* 3 - Manuscript

Srikumar et al.

The Involvement of Cholinergic and Noradrenergic Systems in the Behavioral Recovery Following Oxotremorine Treatment to Chronically Stressed Rats

B.N. Srikumar, 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

*Correspondence should be addressed to: 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: +91-080-2699 5175, Fax: +91-080-2656 2121 / 2656 4830 Email: [email protected] / [email protected]

Section editor: Dr. G. J. Quirk (Behavioral Neuroscience) Running head: Behavioral recovery in stress

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ABBREVIATIONS ACh

acetylcholine

AChE

acetylcholinesterase

ACTH

adrenocorticotrophic hormone

BDNF

brain derived neurotrophic factor

HPLC

high performance liquid chromatography

MHPG

4-Hydroxy 3-methoxy phenylglycol

NE

norepinephrine

RAM

radial arm maze

RME

reference memory errors

WME

working memory errors

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ABSTRACT Chronic stress in rats has been shown to impair learning and memory, and precipitate several affective disorders like depression and anxiety. The mechanisms involved in these stress-induced disorders and the possible reversal are poorly understood, thus limiting the number of drugs available for their treatment. Our earlier studies suggest cholinergic dysfunction as the underlying cause in the behavioral deficits following stress. Muscarinic cholinergic agonist, oxotremorine is demonstrated to have a beneficial effect in reversing brain injury-induced behavioral dysfunction. In this study, we have evaluated the effect of oxotremorine treatment on chronic restraint stressinduced cognitive deficits. Rats were subjected to restraint stress (6h/day) for 21 days followed by oxotremorine treatment for 10 days. Spatial learning and memory was assessed in a partially baited 8-arm radial maze task. Stressed rats exhibited impairment in performance, with decreased percentage of correct choices and an increase in the number of reference memory errors (RMEs). Oxotremorine treatment (0.1 or 0.2mg/kg, i.p.) to stressed rats resulted in a significant increase in the percent correct choices and a decrease in the number of RMEs compared to stress as well as the stress + vehicle treated groups. In the retention test, oxotremorine treated rats committed less number of RMEs compared to stress group. Chronic restraint stress decreased acetylcholinesterase (AChE) activity in the hippocampus, frontal cortex and septum, which was reversed by both the doses of oxotremorine. Further, oxotremorine treatment also restored the norepinephrine levels in the hippocampus and frontal cortex. Thus, this study demonstrates the potential of cholinergic muscarinic

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Srikumar et al. agonists and the involvement of both cholinergic and noradrenergic systems in the reversal of stress-induced learning and memory deficits.

Keywords: Restraint stress; cholinergic dysfunction; radial arm maze; spatial memory; cholinomimetics

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INTRODUCTION Stress is any perturbation of either physiological or psychological homeostasis. While appropriate stress responses help in resisting the diseases and are beneficial, severe and prolonged stress can be harmful (McEwen, 2002). There is a wealth of evidence indicating that stress can precipitate affective disorders and cause impairment in learning and memory (McEwen, 2000). Earlier, we have demonstrated that 21 days of restraint stress impairs acquisition of a T-maze task (Sunanda et al., 2000a). In the radial arm maze (RAM), it is demonstrated that restraint stress can impair acquisition of a spatial memory task (Luine et al., 1994). In a later study, the same group has shown that stress for lesser duration (7 days) causes an enhancement of spatial memory performance, which is explained by adaptive changes following short duration of stress that falter when the stress is severe and prolonged (Luine et al., 1996). In a 14 arm radial maze, it has been shown that psychological stress impairs working memory. Twelve weeks exposure to forced swim stress once a day or 5 weeks of psychosocial stress affects acquisition of a RAM task (Diamond et al., 1996; Nishimura et al., 1999). More recently, the RAM has been used to demonstrate the sexual dimorphism in the effects of stress. While male rats show an impairment following stress, in female rats there is an enhancement of performance (Luine, 2002). Thus, the RAM is extensively used to evince the effects of different types of stressors. Furthermore, stress or chronic corticosterone treatmentinduced 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).

