Norepinephrine

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A MORPHOLOGICAL AND QUANTITATIVE STUDY OF MITRAL CELLS IN THE RAT OLFACTORY BULB AFTER ADMINISTRATION OF PROPANOLOL

ABSTRACT

Objective: The present study on the histological changes in the rat olfactory bulb after the administration of propranolol was carried out with emphasis on its neuromodulatory effect on norepinephrine. Study Design: Experimental study. Place and Duration of Study: The study was conducted in the Department of Anatomy, University of Health Sciences, Lahore from January 2006 to January 2007. Methodology: Twenty samples were obtained from two groups of rats, each comprising ten animals for control and experimental work respectively. Each group was treated with normal saline (5 ml/kg) and propranolol (1 mg/kg) respectively for one month. The skull was fixed in 20% formalin for 10 days and decalcified in 10 % formalin/ 10% nitric acid. The olfactory bulb along with olfactory cortex was dissected. After processing, ten microns thick sections were obtained. The slides were stained with Hematoxylin & Eosin and Bielschowksy’s silver stain (Glees-Marsland modification) and studied under light microscope. The morphology, quantitative analysis of mitral cell layer and the number of mitral cells were studied in the histological study. Results: In the propranolol treated group changes observed in the morphology of the mitral cells included presence of cytoplasmic vacuoles at the periphery of the cells. “t” test showed significant increase in the thickness of mitral cell layer and number of the mitral cell in the propranolol treated group (p-value<0.05). Conclusion: Propranolol is an important neuromodulator of norepinephrine. This study showed morphological and quantitative changes in the olfactory bulb in response to treatment with propranolol, hence its implications in odor induced learning.

INTRODUCTION

Odors play an important role in regulating social behaviour, they can signal the social status of an individual, affect reproductive development, instigate mating behaviour, and are used to identify kin in a variety of species.1

The principal relay neurons of the bulb send their axons through the lateral olfactory tract to project to several parts of the telencephalon.2 A lot of importance has been given to olfaction, clinically. Studies have reported that olfactory impairment may precede the clinical appearance of cognitive impairment in Alzheimer’s disease (AD) and further it was postulated that it may be an early sign of brain change. People who stood at a high risk for AD had demonstrated poor performance on olfactory tests than controls.3-5 Those who showed olfactory impairment were more likely to progress to AD as compared to other subjects.3

Efforts need to be undertaken to study mechanisms of enhancing olfaction. Recently, a lot of work has been done to establish the role of norepinephrine in odor induced learning. It has been shown to be important and often necessary for odor preference learning in rats.6 Veyrac et al.7 showed a decrease in the olfactory induced learning in mice by injecting them with propranolol and prazocin they further implicated that these findings indicate a role of norepinephrine on short term olfactory recognition. In another study on the anti predatory response of rodents to cat odour, propranolol reduced the defensive response to cat odour.8

Paradoxically Gray et al.9 found no change in the olfactory discriminatory ability of rabbits after infusing propranolol. Moreover, a role of combined blockage of α and β adrenoceptors to change the olfactory ability of mice has been postulated and a single receptor blockage doesn’t affect odour discriminatory abilities.10 A change in olfactory induced learning may therefore be associated with structural changes in the olfactory system; hence, this study was designed to see the morphological and quantitative changes in the rat olfactory bulb after administration of propranolol.

MATERIALS AND METHODS

Twenty Albino (Sprague-Dawley) adult male rats were obtained from National Institute of Health, Islamabad. Two groups were made according to administration of drug, comprising 10 rats each. The rats were kept in cages (3-4 rats in each cage). Each group was kept separate from the other, and was provided with a 12-hour light dark cycle and access to food and water ad libitum. The first group of rats (control group) was injected intraperitoneally with saline 5 ml/kg. The second group (experimental group) was injected with propranolol (Sigma, MO) 1 mg/kg daily. These injections were as a single dose into lower left quadrant of abdomen for a period of 30 days. All experimental protocols were conducted in compliance with the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals.11

After the experimental period the animals were sacrificed. The skin was removed and the skull was put into a solution of 20% formalin for ten days for fixation. For decalcification, the decapitated head was placed in a jar containing 10% formalin / 10% nitric acid.12 The solution was changed daily for a period of five days until the skull was decalcified.

