Indian Journal of Medical Sciences Medknow Publications on behalf of Indian Journal of Medical Sciences Trust ISSN: 0019-5359 EISSN: 1998-3654 Vol. 57, Num. 11, 2003, pp. 487-492

Indian Journal of Medical Sciences, Volume 57, Number 11, November 2003, pp. 487-492

Effect of acute noise stress on acetylcholinesterase activity in discrete areas of rat brain

K Sembulingam, Prema Sembulingam, A Namasivayam*

School of Health Sciences, University Science Malaysia, Kubang Kerian, Kelantan, 16150, Malaysia; and ^*Department of Physiology, Dr. ALM. PG Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai-600113. India.

Correspondence: Dr. K. Sembulingam, School of Health Sciences, University Science Malaysia, Kubang Kerian, Kelantan, 16150, Malaysia. E-mail: ksembu@yahoo.com

Accepted Date: 05-11-2003

Code Number: ms03036

ABSTRACT

Effect of various stressor agents on the adrenergic system in brain had been studied extensively. However, reports on the effect of stress on various parameters of central cholinergic system are scanty. And very little is known about the effect of noise stress on the cholinergic system in brain. Hence, it was decided to elucidate the effect of acute noise stress on the activity of the enzyme acetylcholinesterase in discrete areas of brain in albino rats. Male albino rats of Wistar strain were subjected to acute noise stress for 30 minutes. The noise of pure sine wave tone was produced by using a function generator and was amplified. The frequency of noise generated was 1 kHz and the intensity was set at 100 dB. The total acetylcholinesterase activity was determined in the tissues of cerebral cortex, corpus striatum, hypothalamus and hippocampus of brain in these rats. The enzyme activity was estimated by colorimetric method using acetylthiocholine iodide as the substrate. The values were compared with the enzyme activity in the control rats. The activity of the enzyme increased significantly in all the four regions of the brain in rats after exposure to noise stress for 30 minutes. The results of the study indicate that the exposure to acute noise stress could modulate the cholinergic system in these areas of brain in rat.

KEY WORDS: Acetylcholinesterase, Brain, Cholinergic system, Noise, Stress, Rat.

INTRODUCTION

Pioneer stress physiologists like Selye¹ had documented most of the stress induced physiological changes. Many processes intervene between stressful stimulus and subsequent responses in the body. A variety of neurochemical reactions are also involved during these processes both in centraland autonomic nervous system. Acetylcholinesterase (AchE) is an important cholinergic enzyme that plays a key role during these processes.²

Extensive studies have been reported on the changes in adrenergic system in brain after exposure to stress.^3,4 But, little is known about stress-induced changes in the central cholinergic system. Recent investigations indicate that stress conditions could modulate the central cholinergic system in animals.^5,6 And few reports are available to show the changes in the activity of AchE in discrete areas of brain in animals induced by various stress agents like electric shock,^7-9 cold stress⁵ and immobilization or restraint stress.^6,10 However, some controversy exists in the results of these studies. The activity of AchE in various areas of brain in animals was noticed to increase,^5,7,10 decrease^4,8 or remain unaltered¹¹ after exposure to stress.

Also, the reports regarding the effects of noise stress on neurological system are very scanty. Noise in public places especially in occupational environments is considered as health hazard. The problem is more pertinent, particularly in occupational settings where exposure to noise for short periods is unavoidable.

The relationship between noise and central cholinergic system in animals has not been studied much. Lai (1987) reported the changes in high affinity choline uptake in brain regions induced by 30 minutes exposure to noise (100 dB).¹² The effect of noise on the receptors mediating the central cholinergic activity was noticed in discrete areas of rat brain.¹³

Recently we have observed the reduction in the acetylcholine content in discrete regions of rat brain after exposure to acute noise (1 kHz; 100 dB) stress.^14,15 It was thought that the determination of effect of noise stress on AchE activity would be essential to understand the mechanism of noise induced changes in the acetylcholine content in discrete areas of brain. The perusal of literature reveals that, so far, no attempt was made to study the effect of exposure to noise stress on the activity of the enzymes in cholinergic system in whole brain or in discrete areas of the brain in animals. Hence, in this study an attempt was made to elucidate the effect of acute noise stress on the activity of AchE in discrete areas viz. cerebral cortex, corpus striatum, hypothalamus and hippocampus of brain in albino rats.