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Srikumar et al. Although the stress-induced impairment in learning is extensively studied, very few studies have looked into the possible ways of reversal of stress-induced deficits. For instance, tianeptine an antidepressant that lowers extracellular serotonin levels and phenytoin that prevents excitatory amino acid’s action blocks the learning impairment following chronic restraint stress (Luine et al., 1994). Further, another drug, moclobemide improved the chronic stress-induced deficits (Nowakowska et al., 2001). Cholinergic transmission appears to play a predominant role in memory processes (Gold, 2003). The alterations in the cholinergic system are perhaps the most extensively studied neurochemical consequences of stress. An earlier study from our lab has shown that 21 days of restraint stress decreases acetylcholinesterase (AChE) activity in the hippocampus (Sunanda et al., 2000b). In rats subjected to chronic unpredictable stress or chronic corticosterone administration, AChE stained neurons in the medial septum were decreased indicating cholinergic dysfunction (Tizabi et al., 1989). In mice, scopolamine induced amnesia was antagonized by 10 days of restraint stress without any impairment per se. However, 30 days of stress resulted in impairment and was also much sensitive to scopolamine-induced amnesia (Zerbib and Laborit, 1990). Thus, there are evidences for cholinergic dysfunction in stress and we hypothesized that this might contribute to impaired learning in stress. In light of the above reports, the present study examined the effects of oxotremorine (a muscarinic receptor agonist) treatment on chronic restraint stress-induced deficits in a partially baited radial arm maze task. Further, in order to understand the neurochemical basis of the stress-induced deficits and the actions of oxotremorine, we assessed the AChE activity and norepinephrine (NE) levels following stress and drug treatment.

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EXPERIMENTAL PROCEDURES Experimental animals Adult male Wistar rats (200-250g; 2 to 2.5 months old) obtained from Central Animal Research Facility, 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. The experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996 and Institutional 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 wire mesh restrainer, 6h/day for 21 days as described earlier (Shankaranarayana Rao et al., 2001). This form of restraint stress produces gastric ulcers, increases the adrenal weights (Sunanda et al., 1997; 2000a) and plasma corticosterone levels (Luine et al., 1996). Oxotremorine methiodide (ICN Biomedicals Inc, Aurora, USA) was freshly prepared in distilled water. After the completion of stress protocol, stress + oxotremorine group of rats received 10 daily injections of oxotremorine (0.1mg/kg or 0.2mg/kg, i.p.). The stress + vehicle group received 10 daily injections of the vehicle alone. In another set of experiments, rats that were not subjected to stress were given 10 daily injections of either

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Srikumar et al. vehicle alone or oxotremorine at both doses. The number of animals in each group was 912 in case of behavioral experiments and was 6 for neurochemical experiments.

Evaluation of behavior 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., 2004a). The eight arm radial maze (RAM) consisted of a computer monitored plexiform maze (Columbus Instruments, 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 Prior to the acquisition, all the arms were baited and rats were allowed to explore the maze for 10 minutes and were subjected to two such acclimatization 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). 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 maze 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

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Srikumar et al. elapsed. At the end of the trial, the rat was returned to the home cage and was given the second trial after an inter-trial interval of 1h. Training was continued until the rats attained the criteria of 80% correct choice (at least 4 correct entries out of 5). 10 days after acquisition, rats were evaluated for retention of the task. Rats were given two trials and the average was taken for analysis. Evaluation criteria Data from 4 trials were averaged and expressed as blocks. The data was analyzed 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 or an unbaited arm was considered as working memory error correct (WME correct) or working memory error incorrect (WME incorrect), respectively.

Assay of acetylcholinesterase activity Acetylcholinesterase activity was measured by Ellman’s method (Ellman et al., 1961). After the stress and drug treatment protocols, the hippocampus, frontal cortex and septum were quickly dissected out and homogenized in 0.1 M phosphate buffer, pH 8. The reaction mixture consisted of 2.6 ml of phosphate buffer (0.1 M, pH 8.0), 0.4 ml aliquot of homogenate, and 0.1 ml of 0.01 M dithiobisnitrobenzoic acid (DTNB). After the addition of the substrate acetylthiocholine iodide (0.075 M), change in the absorbance was noted every 2 min for 10 min at 412 nm using a spectrophotometer. The activity was expressed as micromoles hydrolyzed per min per gram of tissue (Shankaranarayana Rao et al., 1998; Srikumar et al., 2004b; Sunanda et al., 2000b).

