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ORIGINAL ARTICLE

Restoration of acetylcholinesterase activity by Euphorbia hirta in discrete brain regions of chronically stressed rats H. Anuradha1, B. N. Srikumar2,3, N. Deepti2, B. S. Shankaranarayana Rao2, and M. Lakshmana1 Department of Pharmacology, Government College of Pharmacy, Bangalore, India, 2Department of Neurophysiology, National Institute of Mental Health and Neuro Sciences, Bangalore, India, and 3current address: Laboratoire “Physiologie Cellulaire de la Synapse”, UMR 5091 CNRS, Université Bordeaux 2, Institut François Magendie, Bordeaux, France 2009

1

Abstract Several drugs of herbal origin are known to possess anxiolytic and antidepressant effects. In a recent study, we showed that extracts from Euphorbia hirta L. (Euphorbiaceae) (Eh) demonstrated anxiolytic effects in rats subjected to chronic immobilization stress (CIS) but not in rats that underwent forced swim stress (FSS). Acetylcholine and the cholinergic system are known to be involved in anxiety. However, whether the cholinergic system is involved in the anxiolytic actions of Eh are not known. In the current study, we evaluated the effects of Eh treatment of rats subjected to either CIS or FSS on acetylcholinesterase (AChE) activity in the frontal cortex, hippocampus, and septum. CIS increased the AChE activity in all three regions, while Eh treatment restored it to normal levels. FSS increased the AChE activity only in the septum, and Eh treatment marginally restored this to normal levels. Thus, these results indicate the involvement of the cholinergic system in the behavioral effects of Euphorbia hirta. Keywords: Acetylcholinesterase activity; anxiety; cholinergic transmission; Euphorbia hirta; herbal drugs; chronic stress

Introduction A broad range of findings obtained during the past several decades support the view that forebrain acetylcholine (ACh) modulates several cognitive functions (Gold, 2003). Particularly, ACh is thought to play a role in anxiety. In models of experimental anxiety, physostigmine, an acetylcholinesterase (AChE) inhibitor, demonstrated anxiolytic properties (Sienkiewicz-Jarosz et al., 2000). Further, muscarinic antagonists increase, while nicotinic agonists decrease anxiety (Brioni et al., 1993; Rodgers & Cole, 1995). In clinical studies, AChE inhibitors decreased anxiety in Alzheimer’s patients

(Weiner et al., 1997) and a cholinesterase-inhibiting sage (Salvia officinalis L., Lamiaceae) demonstrated anti-anxiety effects in human subjects (Kennedy et al., 2006). These several lines of evidence show that ACh or the cholinergic system plays a role in mediating anxiety. Stress is increasingly being recognized as the precipitant of several psychiatric illnesses including anxiety and depression (McEwen, 2000). In earlier studies, rats subjected to chronic immobilization stress (CIS) or forced swim stress (FSS) showed anxiety in the elevated plus maze (EPM) and the open field test (OFT) (Anuradha et al., 2008; Govindarajan et al.,

Address for Correspondence: 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. Tel: +91-80-2699 5175. Fax: +91-80-2656 2121/2656 4830. E-mail: [email protected]. nic.in/[email protected] (Received 02 October 2008; revised 23 February 2009; accepted 02 March 2008) ISSN 1388-0209 print/ISSN 1744-5116 online © 2009 Informa UK Ltd DOI: 10.1080/13880200903188534

