CONDITIONED PLACE PREFERENCE IN MICE EXPOSED TO WEAK LOW FREQUENCY MAGNETIC FIELDS. Prepared By: Md. Mansur ul Mulk. Supervised By: Prof.

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1 CONDITIONED PLACE PREFERENCE IN MICE EXPOSED TO WEAK LOW FREQUENCY MAGNETIC FIELDS. Prepared By: Md. Mansur ul Mulk Supervised By: Prof. Yves Bureau A Report presented to Prof. Ion McDonald as a Partial fulfillment of the course 3970Z in the University of Western Ontario, London, Ontario. March 30,

2 Introduction: Traditionally, conditioned place preference (CPP) is used to confirm whether a drug is rewarding [1]. In this regime the effect of drug (unconditioned stimulus) is paired with one distinct environment. Classical conditioning, used in CPP is the process of pairing a neutral stimulus and a reflex extracting stimulus so that the neutral stimulus comes to bring out a version of the reflex. CPP is the pairing of a novel environment stimulus (Place) with a reflex-eliciting stimulus (drug) [1,3]. Conditioned place preference is considered to be a classical conditioning. Conditioning a mouse to a novel environment is conducted by singly or repeatedly placing it in a new environment while exposing it to a drug or PLFMF. If the stimulus is positive the mouse will choose that environment when given a choice. If the stimulus if negative the opposite effect will be observed [2]. PLFMF like many narcotics reduce nociception in mice. Narcotics such as morphine easily condition mice to prefer one environment over another. Although the paradigm has also been criticized because of some inherent methodological problems, but for the past decade condition place preference has become a unique experimental tool which widely used in behavioural science and addiction research [6]. Due to the contiguous association between the context and drug stimulus, conditioned place preference is a test for drug reward. The CPP paradigm has widely been used as an animal model of drug reward. A number of studies have investigated the reward effect of morphine [7, 8, 9]. The main advantages of CPP are that, test animals are in a drug free zone which is sensitive to both reward and aversion. CPP allows simultaneous determination and locomotors activity. It is adaptable to a variety of species. The main limitations of CPP are the interpretation based on the 2

3 notion of novelty Seeking. CPP is difficult to interpret when animals prefer one context prior to drug conditioning. Weak extremely low frequency magnetic fields (ELFMF) are expressed by wavelength or frequency along the electromagnetic radiation spectrum. ELFMF are everywhere in the environment and have been suspected of influencing biology and behaviour since the late 19 th century. Many organisms are capable of perceiving such fields. Birds and even some mammals also respond to ELFMF by changing their orientation in the presence of magnetic fields [14,15,16], while less is known about the proximal mechanisms with respect to elementary perception. Despite of many behavioural experiments involving ELFMF exposure, relatively little information regarding biological mechanisms have been conducted till recently. ELFMF can also be described and earth magnetic field (natural) and 50/60 Hz produced by power lines or electrical appliances [17]. Many Researchers have also investigated the effects of PLEMF on nociceptive response and pain sensitivity. These results have been consistent. It has been observed that when snails, mice and humans are exposed to relatively weak low frequency<1000hz and weak specific pulsed magnetic field (CNP), they produces anti nociception [5]. A conditioning paradigm is one in which the naïve animal (mice) is conditioned to either the non-preferred for positive conditioning or the preferred box for aversion conditioning. This is considered to be a non biased design for condition to place. There are many examples of biased experiment and the results are documented as false positive [23]. The time sequence of drug administration and number of conditioning sessions cannot be underestimated in a CPP experiment. Injecting an animal just before or some time following 3