MATERIAL AND METHODS

Experimental protocol
Twelve adult male albino rats of Wistar strain were selected for the study. All the rats were of same age group weighing 150 to 170 g. The rats were maintained under standard laboratory conditions. The food and water were available freely to all the animals. All the rats were tested for auditory sensation by observing the behavioral response to sound as followed by Dickens.¹⁶ The animals were divided randomly into two groups of six each namely control group and experimental group. The rats of experimental group were exposed to noise stress for 30 minutes and then sacrificed immediately. The rats belonging to control group were sacrificed after being placed inside the noise chamber for 30 minutes without exposure to noise stress. All the experimental procedures were carried out in the morning between 0600 and 0700 h to avoid the influence of circadian fluctuations. The study had been approved by the Animal Ethical committee of the University.

Stress procedure
The exposure of rats to noise stress was carried out by placing them in a noise chamber that was specially fabricated for this type of study. As done in our previous study¹⁴ a pure sine wave tone with a frequency of 10,000 Hz was obtained by using a function generator. The sound waves were amplified by a 40 watts amplifier and fed into a loud speaker fixed at the roof of the noise chamber at a height of 40 cm from the floor. Uniform intensity of noise in the whole area of the floor of the chamber was obtained by using a column speaker. The intensity of noise reaching the floor of the chamber was fixed at 100 dB with the help of a sound level (decibel) meter (Kemplex SLM3). The ambient noise in the room where the noise chamber was placed was <45 dB and that was due to the ventilation system of the room.

Extraction and estimation of AchE activity
All the animals were sacrificed by means of quick decapitation without causing any initial disturbance to them. The brain was isolated from the skull immediately. The dissection for discrete regions of brain was carried out as described by Glowinski and Iverson.¹⁷ Whole of cerebral cortex, corpus striatum, hypothalamus and hippocampus were isolated. Special care was taken to avoid damage or irritation of the tissues of any part of the brain while removing from the skull or while dissecting out the different areas.

The tissues of each brain region were homogenized separately in a Potter Elvehjem homogenizer by using 0.1 M phosphate buffer (pH=8) at a temperature of 0⁰C. The homogenate was centrifuged at 10,000 g for 5 minutes at 4⁰C. The total acetylcholinesterase activity in the aliquot of the homogenate was estimated by the method followed by Ellman et al.¹⁸ The aliquot was mixed with phosphate buffer (pH=8). To this, the substrate acetylthiocholine iodide (Sigma, USA) and dithiobisnitrobenzoic acid (DTNB) reagent (Sigma, USA) were added. Acetylthiocholine iodide was hydrolyzed to thiocholine and acetate by AchE. Thiocholine reacted with DTNB reagent to produce a yellow color. The rate of color development is the measure of the AchE activity. A kinetic profile of the enzyme activity was studied spectrophotometrically at 412 μm at the interval of 15 seconds. The enzyme activity is expressed as the μMol of substrate hydrolyzed/minute/gram tissue. Assay of enzyme activity in each sample was run in duplicate.

Statistical analysis of the results was carried out by the software SPSS 11.0 using Student's `t' test to compare the differences between control and experimental groups.

RESULTS

The data of results are given in the Table 1. Data are presented as means ± S.D of six animals per group. The base line values for the activity of AchE in discrete areas of rat brain in this study were in agreement with the values reported by other investigators.⁸ The exposure to noise stress for 30 minutes resulted in significant elevation of the enzyme activity in all the four areas of the brain viz. cerebral cortex, corpus striatum, hypothalamus and hippocampus.