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2006; Vyas et al., 2002). In addition to anxiety, stress is also known to produce learning and memory deficits. For example, chronic stress impaired learning in the T-maze and radial arm maze (Ramkumar et al., 2008; Srikumar et al., 2006, 2007) or in other paradigms such as the Barnes maze and Morris water maze (Bodnoff et al., 1995; McLay et al., 1998). Several morphological, neurochemical, and neurogenic mechanisms have been proposed to underlie the stress-induced cognitive deficits (McEwen, 2000). Detailed evaluation of the hippocampal morphology over the past several years clearly indicates atrophy of the hippocampal CA3 neurons following stress (Magarinos & McEwen, 1995; Ramkumar et al., 2008). Further, the dopaminergic and cholinergic neurotransmitter systems have been shown to be involved in mediating the stressinduced deficits (Srikumar et al., 2006, 2007; Sunanda et al., 2000). Several studies have examined the possible amelioration of stress-induced cognitive deficits or other types of anxiety (Barros et al., 2007; Rajarao et al., 2007; Srikumar et al., 2006, 2007). In this context, drugs of natural origin are being evaluated in pursuit of better molecules or targets to treat anxiety disorders (Carlini, 2003; Kienzle-Horn, 2002). In clinical studies, Kava-kava (Piper methysticum G. Forst., Piperaceae) demonstrated effective anti-anxiety properties in several randomized controlled trials. St. John’s wort (Hypericum perforatum L., Clusiaceae) and sympathyl were found to possess anti-anxiety properties in a single double-blind, placebo-controlled trial (Saeed et al., 2007). Chrysin, an extract of Passiflora incarnata L. (Passifloraceae) showed anxiolytic properties in the EPM (Brown et al., 2007). Several tests of experimental anxiety indicated the anti-anxiety effects of the inflorescences of Tilia americana L. (Tiliaceae), and β-sitosterol was identified as one of the active constituents (Aguirre-Hernandez et al., 2007). In a recent study, Euphorbia hirta L. (Euphorbiaceae) (Eh) was an effective anxiolytic in CIS-induced anxiety in the EPM and OFT. It is found to mediate its action through the benzodiazepine–γ-aminobutyric acid A (GABAA) receptor–Cl− channel complex (Anuradha et al., 2008). Thus, it is known that stress can precipitate anxiety, and cholinergic mechanisms are involved in both anxiety and in mediating the effects of stress, and Eh is effective in CIS-induced anxiety. However, whether changes in AChE activity occur in response to CIS or FSS, and the effects of Eh treatment on stressed animals are unknown. Accordingly, in the present study, we estimated AChE activity in the frontal cortex, hippocampus, and septum following Eh treatment to rats subjected to either CIS or FSS. PHB 419026

Materials and methods Experimental animals Male Wistar rats weighing 200–225 g (2–2.5 months old) obtained from the Central Animal Research Facility at the National Institute of Mental Health and Neuro Sciences (NIMHANS), Bangalore were used for the experiments. The rats were housed and maintained in standard laboratory conditions. Food and water were provided ad libitum, unless specified otherwise in stress protocols. The experimental protocols were approved by the institutional animal ethics committee. All efforts were made to minimize both the suffering and the number of animals used. Rats were randomly assigned to the following groups consisting of six rats each: normal control (NC), chronic immobilization stress (CIS), CIS + vehicle (Veh), CIS + Eh, forced swim stress (FSS), FSS + Veh, FSS + Eh. NC rats did not undergo any stress or treatments and were housed in standard conditions before sacrifice. CIS and FSS groups of rats underwent 10 days of either chronic immobilization stress or forced swim stress, respectively. CIS + Veh, CIS + Eh, FSS + Veh, and FSS + Eh groups of rats underwent 10 days of the respective stress along with either vehicle or Eh treatment as described below. Induction of stress Two types of stressors were applied to separate groups of animals: CIS, 2 h daily for 10 days (Anuradha et al., 2008; Vyas et al., 2002) and FSS (Roche et al., 2003). All the stress protocols were followed between 10:00 h and 12:00 h. CIS consisted of complete immobilization in rodent immobilization bags as described elsewhere (Vyas et al., 2002). For the FSS, swimming apparatus measuring 60 cm in height and 45 cm in diameter was used. Water was filled to a level of 30 cm. The 30 cm depth allowed rats to swim or float without allowing their tails to touch the bottom of the tank. The temperature was maintained at 23 ± 1°C. On the first day of stress, each animal was forced to swim for a period of 15 min. Later, individual animals were forced to swim for a period of 5 min regularly for 10 days. Immediately after the swim session, rats were removed from the tank, dried, and put in a warming cage that was covered with towels for 10 min. Rats were then returned to their respective home cages (Anuradha et al., 2008; Roche et al., 2003). Drug treatment Hydroalcoholic extract of the whole plant of Euphorbia hirta was supplied by the Himalaya Drug Company, Bangalore, India. Eh was dissolved in distilled water and was administered at a dose of 200 mg/kg orally,