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Srikumar et al. Estimation of norepinephrine levels After, the stress and drug treatment protocols, the hippocampus and frontal cortex were 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 NE and 4-hydroxy 3methoxy phenylglycol (MHPG) were made at a concentration of 1mg/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. NE 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 analysed with Winchrom data station (Indtech Instruments, Mumbai, India) (Deepti 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.

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Srikumar et al. Statistical analysis 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. 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 choices (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 (Fig. 1A).

Oxotremorine treatment reverses stress-induced impairment in acquisition Rats subjected to 21 days of restraint stress were treated with vehicle or oxotremorine for 10 days. In the radial arm maze, stressed rats treated with vehicle did not show any improvement. Rats treated with oxotremorine performed better compared to stress and stress + vehicle treated groups (F4,285= 14.53; p<0.001). Oxotremorine treated rats started performing better than stressed rats from the 3rd block, although it was not statistically significant (p>0.05). In the blocks 7 and 8, while stressed rats showed a clear impairment, the performance of rats treated with oxotremorine was comparable to normal control rats (F4,35= 4.39; p<0.01; Fig 1B and 1C). However, there was no difference in the performance between 0.1mg and 0.2mg doses of oxotremorine (p>0.05; Fig 1B and 1C).

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Srikumar et al. Reference memory impairment in stress and reversal by oxotremorine In normal control rats, the number of reference memory errors (RMEs) showed a steady decline from the block 1 itself. In block 8, the number of RMEs for control rats was 0.40 ± 0.10. In contrast, although stressed rats showed a steady decline, from the 5th block, the number of RMEs started to plateau. In the block 8, the number of RMEs in the stress group was significantly more than control rats (F4,34= 7.70; p<0.01; Fig 2A). Oxotremorine (0.1mg and 0.2mg/kg, i.p.) treated rats committed less number of errors compared to both stress and stress + vehicle treated animals (F4,34= 7.70; p<0.01; Fig 2B and Fig 2C). In the block 7 and 8, the number of RMEs in the oxotremorine treated group was comparable to normal control (Fig 2C). On the contrary, the working memory was not affected by either stress or oxotremorine 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 0.3 in the block 7 and was 0 for all the groups in the 8th block (data not shown).

Effect of stress and oxotremorine treatment on retention In the retention test, both stressed and stress + vehicle treated rats sustained the impairment seen in the acquisition (F4,36= 5.08; p<0.05; Fig 3A). When the percentage correct choice was assessed, apparently, oxotremorine did not produce any reversal of the stress-induced impairment (p>0.05; Fig 3A). However, when the RMEs were considered, the stress-induced impairment was reversed by oxotremorine treatment (F4,34= 4.94; p<0.05; Fig 3B). Although, the lower dose of oxotremorine showed a reversal, it was more pronounced at the higher dose (Fig. 3B). This discrepant finding is explained by an

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Srikumar et al. increase in the number of WMEs correct only in the oxotremorine treated groups (F4,34= 3.01; p<0.05; Fig. 3C).

Oxotremorine per se does not affect learning in the RAM Since oxotremorine produced reversal of stress-induced deficits, we examined the effect of oxotremorine on behavior of rats that were not subjected to stress. There was no difference between the groups in either percentage correct choice (F3,260= 1.73; p>0.05), number of RMEs (F3,250= 2.14; p>0.05) or WMEs correct (F3,267= 0.68; p>0.05) (Table 2). The number of WMEs incorrect was zero for all the groups in the block 8 (data not shown).

