The Neurophysiological, Behavioral and Cognitive Enhancing Effects of Adrafinil in the Aging Canine

Heather M. Callahan

A thesis submitted in conformity with the requirements for the degree of Masters of Science Graduate Department of Physiology University of Toronto

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The Neurophysiological, Behaviord and Cognitive Enhancing Effects of Adrafiil in the Aging Canine Masters of Science, 200 1 Heather M. Callahan Department of Physiology?University of Toronto.

Adratinil is a behavioral stimulant that is currently being used in France to prornote vigilance in the elderly. Most of our undentandinç of adrafinil cornes From it's main metabolite. modafinil. Although the exact mechanism of action of adratinil and modafini l is unknown. most of the research suggests that they are al- adrenergic agonists. When açed beagles were tested on the open field test following the administration of adratinil. the- showed an increase in total distance traveled without the presencr of stereotypical behaviors. On visual discrimination tasks. dogs adrninistered adrafinil made Fewer errors and required fcwer trials to reach a predetermined criterion than dogs given a placebo control. The EEG of dogs administered adratinil revealed a decrease in delta and theta activity and an increase in alpha and slow beta activity. Based on these findings, adrafinil provides excellent promise as a vigilance-prornotinç agent for aged dogs and the elderly human population. 2. Acknowledgernents

In any endeavor. there is always a team of people who make it possible to accomplish the goal you set out to achieve. Since the earliest days of my graduate experience. 1 have had a number of people who stood by me, gave me advice, helped me out. loaned me money. cooked me food and even prevented me from having a very bad fall. I want to take this opportunity to thank al1 of them for making this an adventure 1 will never forget. Fiat of all. I would like to thank my parents, Kathleen Callahan and Wilson Smith. who went für beyond the requirements of parental love and suppon and who showed me what a real challenge was al1 about. Thank you to my siblings. Chns. Rosalind and Shawn. who have always been here for me and with whorn I share an unbreakable bond. Our path has not been easy but 1 am glad we share it together. To Nicos Pelavas. who was there from the beginning. thank you for showing me what real love looks like and for teaching me the phrase "bring it on little man". I would like to thank Patncia Atkinson. a great fnend and Iûb-mate. for putting beagles and friends ahead of sleep and sanity. To Antonio Mendonca (the great guru of ail things) for his excellent surgical skills. his teaching ability. his computer knowledge and mostly for his time and patience. Antonio. you are most definitely one of a kind. At the stan of graduate school. I didn't have much respect for cornputen, but Saman Sadeghi Hosseini and Blaine Condron (the computer guys) have taught me that real research isn't possible without one. I hope Sam never has to spend another Friday night in the Iab. Thank you to my thesis cornmittee: Hon Kwan. John MacDonald and Martin Wojtowicz. for having the patience to read this and offer your expen advice. 1 tmly believe that John MacDonald is partly responsible for this actually getting finished when he went above and beyond his job requirements as graduate watchdog. I would also like to thank a great collaborator. Elizabeth Head. Liz became a collaborator when she realized she loved the Sun in California more than the snow in Toronto. Liz was the one who introduced me to my very fint beagle and who taught me that dogs were not just large rats. Thank you Liz for your support. undentanding, patience, guidance and friendship over all of these yean. Finally. 1 would like to thank the tag team supervison Bill MacKay and Bill Milgram. Bill MacKay took a chance on me when 1 fint showed up on his doorstep and I hope he doesn't regret it. Bill. you have shown me that there is a great deal of integity within the scientific community and 1 truly hope our paths cross again in the future. Bill Milgnm is the person who has taught me so much about science and research. Bill. you've been a great inspiration to me and I'm very happy that you came into my life and gave me this wonderfui opportunity. 1 am also happy that on that first day when you asked me if 1 wanted to work with dogs. rats or humans. 1 chose the right species. For Beth Margolis. You came dong for the joumey and did not give up on me at my darkest moments. It is because of you that this made it to paper and 1 made it at A. 3.Table of Contents

.. 1. ABSTRACT ...... il ... 2. ACKNOWLEDGEMENTS ...... il1

3. DEDICATION ...... v 3. TABLE OF CONTENTS ...... vi 5. LIST OF FIGURES ...... ix 6 . LIST OF APPENDICES ...... xi .. 7. LIST OF ABBREVIATIONS ...... XI1