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Euphorbia hirta restores AChE activity in stressed rats A

Statistical analysis The data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons post hoc test. Values are expressed as mean ± SEM; p < 0.05 was considered statistically significant.

###

###

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***

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NC CIS CIS + Veh CIS + Eh

2 1 0

Assay of acetylcholinesterase activity B micromoles hydrolysed/min/g of tissue

Acetylcholinesterase activity was measured by modified Ellman’s method (Ellman et al., 1961; Ramkumar et al., 2008; Srikumar et al., 2006; Sunanda et al., 2000). At the end of experimentation, rats were decapitated; the frontal cortex, hippocampus, and septum were dissected quickly. The tissues were homogenized in 0.1 M phosphate buffer, pH 8.0. 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 addition of the substrate, acetylthiocholine iodide (0.075 M), the change in absorbance was noted every 2 min for 10 min at 412 nm using an LKB spectrophotometer. The activity was expressed as micromoles hydrolyzed per minute per gram of tissue.

Frontal cortex 5

micromoles hydrolysed/min/g of tissue

for 7 days, i.e. from the 5th through the 10th day of the stress, 1 h prior to stress, and on the 11th day. Since stress-induced anxiety was decreased by this dose of Eh in the earlier study (Anuradha et al., 2008), we used the same dose in the current study. On the 11th and 13th days, they were exposed to a session of anxiety tests in the elevated plus maze and open field test as reported earlier (Anuradha et al., 2008), and the animals were sacrificed on the 14th day for the AChE assay.

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Hippocampus ##

## **

4

2

0

C

Septum 8

micromoles hydrolysed/min/g of tissue

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6

##

# **

4 2 0

Results and discussion Male Wistar rats were subjected to either CIS or FSS for 10 days and were treated with the hydroalcoholic extract of Euphorbia hirta (200 mg/kg, p.o., for 7 days, i.e. from the 5th through the 10th day of the stress, 1 h prior to stress, and on the 11th day). The animals were sacrificed on the 14th day and the frontal cortex, hippocampus, and septum were obtained. AChE activity was estimated using modified Ellman’s method in tissue homogenates (Ramkumar et al., 2008; Srikumar et al., 2006). Ten days of CIS significantly increased the AChE activity in the frontal cortex (F3,20 = 19.14; p < 0.001), hippocampus (F3,20 = 11.11; p < 0.001), and septum (F3,20 = 7.65; p < 0.01) (Figure 1). Eh treatment significantly reduced the CISinduced increase in AChE activity in the frontal cortex, hippocampus, and septum (Figure 1). In contrast, FSS rats demonstrated an increase in AChE activity only in PHB 419026

Figure 1. Restoration of CIS-induced increase in AChE activity in the frontal cortex (A), hippocampus (B), and septum (C) by Euphorbia hirta treatment. NC, normal control; CIS, rats subjected to 10 days of chronic immobilization stress; CIS + Veh, CIS + Eh, rats subjected to CIS followed by vehicle or Euphorbia hirta 200 mg/kg, p.o. treatment, respectively. Values represent mean ± SEM. #p < 0.05, ## p < 0.01, ###p < 0.001 vs. NC; **p < 0.01, ***p < 0.001 vs. CIS; one-way ANOVA followed by Tukey’s post hoc test.