Stress-induced decrease in AChE activity and reversal by oxotremorine Restraint stress significantly (p<0.001) decreased AChE activity in the hippocampus, frontal cortex and septum (Fig. 4 A-C). There was a 36% decrease in AChE activity in both hippocampus and frontal cortex following restraint stress. In the septum, there was a 30% decrease. Oxotremorine treatment significantly restored the AChE activity in the hippocampus (F4,25= 16.66; p<0.001), frontal cortex (F4,25= 17.44; p<0.001) and septum (F4,25= 54.56; p<0.001). The AChE activity following oxotremorine (0.2 mg/kg, i.p.) treatment was increased by 50%, 54% and 53%, in the hippocampus, frontal cortex and septum, respectively when compared to the stressed animals (Fig. 4 AC).

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Srikumar et al. Stress-induced increase in NE levels and reversal by oxotremorine Following 21 days of restraint stress, the NE levels in the hippocampus (F4,24= 86.69; p<0.001) and frontal cortex (F4,25= 69.36; p<0.001) was increased significantly. Oxotremorine treatment at both the doses not only restored the NE levels but also decreased it beyond control levels (Fig. 5A and 5C). To assess the NE metabolism, the MHPG/NE ratio was estimated. One-way ANOVA revealed a significant interaction between stress and drug treatment in the hippocampus (F4,23= 17.10; p<0.001) and frontal cortex (F4,24= 24.68; p<0.001). The MHPG to NE ratio was increased only in the stress + oxotremorine treated groups and there was no difference between the stress and normal control groups (Fig. 5B and 5D).

DISCUSSION In this study, we report that 21 days of restraint stress impairs learning in a partially baited RAM task. This impairment was reversed by treatment with a muscarinic cholinergic receptor agonist, oxotremorine. Numerous investigations have shown that stress impairs learning in various tasks (Conrad et al., 1996; Luine et al., 1994; McLay et al., 1998; Sunanda et al., 2000a). The partially baited RAM task permits us to discern between reference and working memory components of spatial memory. Reference memory deals with memory for information that remains constant over repeated trials and therefore trial independent. Working memory, on the other hand, refers to memory in which the information to be remembered changes in repeated trials, and therefore is trial dependent (Mizuno et al., 2000). Both hippocampus and cerebral cortex have been suggested to contribute to reference and working memory (Durkin, 1994; Jarrard, 1993;

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Srikumar et al. Kesner et al., 1987). This is the first study in our knowledge to demonstrate that chronic restraint stress can impair reference memory component of spatial learning. An earlier report showed that chronic psychosocial stress impairs only working memory with out any change in reference memory (Diamond et al., 1996). However, it has to be noted that there are a lot of methodological differences between the paradigm used in the earlier and the present study. In their study, rats were subjected to a 14-arm radial maze task and rats were allowed to eat in 4 of the 7 baited arms. They were given a delay, at the end of which they were returned to the maze to locate the food in the 3 remaining arms. Rats were exposed to stress during the delay. In our study, the rats were subjected to chronic stress before maze testing. Further, unlike our current findings, the stress-induced changes in learning were reversed by 18 days (Luine et al., 1994) and the hippocampal CA3 atrophy was reversed by 10 days of recovery (Conrad et al., 1999). The reasons for these differences are not clearly understood. However, it can be reasoned that the information obtained from such experiments could be strain, stress and paradigmdependent. Several lines of evidence have shown the involvement of hippocampus in reference memory. In a recent study (Pothuizen et al., 2004), dorsal hippocampus lesion disrupted both reference and working spatial memory confirming an earlier report that the solving of a fixed position of reward task requires an intact hippocampus (He et al., 2002). Further, fimbria-fornix lesion also has been shown to produce a reference memory deficit even after 6.5 months of lesion (Galani et al., 2002) and intra cerebro ventricular infusion of antisense BDNF oligonucleotide produced impairment in reference memory (Mizuno et al., 2000). Thus, there is a clear correlation between hippocampal function