8. INTRODUCTION ...... 1

8.1 CHEMICAL STRUCTURE ...... 1

8.2 MECHANIS M OF A C'Tm...... -3 8.2.1 The al -Adrerzergic Receptor Hvpotliesis ...... 3 8.2.2 Effects of Orher Neiirotransmitters ...... 3 8.2.3 Orexirt as a New Mecliariisni of Action ...... 4

6.3 NON-COGIVrTIVE BEHAVIOR ...... 5 8.3.1 Operi Field Sporliurieoits Activity Test ...... 5 8.3.2 Non-Cognitive Behuvior und Adrafinil ...... 6

8.4 C0GNl'T.ION...... 6 8.1.1 Cognition in [lie Aging Canine ...... 6 8.1.2 Cognition and Adrafinil ...... 7

8.5 EEG...... 8 8.5.1 The Origin of the Electroencephaiograni ...... 8 8.12 nePhysiological Basis of the EEG ...... 9 8.5.3 EEG urid Aging ...... 10 8.5.1 EEG and Canines ...... IO 8.5.5 EEG and Adrafiliil ...... 11 9.EXPEIWIENT 1: ADRAFINIL AS A BEHAVIORAL ACTIVATING AGENT IN AGING CANINES ...... 15

9.2 MATERIALÇ AND METHODS ...... 15 9.2.1 Siibjccts ...... 15 9.2.2 Beliavioraf Procedures ...... 16 9.2.3 Errperirnental Design ...... 16 9.2.3 Data Analysis ...... 17

9.3 RESUL7S...... 17 9.3.1 Eficts of Adrafiriil ori Locomotion ...... 17 9.3.2 Effecrs of Aclrafiiil on Uririarion ...... 20 9.3.3 Effects of Adrafilil or1 Vocaliratiori...... 20 9.3.4 Effects of Adrufinil ori Aaivity Partem ...... 20

9.4 DISCUSSION ...... -24

10.EXPERIMENT 2: ADRAFINIL AS A COGNITIVE ENHANCING AGENT IN AGING BEAGLE DOGS ...... 36

10.2 INTRODUCTION ...... 26

1 O .2 MATERLALS AND METHODS ...... 36 10.2.1 Sicbjecrs ...... -26 10.2.2 Test Apparatus ...... -26 10 .2.3 Pre- Traiuing Procediires ...... -28 10.2.1 Behaviorul Testing ...... 28 10.2.5 E.rper-imertra1 Design ...... 29 10.1.6 Data Amlysis ...... 30

1 0.4 DISCUSSION ...... 35

11.EXPERIMENT 3: THE EFFECTS OF ADRAFINIL ON EEG ACTIVITY IN AN AGED CALVINE:A PILOT STUDY ...... 37

11.2 MATERIALS AND ZbfElXOD...... 38

.uii . 11.2.1 Siibjects ...... 38 1 1.2.2 Elecrrode Bnplautarion Procediires ...... -38 Il .2.3 Erperimental Design ...... 39 11.2.4 EEG Collection ...... 40 1 1.2.5 Data Anabsis ...... JO

1 1.3 RESULTS...... 41 1 1.3.1 Effect of AdraJn il on Resting EEG ...... 41 11.3.2 Effecr of Adrafizil on Individual Freqliency...... 43 1 1.3.3 Conipanson of 10 . 20 and 40 mgkg of Adrafizil ...... -47

1 1 .4 DISCUSSION ...... 49

12.EXPERIMENT 4: THE NEUROPHYSIOLOGICAL MODULATION OF CORTICAL FUNCTION BY .\DRAFINIL ...... 5 1

12.2 MATERMU AND METHODS ...... 51 12.2.1 Sihjects ...... 51 17.2.2 Eiecr rode Implant Procedtires ...... 51 12.3.3 Erperirrie~itulDesign ...... 3,'7 1 2.3.3 EEG Coliect ion ...... 53 12.3.5 Data Anaiysis ...... 53

12.3 RESULTS ...... -54 12.3.1 Resting EEG ...... 54 12.3.2 Effect of Adrufinil 011 Rrsring EEG ...... 54 22.3.3 Effecr of Adrufinil or1 Individtial Freqtiencies ...... 56