The nose, nasal bones and lower jaw was separated from the skull by a section in coronal plane in front of the eyes. A mid-sagittal incision was made along the bone of the remaining skull from the anterior to posterior limit. A third incision was made in coronal plane behind the eyes to the inferior limit of the skull. A fourth incision parallel to and 2 cm posterior to the third was made and the intervening bone was removed. The bone medial to the eyes was slowly chipped off and eyes along with the orbital bones were removed revealing the olfactory bulb. The two olfactory bulbs were separated by cutting along the mid-sagittal plane passing through the nasal septum. The tissues, containing the rostral half of the cerebral cortex, whole of the olfactory bulb and part of the nasal bones were taken en-bloc. The orbital bone was meticulously dissected away and olfactory bulb, and part of the cerebral hemisphere of each side was processed (Histotouch III).

The tissue samples were dehydrated in ascending ethanol series, cleared in xylene and embedded in paraffin wax (56-58oC melting point) according to standard histological procedures.

The specimen was placed with the help of a blunt forceps in such a manner that the part of the tissue adjacent to the septum lied flat on the glass slab. Molten wax was poured into the box and the tissue reoriented appropriately.

Ten microns thick consecutive sections were obtained using Leica rotatory microtome (RM 2125) and stained with Haematoxylin and Eosin for general histological study (A) , and Bielschowsky’s silver stain (Glees-Marsland modification), for demonstration of nerve fibers (B).12

The sections were studied under a light microscope (Leica DM 1000). In addition to the morphology of the mitral cells, quantitative measurements regarding thickness of mitral cell layer and number of mitral cells under 40 X magnifications was made in the mitral cell layer of the olfactory bulb in three separate fields A, B and C respectively. The fields A, B and C were selected in the roof of the bulb at a distance of 250, 275 and 300 microns from the anterior tip of the bulb in all the histological sections.

The method of micrometry as described by Culling13 was adopted because of the unavailability of Neurostereological techniques to measure the thickness of mitral cell layer.

The scale of the eyepiece micrometer was superimposed on the mitral cell layer. Number of divisions from inferior to the superior layer of mitral cells multiplied by 2.4 was taken as actual height of mitral cell layer in microns. The height was measured from three different fields designated Field A, Field B and Field C respectively and mean was calculated.

The number of mitral cells was counted at the diameter of the field under 40 X objective. The diameter under 40 X objective was 400 microns. The number was counted from three different fields designated Field A, Field B and Field C respectively and mean was calculated.

Statistical analysis was conducted using the computer software statistical package for social sciences (SPSS version 13.0). Drug treatment differences between each group of normal saline and Propranolol treated rats were tested for significance using independent sample “t” test.14 The value of  was 0.05, with a confidence interval of 95.

RESULTS

The histology of olfactory bulb of the control group revealed six laminae (Figure 1). The mitral cell layer consisted of pyramidal shaped cells, with abundant cytoplasm and prominent nucleoli (Figure 2). Cells smaller than mitral cells having a dark basophilic nucleus were also observed, these granule cells were scattered throughout the mitral cell layer.

Differences in the morphological characteristics of the mitral cells were seen in the propranolol treated group. Cytoplasmic vacuoles were observed at the periphery of some of the mitral cells. However, the nucleus was rounded in appearance with prominent nucleoli .

The thickness of the mitral cell layer of the propranolol treated group (43.64±3.17µm) was greater as compared to the control group (28.59 ±1.77µm). Similarly, the number of mitral cells in the propranolol treated group (23.9 ±0.67) was greater as compared to the control group (21.2±0.61). T-test showed that there was a significant increase in the thickness of the mitral cell layer and the number of mitral cells in the experimental group p-value < 0.05 (Table I).