the septum (F3,20 = 4.27; p < 0.05), and it was restored following Eh treatment (Figure 2C). Drugs having amnesic or anxiolytic properties are known to influence the septohippocampal cholinergic system (Degroot et al., 2004). Furthermore, several drugs of natural origin have been found to involve the cholinergic system in mediation of the anxiolytic response. Extracts of Hypericum perforatum (St. John’s wort) have been recently demonstrated to mediate the anti-anxiety effect through cholinergic activation

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4 A

Frontal cortex

micromoles hydrolysed/min/g of tissue

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NC FSS FSS + Veh FSS + Eh

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micromoles hydrolysed/min/g of tissue

B

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Hippocampus

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C

Septum 10

micromoles hydrolysed/min/g of tissue

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#

#

6 4 2 0

Figure 2. Effect of FSS and Eh treatment on AChE activity in the frontal cortex (A), hippocampus (B), and septum (C). NC, normal control; FSS, rats subjected to forced swim stress; FSS + Veh, rats subjected to forced swim stress and vehicle treatment; FSS + Eh, rats subjected to forced swim stress and Euphorbia hirta treatment. Values represent mean ± SEM. #p < 0.05 vs. NC; one-way ANOVA followed by Tukey’s post hoc test.

(Vandenbogaerde et al., 2000). A cholinesterase-inhibiting sage (Salvia officinalis) demonstrated anti-anxiety effects in human subjects (Kennedy et al., 2006). In the present study, for the first time, we report that Eh could recruit the cholinergic system in producing anxiolytic activity. The septohippocampal cholinergic pathways are thought to play a pivotal role in mediating the response to novel, stressful, and anxiogenic stimuli (Degroot et al., 2004; Lamprea et al., 2003). This pathway is involved in several cognitive functions such as learning, PHB 419026

anxiety, motivation, exploratory, and ingestive behaviors (McNaughton & Gray, 2000). In spite of several studies, the role of ACh in anxiety remains unclear. Low doses of nicotine produce anxiolytic effects, while high doses have been found to be anxiogenic (File et al., 2000). It has been proposed that the basal level of anxiety and experimental paradigms used are critical in evaluating the effects of cholinergic drugs. For low levels of anxiety, cholinergic antagonists cause anxiety. The endogenous cholinergic tone in the dorsal hippocampus plays a modulatory role in reducing anxiety. It can be speculated that exposure to CIS leads to anxious states that enhance the basal cholinergic tone in the hippocampus as a compensatory mechanism. Our finding that AChE activity in the hippocampus is increased following CIS corroborates this hypothesis. Eh treatment led to decreased anxiety, and, thus, AChE activity was restored. AChE activity in the hippocampus has been shown to correspond to the level of activity of the projections of the medial septum to the hippocampus (Lewis et al., 1967). Septal and fimbria–fornix lesions have been shown to decrease AChE activity in the hippocampus (Lewis et al., 1967; Mellgren & Srebro, 1973). In contrast to CIS, FSS did not modify the AChE activity except in the septum, where there was a marginal increase in AChE activity. Swim stress has been reported not to change the AChE activity in an earlier study (Pung et al., 2006). Further, Eh also did not alter the AChE activity significantly in FSS animals. This lack of action of Eh on AChE activity is similar to the absence of anxiolytic effects of Eh in FSS rats (Anuradha et al., 2008). It has been argued that the serotonergic system plays an important role in FSS-induced anxiety and Eh mediates its action at least in part through the GABAergic system, and thus may be ineffective. The present results show that, to a considerable extent, the cholinergic system also could be involved in the anxiolytic actions of Eh, and FSS might not recruit the cholinergic pathways in a major way, which could explain the ineffectiveness of Eh in FSS-induced anxiety. In conclusion, the present study demonstrates the involvement of the cholinergic system in mediating the anxiolytic effects of Euphorbia hirta in chronically stressed rats.

Acknowledgements This study was partially funded by The Himalaya Drug Company, Bangalore, India. We acknowledge Salesworth Education Foundation, Bangalore, India for a scholarship to one of the authors (H.A.). Declaration of interest:

AQ1

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