12.4 DISCUSSION ...... 56

13.GENERAL DISCUSSION AND CONCLUSIONS ...... -58

APPENDIX A ...... 70

APPENDIX B ...... 71

APPENDIX C ...... -72

.viii . 5. List of Figures

Figure 1. Chernical structure and synthesis of adrafinil. A six step process is used in synthesizing adrafinil. (1) Thiourea is reacted with brornodiplienylnietliane over heat to produce (2) diphenylmetliane-tliiol. Chloroacetic acid is then added to this product to form (3) diphe~rvl>~ietliyl-tliioaceticacid, whic h is al ky lated to (4) etlyl cliplieriylmetlyl- tliioacetate. This compound reacts with hydro.y~anii~teto form (5) diphenylmetliyl- tliioacetoliydru.vani»le acid. A final oxidation step converts this product to (6) (diphe~ty1methyl)sliIfin~~l-acetolzydro-vatnicacid or adrafini 1.

Figure 2. Total distance traveled after the administration of 10,30 or 50 mgkg of adrafinil or a placebo control by aged beagles in Experiment 1. There is a statistically significant difference between adrafinil and the placebo control at each of the dose levels.

Figure 3. The total distance traveled 2,4 or 10 hours after the administration of adrafinil or a placebo control by aged beagle dogs in Experiment 1. The interaction between dose and time following treatment approached statistical significance for each of the tirne penods.

Figure 4. Activity patterns of one animal 2 hours after the administr~tionof 1Orn~A~kgof adnfinil or a placebo control. The dog showed an increase in activity after the administration of adrafinil but there was no change in the overall activity pattern.

Figure 5. Activity patterns of one animal 2.1 or 10 hours following the administration of 50mgkg of adnfinil or a placebo control. There is an increase in activity at eilch of the three time periods after the administration of adrafinil in cornparison to the placebo control. The greatest increase can be seen at the 7 hour time period.

Figure 6. The canine adaptation of the Wisconsin General Test Apparatus.

Figure 7. Mean number of trials and errors during the acquisition of the intensity and size discrimination tasks. A two-tailed test indicated that there was a statistically significant di fference between the two tasks.

Figure 8 Mean erron and trials required to reach criterion on two discrimination tasks following the administration of adrafinil or a placebo control. When the do,as were administered adrafinil, they made fewer errors and required fewer trials than when they were administered the placebo control.

Figure 9. The effect of task order was statistically significant for both the nurnber of trials and errors required to reach criterion. The animals showed a stronger effect in improved Iearning following the administration of adrafinil when they were tested on the size discrimination task first. Figure 10. Response pattem of one dog following the administration of adrafinil or a placebo control. The animal showed a stronger motivation to respond after the administration of adrafinil. When the animal was given a placebo control, there were fewer trials completed for a majority of the test sessions.

Figure 11. Total power as a function of frequency for EEG recordings of control data and following the administration of lOmg/kg of adrafinil. There is an increase in the power for each of the frequencies after the administration of adrafinil, with the greatest increase in the delta frequency.

Figure 12. Changes in the power of delta and high alpha following the administration of LOmgkg of adnfinil. There was a strong decrease in power for delta and an increase in power for high alpha. The change in the frequencies began approximately one half hour after treatment and this Iasted approximatel y four hours after treatment.

Figure 13. Power spectrums prior to the administration of adrafinil and approximately 30 minutes. 1 hour, 2 houn and 4 hours after the administration of 10rn~gklkgof adrafinil. The power spectrums show the shift in power from the low frequencies. prior to treatment. to the higher frequencies after 30 minutes. This pattem continues up to 4 hours and then retums to pre-drug conditions.

Figure 14. Average total power of EEG recordings frorn one channel following the administntion of 10. 20 or JOmzAg of adrafinil. The animal showed the greatest increase in total power at the 2Omgkg dose.

Figure 15. Average total power of EEG recordings for baseline. dm; and washout sessions recorded from one channel for both dogs in Experiment 4. There was a reduction in total power after the administration of 20mzAg of adrafinil. This reduction in power retumed to baseline levels during the washout session.

Figure 16. Changes in the power of delta and high alpha following the administration of 70mgkg of adrafinil. There was a decrease in power for delta approximately 30 minutes after treatment for both Eve and Denny and it lasted approximately 3.5 hours for Eve. The power of delta did not return to pre-treatment conditions for Denny. There was no effect of adrafinil on high alpha for Eve or Denny. 6. List of Appendices

Appendix A. List of dogs used in al1 of the experiments.

