25) = 48.20; p<0.001], frontal cortex [F(4,25) = 106.4; p<0.001] and striatum [F(4,26) = 43.48; p<0.001]. The DOPAC/DA ratio did not differ significantly across groups (data not shown).

Effect of bromocriptine treatment in the absence of stress on dopamine levels Ten days of bromocriptine (5 mg/kg) treatment to unstressed rats decreased the dopamine levels in the frontal cortex [F(3,20) = 97.99; p<0.001; Fig. 7b] and striatum [F(3,20) = 62.14; p<0.001; Fig. 7c] but did not affect the hippocampal dopamine levels (Fig. 7a). In the bromocriptine (10 mg) treated group, the dopamine level was comparable to the normal control in all the three regions (Fig. 7). There was a marginal increase in

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Srikumar et al the frontal cortex DOPAC/DA ratio in the bromocriptine 5 mg group (0.13 ± 0.01) compared to the normal control (0.09 ± 0.01) [F(3,20) = 6.51; p<0.01]. In the vehicle treated group and the bromocriptine (10 mg) treated group, the DOPAC/DA ratio was 0.10 ± 0.01 and was comparable to normal control. In the striatum also, a similar trend was observed, while in the hippocampus, the DOPAC/DA ratio was unaffected (data not shown).

Discussion The salient findings in the current study are that chronic restraint stress impairs learning of the eight arm radial maze task that is reversed by 10 days of bromocriptine treatment. The learning impairment following stress was associated with a decrease in dopamine levels that was restored by the bromocriptine treatment. The modulation of the dopaminergic system has been shown to produce effects on spatial learning. For instance, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) lesion of the substantia nigra produced behavioural deficits in the Morris water maze task (Miyoshi et al. 2002; Da Cunha et al. 2003) and was associated with decreased dopamine levels in the striatum and frontal cortex (Miyoshi et al. 2002). Particularly, the dopaminergic D2 receptor has been implicated in the early memory consolidation processes (Packard and White 1989). In the present study, bromocriptine treatment to rats subjected to 21 days of restraint stress reversed the deficit in performance of a partially baited task. Our present results are in agreement with earlier reports that demonstrate the use of bromocriptine or other dopaminergic D2 receptor agonists to ameliorate cognitive dysfunctions. In traumatic brain injury, bromocriptine showed improvement in a working memory paradigm of the

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Srikumar et al water maze task (Kline et al. 2002). Intra cerebro ventricular quinpirole injection in freely moving animals inhibited depotentiation (Manahan-Vaughan and Kulla 2003) and reversed the impairment in radial-arm maze learning following medial cholinergic pathway lesion (McGurk et al. 1992). It has been shown that the non-selective dopamine agonist apomorphine or selective agonist quinpirole prevents the scopolamine-induced impaired consolidation of passive avoidance behaviour (Sigala et al. 1997) or facilitate learning in an aversively motivated Stone maze (Umegaki et al. 2001). An earlier study found that four weeks of restraint and water immersion stress impaired memory in a T-maze task associated with dopaminergic dysfunction in the prefrontal cortex and administration of the dopaminergic D1 receptor agonist, SKF 81297, reversed this deficit (Mizoguchi et al. 2000). Unlike this study, our present results indicate that there were no working memory deficits (Table 1) following chronic restraint stress. It is likely that the effects of stress manifests in different forms in different learning paradigms. However, the effects of a D1 receptor agonist administration on stress-induced deficits in performance of the radial arm maze task remain to be established. Nevertheless, both the earlier report by Mizoguchi et al (2000) and our present results suggest the role of both dopaminergic D1 and D2 receptors in stressinduced cognitive deficits. In the study by Kline et al (2002), bromocriptine produced recovery in the acquisition of the spatial memory task and it restored the hippocampal CA3 cell loss analogous to the partial restoration of the spatial memory task. In restraint stress, our earlier studies document hippocampal CA3 neuronal dendritic atrophy (Shankaranarayana Rao et al. 2001; Sunanda et al. 1995) and impaired learning in a Tmaze task (Sunanda et al. 2000a). It remains to be investigated whether the reversal of