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Srikumar et al. and reference memory. Studies from our lab (Shankaranarayana Rao et al., 2001; Sunanda et al., 1995; Vyas et al., 2002) and others (Watanabe et al., 1992) have shown that there is hippocampal dendritic atrophy following stress. This could possibly result in the impairment in reference memory as reported in this study. In addition to reference memory, several studies have documented the impairment of working memory in other paradigms of learning like the T maze (Mizoguchi et al., 2000) and radial arm maze (Luine et al., 1994). Oxotremorine treatment to chronically stressed rats ameliorated the learning impairment. The stress-induced increase in the number of RMEs was restored to normal levels by oxotremorine treatment (Fig. 2). For the first time, we report here that treatment with a muscarinic cholinergic receptor agonist produces recovery of restraint stressinduced memory deficits. In a recent study, nicotine administration attenuated the deficits in a water maze task (Aleisa et al., 2005). The potential of oxotremorine to ameliorate learning and memory deficits is documented in several disorders. Oxotremorine (30100g/kg, s.c.) reversed the acetylcholine (ACh) depletor hemicholinum-3-induced behavioral deficits (Hagan et al., 1989) and oxotremorine (0.1mg/kg, i.p.) antagonized morphine-induced memory impairment (Li et al., 2001). In addition, oxotremorine in the same dose also reversed the behavioral impairment in fornix-lesioned rats (Maho et al., 1988). Muscarinic receptor agonists including oxotremorine reversed scopolamineinduced amnesia (Nakahara et al., 1990) and the ethylcholine mustard, AF64A and pirenzepine induced behavioral deficits have been shown to be reversed by oxotremorine treatment (Inagawa, 1994; Ukai et al., 1995; Yamazaki et al., 1991). Our results in both acquisition and retention of the RAM task are in agreement with the above studies.

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Srikumar et al. Oxotremorine is shown to increase the endogenous levels of ACh and decrease ACh turnover in the brains of mice and rats (Cheney and Costa, 1977; Schuberth et al., 1969; Trabucchi et al., 1975). In our study, oxotremorine administration to stressed rats reversed the deficits in AChE activity (Fig. 4). Acute stress activates the septohippocampal pathway and increases ACh levels (Gilad et al., 1985). On the contrary, chronic stress decreases AChE activity in the hippocampus (Sunanda et al., 2000b). However, Mizoguchi et al (2001) have reported that chronic restraint and water immersion stress increases hippocampal ACh levels. The importance of cholinergic system in the regulation of stress responses has been documented. For instance, stressinduced increases in ACTH and corticosterone are enhanced by cholinergic blockade by scopolamine (Bhatnagar et al., 1997). It is well established that the cholinergic system plays an important role in learning and memory processes and drugs influencing the learning and memory or having anxiolytic properties are known to influence the septohippocampal cholinergic system (Degroot et al., 2004). This pathway is involved in a variety of functions like learning, anxiety, motivation, exploratory and ingestive behaviors (McNaughton and Gray, 2000). This notion is further supported by our findings that a decrease in AChE activity is associated with learning and memory deficit following stress and the behavioral reversal by the administration of a cholinergic agonist involves restoration of AChE activity in both the hippocampus and septum. AChE activity has been shown to correspond to the activity levels of the medial septal projections to the hippocampus (Lewis et al., 1967) and septal or fimbria-fornix lesions have been shown to decrease AChE activity in the hippocampus (Lewis et al., 1967; Mellgren and Srebro, 1973). However, in a series of experiments, it

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Srikumar et al. has been shown that in normal animals, acetylcholine may participate but is not necessary for learning (Parent and Baxter, 2004). But in conditions of cholinergic dysfunction, administration of cholinergic agents can be beneficial. The decrease in cholinergic activity could result in changes in the receptor status as well. Mice subjected to prolonged stress for 30 days showed increased sensitivity to scopolamine-induced amnesia, oxotremorine-induced hypothermia, and increased the step through latencies (Orsini et al., 2001; Zerbib and Laborit, 1990). Chronic immobilization stress has been shown to increase the number of muscarinic ACh receptors in the hippocampus (Gonzalez and Pazos, 1992). It can be speculated that in stress, the cholinergic dysfunction might induce an upregulaton of muscarinic receptors thus sensitizing the animals. Such an upregulation could facilitate the actions of oxotremorine and might be responsible for the recovery of stress-induced behavioral dysfunction. Several studies have documented changes in the NE system following stress (Carrasco and Van de Kar, 2003). In fact, the brainstem catecholamine cell body groups (A2/C2, nucleus tractus solitarius; A1/C1, ventrolateral medulla; A6, locus ceruleus) constitute one of the main brain regions involved in the organization of the stress responses (Carrasco and Van de Kar, 2003). Projections from these nuclei to the hippocampus can have a substantial effect on learning and memory (Vizi and Kiss, 1998). While acute stress decreases tissue NE (Hellriegel and D'Mello, 1997; Pol et al., 1992), chronic stress either increases hippocampal NE (Adell et al., 1988; Beck and Luine, 1999; 2002) or produces no change (Campmany et al., 1996). These changes have been demonstrated to occur both at the level of neurotransmitter synthesis and