4 being placed in a novel environment produces conditioning [24]. Also, CPP experiments usually require a number of sessions to successfully condition an animal. The recent findings and developments with respect to the reward systems in the brain are generalized from studies using the conditioning place preference paradigm [5]. No relevant to this study but of collateral interest is that brain areas shown as being important in generating subjective reward include the medial prefrontal cortex, ventral pallidum, amygdala and the pedunculopontine tegmental nucleus to name a few [4]. It is likely that these brain areas are instrumental to the observed effects [10]. In the present study, we examined the condition place preference of morphine induced mice in extremely low-weak magnetic fields. During the conditioning phase, three groups of mice were placed in a defined environment paired with morphine. Following exposure to 60 Hz or sham magnetic fields for 30 min/day, respectively, and were placed in another defined environment paired with physiological saline without exposure to magnetic fields. After finishing of conditioning, preference tests for the morphine-paired place were performed. The exposure to magnetic field motivated morphine-induced place preferences in mice will be investigated. Previous evidence suggests that the action of the addiction of morphine on opiod receptors are rewarding. Exposure to low weak magnetic field with morphine induced condition place preference to mice provided positive feedback, which are consistent with previous results [11] Pain and Nociception is an unpleasant sensory and emotional experience associated with actual potential tissue damage. There is much evidence that explains PLFML influences on pain sensitivity in snails [19], thermal nociceptions and PLFML influenced the vestibular system. 4

5 Various combinations of static and dynamic magnetic fields have also been shown to increase or decrease pain sensitivity [22]. Hypotheses: 1. Narcotics such as morphine will positively condition mice to prefer a previously non preferred box in the CPP apparatus compared to control treatments. 2. As in the first hypothesis, PLFMF will be successfully used to positively condition mice.. Method: Mice were first habituated to a conditioned place preference apparatus. Following Habituation the amount of time mice spent in either box or the bridge for a 15 minute session was recorded. For the next 10 days mice were confined for 30 minutes per day every second day to the box they preferred least while being exposed to either: (1). Morphine (5 mg/kg) (2).Saline or Sham magnetic field (Control) (3). Cnp (4).60 Hz On the alternate days mice were exposed to either saline if drug injected or to a sham magnetic field condition if exposed to PLFMF while in the preferred box. On the test day the mice were given the choice of spending time in either the preferred or non-preferred box for 15 minutes. Apparatus: Each place conditioning apparatus consists of One White and One Black box connected with a bridge. General activity and location of the Box is monitored by video recording. Magnetic field 5

generator: Helmholtz coils surround the individual boxes.

CPP apparatus and Magnetic field generator. Helmholtz coils surround the individual boxes.

along the spectrum of electromagnetic radiation.

6 generator: Helmholtz coils surround the individual boxes. Prior to each session the box and floors are cleaned using a 70% ETOH solution. CPP apparatus and Magnetic field generator. Helmholtz coils surround the individual boxes. Low Frequency Magnetic Field: Electromagnetic wave can be classified based on their frequency and wavelength along the spectrum of electromagnetic radiation. In artificial electromagnetic fields studies on pain sensitivity extremely low frequency magnetic field at 50 or 50Hz is used to expose human or mice. Figure-1: Low frequency (10 5 ~10 7 ) magnetic field is used to expose mice in condition Place preference experiment [24]. 6

$The discontinuous 60 Hz field was identical except for the Sinusoidal form in place of the box-like wave (refractory period identical in both) [2] Statistics: A statistical tool SPSS was used for$

7 Pulsed Low Frequency Magnetic Field: Table-2: PLFMF used in this study. The discontinuous 60 Hz field was identical except for the Sinusoidal form in place of the box-like wave (refractory period identical in both) [2] Statistics: A statistical tool SPSS was used for analysis. A mixed analysis of variance was conducted with pre and post conditioning and box type as the within subject variable and treatment as the between subject variable. Simple simple main effect analysis was conducted for condition at the non preferred box post conditioning. All results were considered significant when the probability of making a Type 1 error was less that 5% (p<.05). All results were interpreted using the Greenhouse Geisser correction. 7