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Srikumar et al RAM performance by bromocriptine in our present study involves the reversal of dendritic atrophy. In the present study, the dopamine level in the frontal cortex, hippocampus and striatum was decreased following 21 days of restraint stress, reiterating our earlier findings (Sunanda et al. 2000b). The decreased dopamine concentration was reversed in a dose-dependent manner in the hippocampus and frontal cortex with the higher dose (10 mg/ kg) being more effective. There was a significant correlation between the performance of the radial arm maze in the eighth block of the acquisition and the dopamine levels in the hippocampus (r = 0.93; p<0.05), frontal cortex (r = 0.93; p<0.05) and striatum (r = 0.88; p<0.05). So it is likely that bromocriptine is reversing the stressinduced impairment in behaviour by enhancing dopamine levels. Berger et al. (2002) have shown that the dopaminergic D2 receptors are upregulated in the CA1 region of the hippocampus following exposure to prenatal stress. It can be reasoned that the hypodopaminergic state following stress could produce an upregulation of the dopaminergic receptors as a compensatory phenomenon. In this context, administration of dopaminergic agonists could produce the behavioural recovery by acting on these receptors in addition to enhancing endogenous dopamine levels. However, the effects of bromocriptine on stress-induced changes in the dopaminergic receptor status that might accompany the behavioural recovery brought about by this agonist need further evaluation. Other possible mechanisms like antioxidant and neurotrophic support may also play a role since bromocriptine decreases lipid peroxidation (Kline et al. 2004), scavenges free radicals (Yoshikawa et al. 1994) and transactivates the phosphoinositide 3-kinase signalling pathway (Nair and Sealfon 2003).

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Srikumar et al Bromocriptine, in the absence of stress had contrasting dose-dependent effects on the acquisition of the RAM task. None of the studies in our knowledge thus far have examined the effect of chronic bromocriptine treatment on performance of a partially baited RAM task. Local infusion of quinpirole (also called as LY171555) into the ventral hippocampus caused an improvement in choice accuracy (Wilkerson and Levin 1999) and post-training intrahippocampal infusions of LY171555 improved win-shift retention without affecting the performance in the win-stay task (Packard and White 1991). However, systemic quinpirole administration produced a dose dependent increase in the latency of choices in a delayed non-match to sample task of the RAM (Chrobak and Napier 1992). In an early study, LY171555 over a broad range of doses increased the latency to finish the maze without significantly affecting the number of choices needed to finish the task (Levin and Bowman 1986). Thus, it appears that the effect of dopaminergic modulation on learning is governed by multiple factors such as the route of drug administration, the dose of the drug used, and the kind of the behavioural task itself. It is not entirely clear why bromocriptine in the present study, exhibits a dosedependent effect on RAM performance. Our results demonstrate that bromocriptine at 5 mg dose, decreases the DA levels in the frontal cortex and striatum while the DA concentration in rats administered with 10 mg of bromocriptine was comparable to normal control. This possibly explains the dose-dependent dichotomy in radial arm maze behaviour. An earlier report shows a dose-dependent dimorphism on bromocriptineinduced striatal extracellular dopamine level with a 5 mg/kg dose increasing DA levels, while at 10 mg/kg, it was decreased (Brannan et al. 1993). Such inverted U-shaped or Ushaped dose-dependent effects on behaviour have been observed with other psychoactive

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Srikumar et al drugs like the cholinesterase inhibitor heptylphysostigmine (Braida et al. 1996) or the D3-preferring agonist 7-OH-DPAT (Khroyan et al. 1995). The frontal cortex and striatum express the dopaminergic D1 and D2 receptors (Bergson et al. 1995; Hersch et al. 1995). The other subtypes have also been shown to be expressed albeit in lesser quantities (Bouthenet et al. 1991; Meador-Woodruff et al. 1992). The D2 receptors are present both postsynaptically and presynaptically and the predominant presynaptic receptors are important in the regulation of dopamine release (Carter and Muller 1991; Lahti et al. 1992). The D2 receptor agonists at lower concentrations are believed to act primarily on these sites to decrease the dopamine release (Fusa et al. 2002). Most of the studies particularly in Parkinson’s disease are limited to behavioural evaluations and none of them examine the effect of chronic dopaminergic agonist administration on dopamine levels. Acute administration of D2 agonists has been shown to decrease firing of the dopaminergic neurons (Yarbrough et al. 1984). It is likely that bromocriptine at low doses such as 5 mg produces effects predominantly on the presynaptic sites where as at higher doses could bring about its effects through actions on the postsynaptic D2 receptors. This could explain the dosedependent effects of bromocriptine on learning of the radial arm maze. Similar effects of dopaminergic agonists have been reported elsewhere. D2 receptor agonists show biphasic effects on sleep-wake cycle, with low doses increasing and high doses decreasing sleep (Monti et al. 1988). Although bromocriptine is a strong agonist of the D2 - like receptors, it is not possible to rule out the role of other dopaminergic receptors or other neurotransmitter systems in these effects since bromocriptine is a partial antagonist of the D1 - like