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Srikumar et al. metabolism. In our study, restraint stress increased norepinephrine levels and these changes were reversed by oxotremorine treatment (Fig. 5). The NE levels were reduced to levels beyond normal control values, which could be explained by an increase in the MHPG to NE ratio only in the oxotremorine treated groups (Fig. 5B & D). The interaction between noradrenergic and cholinergic systems is important for learning and memory processes (Ayyagari et al., 1991) and has been a subject of considerable study. For example, an early study showed that memory facilitation by oxotremorine involves the catecholamines (Huygens et al., 1980). AF64A lesion of the basal forebrain that produces learning deficits also decreased NE levels along with decreased ACh levels and administration of desipramine (an antidepressant drug that increases synaptic NE levels) reversed the deficit (Nakamura et al., 1992). In the present study, the stress-induced increase in NE levels was decreased by oxotremorine, a muscarinic agonist. Nakamura et al (1992) have demonstrated that oxotremorine can decrease NE release. Further, oxotremorine has been shown to increase the metabolism of NE in the hypothalamus (Karoum et al., 1980), striatum, nucleus accumbens and neocortex (Weinstock et al., 1980). These studies substantiate that oxotremorine could have effects on both the release and metabolism of NE to bring about the restoration of stress-induced increase in NE levels. In our study, 10 days of oxotremorine treatment to naïve rats did not affect RAM performance (Table 2). Chronic treatment with oxotremorine has been postulated to downregulate cholinergic receptors and impair performance in a water maze task (Abdulla et al., 1993). Another study reported that chronic oxotremorine (0.5mg /kg/h) for 6 days impaired spatial learning but this was absent in mice that began training 2 days

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Srikumar et al. after cessation of oxotremorine treatment (Wehner and Upchurch, 1989). The doses of oxotremorine used and the treatment period are much higher than what was used in the present study (0.5mg/kg and 10mg/kg as against 0.1 and 0.2mg/kg, in our study). It assumes significance because oxotremorine at doses, which did not affect learning per se, improved performance in stressed rats. Further, our finding is divergent to the other studies that show that administration of oxotremorine post-training facilitates retention of a Y-maze (Gasbarri et al., 1997) or a inhibitory avoidance task (Power et al., 2003a) (for a detailed review please refer (Power et al., 2003b). But it is important to note the methodological differences between these studies and the current study. First, oxotremorine was administered immediately post-training, where as in our study, the training began only after the administration of oxotremorine. Second, the task itself was different. It is possible that these methodological disparities account for the differences between the studies and remains to be established. In the retention test, oxotremorine treatment to stressed rats decreased the RMEs without apparently affecting the percent correct choices (Fig. 3A and 3B). Furthermore, the WMEs correct were increased only in the oxotremorine treated rats (Fig. 3C). Cholinergic drugs are known to modulate the working memory. For example, intraseptal infusion of oxotremorine post acquisition, impairs memory in a delayed-non-matchsample radial maze task (Bunce et al., 2003). A few other studies that used intraseptal administration of cholinergic agonists suggest that it could enhance working memory (Frick et al., 1996; Markowska et al., 1995). Here, we show reversal of stress-induced deficits after systemic administration of oxotremorine. It has to be emphasized that persistent effects of drug treatment include dynamic alterations in receptor density, basal

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Srikumar et al. firing rates, and a host of other intracellular metabolic changes. The cellular status could be further complicated by the presence of stress-induced changes. More studies are needed to understand the complex interplay between stress and drug treatment on receptor status and electrophysiology. In summary, our results demonstrate that oxotremorine produces reversal of stress-induced deficits in learning and this could involve both the cholinergic and noradrenergic mechanisms.