Appendix B. The random assignment of dogs for Experiment 1

Appendix C. The random assignment of dogs for Experiment 2 7. List of Abbreviations

al alpha l -adrenergic CNS central nervous system EEG electroencephalograrn ECoG electrocorticogram DEEG depth electroencephalogram FDA federal drug administration FFT fast fourier transform GABA gamma-aminobutyric acid im. intra-musculcu i.v. intra-ventricular REM rapid eye movement

- xii - 8. Introduction

Adrafinil is a novel pharmaceutical that is currently being used as a vigilance- promoting agent in elderly patients throughout France (Dewailly et al.. 1989: Israel et al.. 1989; Fontan et al., 1990: Kohler and Lubin, 1990; DeFrance et ai., 1991; Boyer et al.. 1994). Unli ke amphetamine and other behavioral stimulants, adrafinil increases arousal without disrupting the phasic activity of sleep or inducing stereotypical behavior. Although adrafinil has widespread interest throughout Europe. in the United States adrafiil is known mostly by its main metabolite, modafinil. Modafinil has recently received approval by the FDA for the treatment of narcolepsy: a CNS disorder that is mainly characterized by excessive daytime sleepines. Although adrafinil's exact mechanism of action is unknown. studies have suggested that it may be a central al-adrenergic agonist. Recently. investigators have begun to study a possible new mechanism of action for adrafinil and modafinil through their effects on the neuropeptide orexin. Orexin (also known as hypocretin) is a recently discovered neuropeptide that is believed to play a role in sleep regulation. Lin et al., (1999) have found that disruption of the hypocretin receptor causes narcolepsy in canines. Whe~mice were given modafinil and their cells were immunostained for orexin and Fos. there was a 9-fold increase in the number of immunoreactive cells and this may be evidence of modafini 1's activity on orexin neurons (Chemelli et al.. 1999). Adrafinil's ability to increase activity and act as a vigilance-enhancing agent in the elderly human population suggests it may be an excellent substance for treatment of elderly pets. The present investigation is a funher study of adrafinil's effecis on behavioral activity, cognition and EEG in an aging population of canines.

8.1 Chemical Structure

Adrafinil was synthesized by Lafon Laboratories in France. It is a white-to-rosy beige crystalline powder with a high sulfurous odor. It has a molecular weight of 189.85. a melting point of 154°C and is slightly soluble in water. more soluble in ethanol. and soluble in methanol. The proprietary narne of adrafinil is Olrnifon @ and its chernical name is (diplienylmetlzyl)siiZfiny1-2-acetolr$ro.ramic acid. The synthesis of adrafinil was previously descnbed by Milgram et al., (1999) and is summarized in Figure 1. Thiourea (1) is reacted with broniodiplienylmetlinne over heat to produce diplienylmetlimze-tlliol (2). Cliloroucetic acid is then added to this product to form diplienylmetlivl-tlzioacetic acid (3). which is alkylated to ethyl diplienylnietliyl- tliioacetate (4). This compound reacts wi th liydro-ylaniirie to fom diphenylnietliyl- tliioacetohydro.rnmine acid (5). A final oxidation step converts this product to (diphenylnietlzy1)siilfinyZ-acetol~ydro-~aniic acid or adnfini 1. The final compound is crystallized. recovered by filtration, and then purified by recrystallization in ethyl acetate and isopropyl ethanol. Adrafinil has two metabolites, an amide (CRL 40476, also known as modafinil) and an acid (CRL 40467). The amide form (modafinil) is the main metabolite.