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Srikumar et al receptors, and has shown affinity to non-dopaminergic receptors, particularly 5-HT1, 5HT2, and α2 - adrenoceptors (Closse et al. 1984; Gibson and Samini 1979; McPherson and Beart 1983; Fukuzaki et al. 2000). To summarize, we demonstrate the ameliorative effects of bromocriptine administration on radial arm maze learning in restraint stressed rats. Further, we show that bromocriptine produces differential dose-dependent effect on learning following administration to stress-naïve rats. And these alterations in behaviour were attributable at least in part to the changes in the dopamine levels. Earlier studies show that D2 receptor agonists like bromocriptine or quinpirole have antidepressant (Muscat et al. 1992; Borsini et al. 1988) or anxiolytic activity (Bruhwyler et al. 1991). In this context, our present study underscores the role of dopaminergic system in stress and reversal of stress disorders.

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Srikumar et al Devi L, Diwakar L, Raju TR, Kutty BM (2003) Selective neurodegeneration of hippocampus and entorhinal cortex correlates with spatial learning impairments in rats with bilateral ibotenate lesions of ventral subiculum. Brain Res 960:9-15 Finlay JM, Zigmond MJ (1997) The effects of stress on central dopaminergic neurons: possible clinical implications. Neurochem Res 22:1387-1394 Fukuzaki K, Kamenosono T, Kitazumi K, Nagata R (2000) Effects of ropinirole on motor behavior in MPTP-treated common marmosets. Pharmacol Biochem Behav 67:121-129 Fusa K, Saigusa T, Koshikawa N, Cools AR (2002) Tyrosine-induced release of dopamine is under inhibitory control of presynaptic dopamine D2 and, probably, D3 receptors in the dorsal striatum, but not in the nucleus accumbens. Eur J Pharmacol 448:143-150 Gamaro GD, Manoli LP, Torres IL, Silveira R, Dalmaz C (2003) Effects of chronic variate stress on feeding behavior and on monoamine levels in different rat brain structures. Neurochem Int 42:107-114 Gibson A, Samini M (1979) The effects of bromocriptine on pre-synaptic and postsynaptic alpha-adrenoceptors in the mouse vas deferens. J Pharm Pharmacol 31:826-830 Goldman-Rakic PS (1995) Cellular basis of working memory. Neuron 14:477-485 Herbert J, Goodyer IM, Grossman AB, Hastings MH, de Kloet ER, Lightman SL, Lupien SJ, Roozendaal B, Seckl JR (2006) Do corticosteroids damage the brain? J Neuroendocrinol 18:393-411 Hersch SM, Ciliax BJ, Gutekunst CA, Rees HD, Heilman CJ, Yung KK, Bolam JP, Ince E, Yi H, Levey AI (1995) Electron microscopic analysis of D1 and D2 dopamine receptor proteins in the dorsal striatum and their synaptic relationships with motor corticostriatal afferents. J Neurosci 15:5222-5237 Khroyan TV, Baker DA, Neisewander JL (1995) Dose-dependent effects of the D3preferring agonist 7-OH-DPAT on motor behaviors and place conditioning. Psychopharmacology (Berl) 122:351-357 Kline AE, Massucci JL, Ma X, Zafonte RD, Dixon CE (2004) Bromocriptine reduces lipid peroxidation and enhances spatial learning and hippocampal neuron survival in a rodent model of focal brain trauma. J Neurotrauma 21:1712-1722 Kline AE, Massucci JL, Marion DW, Dixon CE (2002) Attenuation of working memory and spatial acquisition deficits after a delayed and chronic bromocriptine treatment regimen in rats subjected to traumatic brain injury by controlled cortical impact. J Neurotrauma 19:415-425