DISCUSSION

Noise is generally considered as a common and strong environmental stress factor for both human beings and animals. Several reflex responses in the body, which are evoked by the exposure to noise stress become more pronounced if the noise is of unknown character or unexpected. The non-familiar noise can produce a fully developed defense alarm reaction with hemodynamic, hormonal, behavioral and various other changes leading to fight and flight reactions.¹

Estimation of the activity of AchE was thought worth doing in this study because this enzyme is considered as the classical enzyme in the cholinergic neurohormonal system. It plays an important role in cholinergic neurohormonal system particularly during stress conditions.² It is established that AchE inhibits the prolonged action of the cholinergic neurotransmitter acetylcholine by hydrolyzing it.¹⁹ Also, AchE ranks as one of the most efficient enzymes, since one molecule of the enzyme can hydrolyze 5000 molecules of acetylcholine per second.²⁰ AchE was found to be present in all the areas of cholinergic system in brain, which contain acetylcholine. Of course it is reported to be present also in the brain areas, which are devoid of acetylcholine.²¹

Among the brain regions, cerebral cortex, corpus striatum, hypothalamus and hippocampus were chosen for investigation in this study because of few interesting facts. First, it was established that only these four regions of brain contained highest amount of cholinergic innervations.⁸ Second, it was known that the activity of AchE was very much related to the content of total acetylcholine in these four areas of the brain.^8,11

Also, the cholinergic neurons in these areas of brain play some specific and unique role in modulating various physiological, biochemical and behavioral responses particularly during exposure to stress.²² The cholinergic nerve fibers in cerebral cortex are involved in cortical activation and selective awareness that are necessary for information and acquisition. The corpus striatum was reported to be extremely rich in acetylcholine content and AchE activity than any other part of brain in animals, particularly rats.⁸ Functionally, the cholinergic fibers of corpus striatum are responsible for the extrapyramidal control of all the activities.⁸ The involvement of cholinergic fibers in hypothalamus in stress conditions had been well established. Hypothalamus is known for its role in the commencement and maintenance of various reactions during general adaptation syndrome by activating both sympathetic adrenomedullary and pituitary adrenocortical pathways. Hippocampus is involved in mediating various mechanisms concerned with adaptation, habituation in novel environment and behavioral responses, especially during stress conditions.²²

The result of the present study i.e., the elevation of AchE activity in all the four areas of rat brain viz. cerebral cortex, corpus striatum, hypothalamus and hippocampus after exposure to 30 minutes noise (10,000 Hz; 100 dB) stress supports the results of our previous studies. Earlier we had noticed the significant reduction in the acetylcholine content of the same four areas of brain in rats exposed to 30 minutes noise (10,000 Hz; 100 dB) stress.^14,15

In view of the results of our previous studies and the present study, it could be speculated that the fall in acetylcholine content in discrete areas of brain in animals exposed to acute noise stress might be due to the release of acetylcholine from the cholinergic nerve terminals and its subsequent hydrolysis by AchE. Literature reveals this type of inverse relationship between the activity of AchE and the content of acetylcholine in brain regions of animals subjected to few other types of stress agents like cold stress⁵ and electric shock.⁹

It was not known whether the changes in the activity of the AchE and acetylcholine content in different regions of brain after exposure to acute noise stress could be beneficial or harmful to the organism. But, the exposure to noise could lead to more detrimental behavioral and physiological functions by modulating central cholinergic system in the brain of the organism.

The neural mechanism, through which the noise stress could affect the activity of cholinergic system in brain is also not known. Because of the nonexistence of direct connections of central auditory pathway with hippocampus, hypothalamus, striatum and different lobes of cerebral cortex other than temporal lobe, the effect of noise could be an indirect one. Perhaps, the arousal effects of noise could influence the reticular formation, which in turn might modulate the cholinergic pathway.¹² So, further investigations are essential to understand the mechanisms involved in the noise induced changes in the central cholinergic system.

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