DISCUSSION

The histological changes in the rat olfactory bulb were explored by administering propranolol. The rat was used as an experimental animal as ‘the rat pup odor preference learning model’ and adult rat olfactory learning, offers an advantage because as an unconditioned stimulus the role of  adrenoceptors activation is well established.15,16 In the infant rat pup, learning to prefer odors associated with maternal care helps the pup maintain proximity to the odor. Stroking and licking the pup produced a prolonged activation of locus coeruleus neurons17,18 and as a result norepinephrine was released in the olfactory bulb.19 Exposure to a novel odor prior to stroking induced a preference for the novel odor.20

In the present study, significant morphological and quantitative changes were observed in the mitral cells, such a significant change on light microscopic level has been scarcely mentioned in the literature. Therefore further work needs to be done to find out the exact significance of these histological findings.

Sullivan et al.21 examined the adequacy of pairing an odor with either intrabulbar activation of noradrenergic -receptors or pharmacological stimulation of the locus coeruleus to document learned odor predilection in 6-7 days postnatal rats. Odor yolked with -receptor stimulation displayed an adapted approach response on subsequent exposure to that odor. Stimulation of the locus coeruleus also produced a conditioned approach to the odor. The effects of locus coeruleus stimulation were blocked by pretreatment with propranolol at 20 mg/kg dose. In the present study a low dose of propranolol was used, it also showed significant histological changes in the number of mitral cells and thickness of mitral cell layer.

Gray et al.22 studied the characteristic EEG pattern in the olfactory bulb aroused by an odor in rabbits and observed that it was altered by olfactory learning. This modification was averted during training by blockage of the -adrenergic receptors by propranolol. Sullivan et al.23observed that the postnatal olfactory learning created a conditioned behavioral response as well as a reformed olfactory bulb neural response to the learnt odor. They studied the role of norepinephrine on both of these responses and observed that propranolol injected prior to odor-stroke training blocked attainment of both the learnt behavior and olfactory bulb neural response. Due to the learnt behavior there was an increase in the uptake of 14C-2DG in the odor-specific foci within the bulb and the neural response modified output signal from the bulb determined by a single cell recording of mitral/tufted cells. Wilson and Sullivan24 observed the response design of mitral-tufted cells reformed by correlated adaptation during the early postnatal period. They suggested that norepinephrine -receptor activation is mandatory for early olfactory learning using medial forebrain bundle-lateral hypothalamus (MFB-LH) stimulus as a reward. Propranolol injected intraperitoneally before odour-MFB-LH pairing blocked the acquirement of conditioned behavioral reactions and their neural responses were associated with the conditioned odor. In olfactory affiliated learning citral odor can act as the conditioned stimulus (CS) and intraoral infusion of milk as the unconditioned stimulus (UCS). Rat pups injected with propranolol after a thirty-minute training period, were tested 24 hours later for an attained associated odor preference for the conditioned stimulus. Propranolol flawed memory for the conditioned stimulus in a dose dependant conduct.25 However, our results show an increase in the number of mitral cells and thickness of mitral cell layer after the administration of propranolol.
Although, the present study did not aim at any molecular mechanism involved, however, it can be postulated that -adrenoceptor blockage or facilitation is a molecular mechanism in olfactory induced learning and there are significant histological changes involved in such learning reflected by a significant increase in number of mitral cells and a significant increase in the thickness of mitral cell layer.

CONLUDING REMARKS

The current study assessed the morphological and quantitative changes in the rat olfactory bulb after the administration of propranolol. Histological changes were evident in the mitral cells showing vacuolization. The quantitative studies showed an increase in the thickness of mitral cell layer and increased number of mitral cells in the propranolol treated group. Previously behavioral and molecular studies have shown propranolol to decrease odor induced learning in rats. This study will provide an impetus to carry out investigation further, using an Electron microscope with the hope to elucidate the possible ultrastructural changes in the olfactory bulb in response to treatment with propranolol.

REFERENCES

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5. Schiffman SS, Graham BG, Sattely-Miller EA, Zervakisa J, Welsh-Bohmera K. Taste, smell and neuropsychological performance of individuals at familial risk for Alzheimer’s disease. Neurobiol Aging 2002; 23: 397-404.

6. Aghajanian GK. Modulation of a transient outward current in serotonergic neurones by alpha 1-adrenoceptors. Nature 1985; 315: 501-503.

7. Veyrac A, Nguyen V, Marien M, Didier A Jourdan F. Noradrenergic control of odor recognition in a nonassociative olfactory learning task in the mouse. Learn Mem. 2007; 14(12):847-54.