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Srikumar et al Krugers HJ, Goltstein PM, van der LS, Joels M (2006) Blockade of glucocorticoid receptors rapidly restores hippocampal CA1 synaptic plasticity after exposure to chronic stress. Eur J Neurosci 23:3051-3055 Lahti RA, Figur LM, Piercey MF, Ruppel PL, Evans DL (1992) Intrinsic activity of DA agonists. Clin Neuropharmacol 15 Suppl 1 Pt A:182A-183A Levin ED, Bowman RE (1986) Effects of the dopamine D-2 receptor agonist, LY 171555, on radial arm maze performance in rats. Pharmacol Biochem Behav 25:1117-1119 Liu XH, Kato H, Chen T, Kato K, Itoyama Y (1995) Bromocriptine protects against delayed neuronal death of hippocampal neurons following cerebral ischemia in the gerbil. J Neurol Sci 129:9-14 Luine V, Martinez C, Villegas M, Magarinos AM, McEwen BS (1996) Restraint stress reversibly enhances spatial memory performance. Physiol Behav 59:27-32 Luine VN, Spencer RL, McEwen BS (1993) Effects of chronic corticosterone ingestion on spatial memory performance and hippocampal serotonergic function. Brain Res 616:65-70 Lupien SJ, Wilkinson CW, Briere S, Menard C, Ng Ying Kin NM, Nair NP (2002a) The modulatory effects of corticosteroids on cognition: studies in young human populations. Psychoneuroendocrinology 27:401-416 Lupien SJ, Wilkinson CW, Briere S, Ng Ying Kin NM, Meaney MJ, Nair NP (2002b) Acute modulation of aged human memory by pharmacological manipulation of glucocorticoids. J Clin Endocrinol Metab 87:3798-3807 Magarinos AM, McEwen BS (1995) Stress-induced atrophy of apical dendrites of hippocampal CA3c neurons: involvement of glucocorticoid secretion and excitatory amino acid receptors. Neuroscience 69:89-98 Manahan-Vaughan D, Kulla A (2003) Regulation of Depotentiation and Long-term Potentiation in the Dentate Gyrus of Freely Moving Rats by Dopamine D2-like Receptors. Cereb Cortex 13:123-135 McEwen BS, Conrad CD, Kuroda Y, Frankfurt M, Magarinos AM, McKittrick C (1997) Prevention of stress-induced morphological and cognitive consequences. Eur Neuropsychopharmacol 7 Suppl 3:S323-S328 McGurk SR, Levin ED, Butcher LL (1992) Dopaminergic drugs reverse the impairment of radial-arm maze performance caused by lesions involving the cholinergic medial pathway. Neuroscience 50:129-135 McLay RN, Freeman SM, Zadina JE (1998) Chronic corticosterone impairs memory performance in the Barnes maze. Physiol Behav 63:933-937 Page 22 of 36

Srikumar et al McPherson GA, Beart PM (1983) The selectivity of some ergot derivatives for alpha 1 and alpha 2-adrenoceptors of rat cerebral cortex. Eur J Pharmacol 91:363-369 Meador-Woodruff JH, Mansour A, Grandy DK, Damask SP, Civelli O, Watson SJ, Jr. (1992) Distribution of D5 dopamine receptor mRNA in rat brain. Neurosci Lett 145:209-212 Miura H, Qiao H, Kitagami T, Ohta T, Ozaki N (2005) Effects of fluvoxamine on levels of dopamine, serotonin, and their metabolites in the hippocampus elicited by isolation housing and novelty stress in adult rats. Int J Neurosci 115:367-378 Miyoshi E, Wietzikoski S, Camplessei M, Silveira R, Takahashi RN, Da Cunha C (2002) Impaired learning in a spatial working memory version and in a cued version of the water maze in rats with MPTP-induced mesencephalic dopaminergic lesions. Brain Res Bull 58:41-47 Mizoguchi K, Yuzurihara M, Ishige A, Sasaki H, Chui DH, Tabira T (2000) Chronic stress induces impairment of spatial working memory because of prefrontal dopaminergic dysfunction. J Neurosci 20:1568-1574 Monti JM, Hawkins M, Jantos H, D'Angelo L, Fernandez M (1988) Biphasic effects of dopamine D-2 receptor agonists on sleep and wakefulness in the rat. Psychopharmacology (Berl) 95:395-400 Muscat R, Papp M, Willner P (1992) Antidepressant-like effects of dopamine agonists in an animal model of depression. Biol Psychiatry 31:937-946 Nair VD, Sealfon SC (2003) Agonist-specific transactivation of phosphoinositide 3kinase signaling pathway mediated by the dopamine D2 receptor. J Biol Chem 278:47053-47061 O'Neill MJ, Hicks CA, Ward MA, Cardwell GP, Reymann JM, Allain H, Bentue-Ferrer D (1998) Dopamine D2 receptor agonists protect against ischaemia-induced hippocampal neurodegeneration in global cerebral ischaemia. Eur J Pharmacol 352:37-46 Packard MG, White NM (1989) Memory facilitation produced by dopamine agonists: role of receptor subtype and mnemonic requirements. Pharmacol Biochem Behav 33:511-518 Packard MG, White NM (1991) Dissociation of hippocampus and caudate nucleus memory systems by posttraining intracerebral injection of dopamine agonists. Behav Neurosci 105:295-306 Piekarzewska A, Sadowski B, Rosochacki SJ (1999) Alterations of brain monoamine levels in pigs exposed to acute immobilization stress. Zentralbl Veterinarmed A 46:197-207