8 Results: There was a tendency toward a pre/post conditioning by box type by treatment interaction, (F (5,74)=1.8, p=.120) (Figures 3, 4). Due to sample size this effect was not significant. However, a simple simple main effect analysis on treatment at the non preferred boxes post conditioning was conducted in spite of this negative result. A significant group difference was observed (F(3,43)=2.96, p<.05). A post hoc Tukey test did not show any differences suggesting that the simple simple main effect was due a combination of treatment groups that differed from the control group. All-in-all the control group was different from all the treatment groups combined (Figure 4). Time Spend Before Conditioning Control 60 Hz Cnp Morphine Time (sec) Pref Pre Ntrl Pre Nonpre Pre Figures 3: Time spent in either the preferred, neutal (bridge), or non preferred box prior to conditioning using Morphine, Cnp, 60 Hz, or Saline. 8

9 Time spend after Conditioning Control 60 Hz Cnp Morphine Time (sec) Pref Post Ntrl Post Nonpre Post Figures 4: Time spent in either the preferred, neutal (bridge), or non preferred box after condition using Morphine, Cnp, 60 Hz, or Saline. Discussion: We did show that morphine and PLFMF of all kinds produced conditioning. Figure 3 and 4 indicates that Time spent in either the preferred, neutral (bridge), or non preferred box postconditioning was influenced by Morphine, Cnp, and 60 Hz compared to the Saline/sham condition. Time spent in neutral (bridge), pre-conditioning was 240 second and postconditioning was 160 second and the effect of using Morphine was significant. The effect produced by PLFMF, were weak but suggestive of an effect. We did not have sufficient numbers to significantly show a box type by treatment interaction. However, we are confident that with greater sample sizes, we would have observed significance. In spite of this lack of effect, we did proceed with a simple main effect analysis. We did show an effect of all treatment groups combined to be different from the control group. This liberal analysis suggests that all treatment groups were not different from one another but were influential in the CPP paradigm. 9

10 We feel that with a replication study in which a greater number of subjects are used that some of the groups, morphine injected in particular with the Cnp in tow, will be different from the control group. From this analysis, we can suggest that PLFMF are potentially rewarding. The possible application of the PLFMF as shown by this experiment, would be as an Antidepression treatment, or of course an anti pain treatment [21]. We are of course not suggesting this without experimental verification. Nonetherless, there is one group that has shown that weak magnetic field used during magnetic spectroscopy imaging attenuates depressive symptomology in a clinically depressed population [6]. We are hopeful that this treatment will eventully reach the clinic. Conclusion: This study did show weak effects suggesting that PLFMF are rewarding in nature. At this time we cannot conclude that PLFMF are habit forming as our paradigm did not investigate that concept directly. We are of the opinion, however, that PLFMF may have the potential to alleviate anhedonia in pathologies such as depression since depression has as a defining characteristic an attenuated ability to experience positive affect. In order to answer these questions, we feel that a replication of this study is essential. We also feel that we should implement other designs to study the potential for habit formation. The finding of the experiment can be implemented in living organisms and PLFML can be a positive driving force for future pain related treatment in humans. 10

11 Acknowledgments: I would like to express my gratitude to Prof. Yves Bureau for his extensive support in condition place preference paradigm, without him this work would be impossible. This project gave me a solid base of understanding of behavioural research and one step ahead of thinking the correlation between CPP and the physiology of brain activity. References: [1]. V. Vindenes et al. Conditioned place preference induced by morphine and morphine-6- glucuronide in mice, Pharmacology, Biochemistry and Behavior 85 (2006) [2]. Presentation Slides from Prof. Yves Bureau,, LHRI, St. Joseph Hospital, London ON, April 01, [3]. A.W. Thomas, J. Robertson, Frank Prato, Neuromodulation by exposure to a pulsed low frequency magnetic field. Imaging program, LHRI, St. Joseph Hospital London, ON N6H 4V2. [4]. Thomas M. Tzschentk, Measuring Reward with the Condition Place Preference Paradigm: A Comprehensive Review of Drug Effects, Recent Progress and New Issues. ISSUES., Neurobiology Vol. 56, pp. 613 to 672, [5]. A.W. Thomas, J. Robertson, Frank Prato, Neuromodulation by exposure to a pulsed low frequency magnetic field. Imaging program, LHRI, St. Joseph Hospital London, ON N6H 4V2. [6]. Rohan et al. Bipolar disorders and current depression. American Journal of Phychiatry2004;161(1):93-8. [7]. Kuo CK, Hanioka N, Hoshikawa Y, Oguri K, Yoshimura H. Species difference of siteselective glucuronidation of morphine. J Pharmacobiodyn 1991;14: [8]. Dambisya YM, Chan K, Wong CL. Further metabolic studies of codeine and morphine in mice pre-treated with sympathomimetics. Methods Find Exp Clinical Pharmacology 1992;14: [9]. Handal M, Grung M, Skurtveit S, Ripel A, Morland J. Pharmacokinetic differences of morphine and morphine-glucuronides are reflected in locomotor activity. Pharmacol Biochem Behav 2002;73: [10]. A.W. Thomas, J. Robertson, Frank Prato, Neuromodulation by exposure to a pulsed low frequency magnetic field. Imaging program, LHRI, St. Joseph Hospital London, ON N6H 4V2. 11