8. Do Monte FH, Canteras NS, Fernandes D, Assreuy J, Carobrez AP. New perspectives on beta-adrenergic mediation of innate and learned fear responses to predator odor. J Neurosci. 2008; 28(49): 13296-302.

9. Gray CM, Freeman WJ, Skinner JE. Chemical dependencies of learning in the rabbit olfactory bulb: Acquisition of the transient spatial pattern change depends on norepinephrine. Behav. Neurosci. 1986; 100: 585-596.

10. Doucette W, Milder J, Restrepo D, Adrenergic modulation of olfactory bulb circuitry affects odor discrimination. Learn. Mem. 2007 14: 539-547

11. International Helsinki Federation for Human Rights. International Helsinki Federation for Human Rights.). Vienna: International Helsinki Federation for Human Rights.

12. Bancroft JD, Gamble M. Theory and practice of histological techniques. 5th ed. Edinburgh: Churchill Livingstone; 2002.

13. Culling CFA. Handbook of histopathological and histochemical techniques:(including museum techniques). 3rd ed. London: Butterworth; 1974.

14. Kuzma JW, Bohnenblust SE. Basic statistics for the health sciences. 4th ed. Mountain View: Mayfield Publishing Company; 2001.

15. Sullivan RM, McGaugh JL, Leon M. Norepinephrine-induced plasticity and one-trial olfactory learning in neonatal rats. Brain Res Dev Brain Res 1991; 60: 219-228.

16. Langdon PE, Harley CW, McLean JH. Increased beta adrenoceptor activation overcomes conditioned olfactory learning deficits induced by serotonin depletion. Brain Res Dev Brain Res 1997; 102: 291-293.

17. Kimura F, Nakamura S. Locus coeruleus neurons in the neonatal rat: electrical activity and responses to sensory stimulation. Brain Res 1985; 355: 301-305.

18. Nakamura S, Kimura F, Sakaguchi T. Postnatal development of electrical activity in the locus ceruleus. J Neurophysiol 1987; 58: 510-524.

19. Rangel S, Leon M. Early odor preference training increases olfactory bulb norepinephrine. Brain Res Dev Brain Res 1995; 85: 187-191.

20. Sullivan RM, Hall WG. Reinforcers in infancy: classical conditioning using stroking or intra-oral infusions of milk as UCS. Dev Psychobiol 1988; 21: 215-223.

21. Sullivan RM, Stackenwalt G, Nasr F, Lemon C, Wilson DA. Association of an odor with activation of olfactory bulb noradrenergic beta-receptors or locus coeruleus stimulation is sufficient to produce learned approach responses to that odor in neonatal rats. Behav Neurosci 2000; 114: 957-962.

22. Gray CM, Freeman WJ, Skinner JE. Chemical dependencies of learning in the rabbit olfactory bulb: acquisition of the transient spatial pattern change depends on norepinephrine. Behav Neurosci 1986; 100: 585-596.

23. Sullivan RM, Wilson DA, Leon M. Norepinephrine and learning-induced plasticity in infant rat olfactory system. J Neurosci 1989; 9: 3998-4006.

24. Wilson DA, Sullivan RM. Olfactory associative conditioning in infant rats with brain stimulation as reward: II. Norepinephrine mediates a specific component of the bulb response to reward. Behav Neurosci 1991; 105: 843-849.

25. Wilson DA, Pham TC, Sullivan RM. Norepinephrine and posttraining memory consolidation in neonatal rats. Behav Neurosci 1994; 108: 1053-1058.

TABLE
Table I: Statistical Analysis of Thickness of Mitral Cell Layer and Number of Mitral Cells among the Control and Experimental Group

T Df Significance (2 tail) Mean difference Std. Error difference 95 % confidence interval of the difference
Lower Upper
Thickness of mitral cell layer (μm) -4.14 18 .001 -15.05 3.63342 -22.68 -7.42
-4.14 14.10 .001 -15.05 3.63342 -22.84 -7.26
Number of Mitral Cells -2.96 18 .008 -2.7 .909 -4.61 -.78
-2.96 17.82 .008 -2.7 .909 -4.61 -.78

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