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Srikumar et al Ploj K, Roman E, Nylander I (2003) Long-term effects of maternal separation on ethanol intake and brain opioid and dopamine receptors in male Wistar rats. Neuroscience 121:787-799 Puri S, Ray A, Chakravarti AK, Sen P (1994) Role of dopaminergic mechanisms in the regulation of stress responses in experimental animals. Pharmacol Biochem Behav 48:53-56 Samini M, Moezi L, Jabarizadeh N, Tavakolifar B, Shafaroodi H, Dehpour AR (2002) Evidences for involvement of nitric oxide in the gastroprotective effect of bromocriptine and cyclosporin A on water immersion stress-induced gastric lesions. Pharmacol Res 46:519-523 Shankaranarayana Rao BS, Madhavi R, Sunanda, Raju TR (2001) Complete reversal of dendritic atrophy in CA3 neurons of the hippocampus by rehabilitation in restraint stressed rats. Curr Sci 80:653-659 Sigala S, Missale C, Spano P (1997) Opposite effects of dopamine D2 and D3 receptors on learning and memory in the rat. Eur J Pharmacol 336:107-112 Srikumar BN, Bindu B, Priya V, Ramkumar K, Shankaranarayana Rao BS, Raju TR, Kutty BM (2004) Methods of assessment of learning and memory in rodents. In: Raju TR, Kutty BM, Sathyaprabha TN, Shankaranarayana Rao BS (eds) Brain and Behavior, NIMHANS, Bangalore, pp 145-151 Srikumar BN, Raju TR, Shankaranarayana Rao BS (2006) The involvement of cholinergic and noradrenergic systems in behavioral recovery following oxotremorine treatment to chronically stressed rats. Neuroscience 143:679-688 Sunanda, Meti BL, Raju TR (1997) Entorhinal cortex lesioning protects hippocampal CA3 neurons from stress-induced damage. Brain Res 770:302-306 Sunanda, Rao MS, Raju TR (1995) Effect of chronic restraint stress on dendritic spines and excrescences of hippocampal CA3 pyramidal neurons--a quantitative study. Brain Res 694:312-317 Sunanda, Shankaranarayana Rao BS, Raju TR (2000a) Chronic restraint stress impairs acquisition and retention of spatial memory task in rats. Curr Sci 79:1581-1584 Sunanda, Shankaranarayana Rao BS, Raju TR (2000b) Restraint stress-induced alterations in the levels of biogenic amines, amino acids, and AChE activity in the hippocampus. Neurochem Res 25:1547-1552 Torres IL, Gamaro GD, Vasconcellos AP, Silveira R, Dalmaz C (2002) Effects of chronic restraint stress on feeding behavior and on monoamine levels in different brain structures in rats. Neurochem Res 27:519-525

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Srikumar et al Umegaki H, Munoz J, Meyer RC, Spangler EL, Yoshimura J, Ikari H, Iguchi A, Ingram DK (2001) Involvement of dopamine D(2) receptors in complex maze learning and acetylcholine release in ventral hippocampus of rats. Neuroscience 103:27-33 Wilkerson A, Levin ED (1999) Ventral hippocampal dopamine D1 and D2 systems and spatial working memory in rats. Neuroscience 89:743-749 Yarbrough GG, McGuffin-Clineschmidt J, Singh DK, Haubrich DR, Bendesky RJ, Martin GE (1984) Electrophysiological, biochemical and behavioral assessment of dopamine autoreceptor activation by a series of dopamine agonists. Eur J Pharmacol 99:73-78 Yoshikawa T, Minamiyama Y, Naito Y, Kondo M (1994) Antioxidant properties of bromocriptine, a dopamine agonist. J Neurochem 62:1034-1038 Ziabreva I, Poeggel G, Schnabel R, Braun K (2003) Separation-induced receptor changes in the hippocampus and amygdala of Octodon degus: influence of maternal vocalizations. J Neurosci 23:5329-5336