12 [11]. O.V. Rice, N. Gordon, N.G. Andrew, Conditioned place preference to morphine in cannabinoid CB1 receptor knockout mice., Brain Res. 945 (2002) [12]. S.S. Negus, S.J. Henriksen, A. Mattox, G.W. Pasternak, P.S. Portoghese, A.E. Takemori, M.B. Weinger, G.F. Koob, Effect of antagonists selective for and -opioid receptors on the reinforcing effects of heroin in rats, J. Pharmacol. Exp. Ther. 265 (1993) [13]. M.V.R. Jan, M.A.F.M. Gerrits, L.J.M.J.V. Vanderschuren, Opioids, reward addiction: an encounter of biology, psychology, and medicine, J. Pharmacol. Rev. 51 (1999) [14]. Johnson. S., Lohmann. K. J. The Physics and Neurobiology of magnetoreceiption. Nature J. Reviews neuroscience 6, 2005, [15]. Mouritsen. H., Ritz. T., Magnetoreceiption and its use in bird navigation. Current opinion in Meurobiology. 2005, 15, [16]. Wiltschko. W., Wiltschko. R., Magnetic orientation and magnetoreceiption in birds and other animals. Journal of Comparative Physiology. 2006, A(9), [17]. Cristina Del Seppia et all. Pain perception and electromagnetic fields., J.Neuroscience and [18]. Schechter MD., Calcagneti DJ, Trends in Place preference conditioning with a cross indexed bibliography, Neuroscience Biobehav, Rve 17:21-43, ehavioural Reviews, 31(2007) [19]. Kavaliers, M., Choleris, E., Prato, F.S.,Ossenkopp, K.P., Evidence for the involvement of Nitric Oxide and Nitric Oxide synthase in the modulation of opoid induced antinociception and the inhibitoryeffects of exposure to 60 H magnetic fields in the land snails. Brain Research, 1998a, 809, [20]. Barkley, K.J,. Sex difference in pain. Behavioural and Brain Science 1997, 20, [21] Hagen et al., Does Hypertension protect against chronic musculoskeletal complaints. 2005, Archives of Internal Medicine 165, [22]. Radzievsky et al., Hypoalgesic effect of millimetre waves in mice; dependence on the site of exposure. 2000, Life Science 66, [23]. Papp, M. et al. Differential effects of agents acting at various sites of the NMDA receptor complex in a place preference conditioning model, J. Pharmacol, 317, [24] Presentation Slides from Prof. Jerry Batista, Radiation Biology, Medical Biophysics, UWO, London ON,

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Ultra-low-dose naltrexone suppresses rewarding effects of opiates and aversive effects of opiate withdrawal in rats

Psychopharmacology (2005) 181: 576 581 DOI 10.1007/s00213-005-0022-7 ORIGINAL INVESTIGATION Mary C. Olmstead. Lindsay H. Burns Ultra-low-dose naltrexone suppresses rewarding effects of opiates and aversive