Acknowledgements 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.

FIGURE LEGENDS Figure 1: The effect of stress and oxotremorine 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 blocks 7 and 8. Stress: rats subjected to 21 days of restraint stress (n = 9). Stress + Veh: rats subjected to 21 days of stress followed by 10 days of vehicle treatment (n = 9). Stress + Ox 0.1 (n = 9) and stress + Ox 0.2 (n = 9): stressed rats subjected 10 days of treatment with oxotremorine 0.1mg/kg, i.p. and 0.2mg/kg, i.p., respectively. #p<0.05, ###

##

p< 0.01,

p< 0.001 vs. normal control (n = 12), *p<0.05 and **p<0.01 vs. stress; One-

way ANOVA followed by Tukey’s post-hoc test.

Figure 2: The effect of stress and oxotremorine 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 blocks 7 and 8. Groups are as described in figure 1. #p< 0.05,

##

p<0.01,

###

p< 0.001 vs. normal control, **p<0.01 vs. stress; One-

way ANOVA followed by Tukey’s post-hoc test.

Figure 3: The effect of stress and oxotremorine treatment on % correct choice (A), number of RMEs (B) and 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 described in Page 32 of 33

Srikumar et al. figure 1. Data is represented as Mean  SEM.

##

p< 0.01,

###

p<0.001 vs. normal

control, *p<0.05, **p<0.01 vs. stress; One-way ANOVA followed by Tukey’s post-hoc test.

Figure 4: The effect of stress and oxotremorine treatment on acetylcholinesterase activity in the hippocampus (A), frontal cortex (B) and septum (C). Groups are as described in figure 1 and n=6 in each group. Data is represented as Mean  SEM. ###

p<0.001 vs. normal control, *p<0.05, **p<0.01, ***p<0.001 vs. stress; One-

way ANOVA followed by Tukey’s post-hoc test.

Figure 5: The effect of stress and oxotremorine treatment on norepinephrine levels (A & C) and MHPG to NE ratio (B & D) in the hippocampus and frontal cortex, respectively. Groups are as described in figure 1 and n=6 in each group. Data is represented as Mean  SEM. #p<0.05,

##

p<0.01,

###

p<0.001 vs. normal control,

***p<0.001 vs. stress; One-way ANOVA followed by Tukey’s post-hoc test.

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Figure 1 Click here to download high resolution image

Figure 2 Click here to download high resolution image

Figure 3 Click here to download high resolution image

Figure 4 Click here to download high resolution image

Figure 5 Click here to download high resolution image

4 - Table 1& 2

Srikumar et al Table 1: Working memory errors (correct) in rats subjected to stress and oxotremorine treatment

Block 7 Block 8

Normal control (n = 13)

Stress (n = 11)

0.40 ± 0.13 0.16 ± 0.06

0.43 ± 0.15 0.25 ± 0.08

Stress + Vehicle (n = 9) 0.39 ± 0.21 0.20 ± 0.07

Stress + Oxotremorine 0.1mg (n = 8) 0.25 ± 0.13 0.22 ± 0.13

Stress + Oxotremorine 0.2mg (n = 8) 0.34 ± 0.14 0.22 ± 0.12

Data is represented as Mean  SEM.

Table 2: Oxotremorine treatment in the absence of stress did not affect RAM performance

% Correct choice

Normal control (n = 13) 89.17 ± 1.78

Vehicle (n = 9) 87.62 ± 3.85

Oxotremorine 0.1mg (n = 8) 93.71 ± 3.28

Oxotremorine 0.2mg (n = 8) 82.76 ± 3.16

Number of RMEs

0.41 ± 0.11

0.39 ± 0.11

0.41 ± 0.11

0.39 ± 0.11

Number of WMECs

0.16 ± 0.06

0.07 ± 0.07

0.04 ± 0.04

0.36 ± 0.09

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