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Srikumar et al Figure legends Figure 1: The effect of stress and bromocriptine treatment on percentage correct choice (Mean ± SEM) in a partially baited radial arm maze task. a and b show the acquisition of the RAM task across trials (expressed as block of 4 trials; see methods for details). c shows performance in block 8. Stress: rats subjected to 21 days of restraint stress. Stress + Veh: rats subjected to 21 days of stress followed by 10 days of vehicle treatment. Stress + Br 5 and stress + Br 10: stressed rats subjected to 10 days of treatment with bromocriptine 5 and 10 mg/kg, i.p., respectively. #p<0.05, ##p< 0.01 and ###p<0.001 vs. normal control, **p<0.01 and ***p<0.001 vs. stress; One-way ANOVA followed by Tukey’s post-hoc test (n=9-12).

Figure 2: The effect of stress and bromocriptine treatment on the number of RMEs (Mean ± SEM) in a partially baited radial arm maze task. a and b show the acquisition of the RAM task across trials (expressed as block of 4 trials; see methods for details). c shows performance in block 8. Groups are as described in figure 1. ##p<0.01, ###p<0.001 vs. normal control, *p<0.05 vs. stress; One-way ANOVA followed by Tukey’s post-hoc test (n=9-12).

Figure 3: The effect of stress and bromocriptine treatment on percentage correct choice (a), number of RMEs (b) and number of WMEs correct (c) in the retention test of the RAM task. 10 days after the last day of the acquisition of the RAM task, rats were subjected to two trials and the results were averaged. Groups are as

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Srikumar et al described in figure 1. Data is represented as Mean ± SEM. ###p<0.001 vs. normal control, *p<0.05 and **p<0.01 vs. stress; One-way ANOVA followed by Tukey’s post-hoc test (n=9-12).

Figure 4: The effect of bromocriptine treatment in the absence of stress on percentage correct choice (a and b) and the number of RMEs (c and d) in a partially baited radial arm maze task. a and c show the acquisition of the RAM task across trials (expressed as block of 4 trials; see methods for details). b and d shows the performance in block 8. Vehicle: rats subjected to 10 days of vehicle treatment. Br 5 and Br 10: rats subjected to 10 days of treatment with bromocriptine 5 and 10 mg/kg, i.p., respectively. Data is expressed as Mean ± SEM (n=9-12). #p<0.05, ###p<0.001 vs. normal control, One-way ANOVA followed by Tukey’s post-hoc test.

Figure 5: The effect of bromocriptine treatment in the absence of stress on percentage correct choice (a), number of RMEs (b) and number of correct WMEs (c) in the retention test of the RAM task. 10 days after the last day of the acquisition of the RAM task, rats were subjected to two trials and the results were averaged. Groups are as described in figure 4. Data is represented as Mean ± SEM. #p<0.05 vs. normal control, One-way ANOVA followed by Tukey’s post-hoc test (n=9-12).

Figure 6: The effect of stress and bromocriptine treatment on dopamine levels in the hippocampus (a), frontal cortex (b) and striatum (c). Groups are as described in

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Srikumar et al figure 1. Data is represented as Mean ± SEM (n=6). ##p<0.01 and ###p<0.001 vs. normal control, **p<0.01 ***p<0.001 vs. stress; One-way ANOVA followed by Tukey’s post-hoc test.

Figure 7: The effect of bromocriptine treatment in the absence of stress on dopamine levels in the hippocampus (a), frontal cortex (b) and striatum (c). Groups are as described in figure 4. Data is represented as Mean ± SEM (n=6). ###p<0.001 vs. normal control, one-way ANOVA followed by Tukey’s post-hoc test.

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Table 1: Working memory errors (correct) in rats subjected to stress and drug treatment

Block 7 Block 8

Normal control

Stress

Stress + Vehicle

0.40 ± 0.13 0.16 ± 0.06

0.43 ± 0.15 0.25 ± 0.08

0.33 ± 0.10 0.33 ± 0.17

Stress + Bromocriptine 5mg

Stress + Bromocriptine 10mg

0.15 ± 0.08 0.21 ± 0.14

0.19 ± 0.11 0.03 ± 0.03

Data is represented as Mean ± SEM.

Table 2: Working memory errors (correct) in rats subjected to bromocriptine treatment

Number of WMEs correct

Normal control

Vehicle

Bromocriptine 5mg

Bromocriptine 10mg

0.16 ± 0.06

0.07 ± 0.07

0.41 ± 0.09

0.33 ± 0.10

Performance in block 8 is shown; Data is represented as Mean ± SEM.

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