O H?N \ a.0CHBr 0, Cl - CH2 - COHII C=S + b 1 CH - SH HIN /'

CH-S-CHI-COH O/

Figure 1: Chernical structure and synthesis of adrrifinil. A six step process is used in synthesizing adrrifinil. ( 1) Thiourea 1s reacted with brontodiplrenylmetltane over hesit to produce (2)dipllenyltnerliane-ritiol. Cldoroacetic acid is then added to this product to form (3)dipltenyltrretliyl-tlzioaceric acid. which is rilkylated to (4) etlzyl diplreti~lmetlzyl-tiiioacetate.This compound rems with Izyiro.rylantine to form (5) diplrenylrrretlz~l-rttioncetoirydro.ranzineacid. A final oxidation step converts this product to (6)(diplrenyftnetlr~l)s~i~n_vl-acetoI~ydroOwntic acid or adraiinil. 8.2 Mechanisrn of Action

8.2.1 The ai-Adrenergic Receptor Hypothesis

The exact mechanism of action of adrafinil and modafinii is unknown. Several studies have suggested that an intact centrd ul-adreneqic system is necessary and the compounds rnay be ul-adrenergic receptor agonists (Rambert, 1986: Duteil et al.. 1990: Akaoka et al.. 199 1; Lir. et al.. 1992: Mignot et al., 1994). Adrafinil's ability to increase vigilance and behavioral activity can be blocked by ul- adreneqic antagonists such as prazosin, phenox ybenzamine and yohimbine (Duteil et al..

1990) but not by u1- adrenergic or dopamine antagonists (Duteil et al.. 1990: Hermant et al.. 199 1; Simon et al., 1994). Adrafinil and modafinil's action also appean to be of central origin because phentolamine. a peripherally acting adrenergic antagonist, was unable to block the behaviorai effects of these substances (Duteil et al.. 1990). Phentolamine is unable to cross the blood brain bmier (Ander and Strornbor. 1974). Furthemore. at therapeutic dosases. adrafinil and rnodafinil have only transient and very limited peripheral sympathomimetic side effects. Unlike other stimulants. there was no evidence of nausea. headac he. hypersalivation. i nsomnia. motor overactivity or tachycardia (Duteil et al.. 1979: Ramben et al., 1986: Bastuji and Jouvet. 1988). The noradrenergic system has been hypothesized to be involved in the regulation of the sleep-waking cycle (Fuxe et al.. 1974: De Sam et al., 1987) and modafinil's ability to increase arousal in narcoleptic patients has been suggesred to act through this noradrenergic arousal system (Menkes et al.. 198 1).

8.2.2 Effects on Other Neurotransmitters Other mechanism of action include the reduction of y-aminobutytic acid (GABA) release via serotonegic pathways (Tanganelli et al.. 1995) and the enhanced release of

5alutarnate ( Ferraro et al.. 1996: Ferraro et al.. 1997). Tanganelli et al.. ( 1997) were the fint to propose that modafinil inhibited the release of GABA in the cortex and this effect was not blocked by the adrenergic antagonist prazosin. Inhibited release of GABA was also found in the hypothalamus (Ferraro et al., 1996). the neocortex (Tanganelli et al.. 1995), nucleus accumbens (Ferraro et al., 1996), and striatum (Ferraro et al., 1998). In the posterior hypothalamus and the media1 preoptic area the GABAergic system may play a role in sleep regulation (Lin et al., 1989) and the vigilance promoting action of modafinil may be related to an inhibitory effect of GABAergic transmission (Ferraro et al.. 1996). The suppressed release of GABA has also been suggested to facilitate the release of dopamine. Ferraro et al., (1996) found that modafinil enhanced the basal release of dopamine in the nucleus accumbens and this effect was counteracted by the GABA agonist muscimol and the GABA uptake blocker SKF 89976. The increase in dopamine may account for sorne of the enhanced arousal of modafinil and adrafinil but the effects do not appear to be due to their direct action on dopamine. Modafinil has no effect on the firing of dopaminergic neurons (Akaoka et al.. 199 1) and it does not appear to significantly alter the release of dopamine or norepinephrine directly (Akaoka et al.. 1991: Lin et al., 1992; Deserveille et al., 1994: Mignot et al.. 1994: Simon et al.. 1995). Modafinil has also been shown to increase glutamate release in the ventro-media1 and lateral thalamus. hippocampus (Ferraro et al.. 1997) and striatum (Ferraro et al.. 1998) but there is very little work that has been done to investigaie how this release may occur. De La Mora et al.. ( 1999) have found that modafinil does not effect the overall synthesis of glutamate or GABA in the rat hypothalamus and the regional effects on these neurotransmitters may be a consequence of the indirect action of rnodafinil.

8.2.3 Orexin as a New Mechanism of Action Recentl y. investigators have suggested that modafinil 's mechanism of action may be its effect on the orexin neurons in the lateral hypothalamus (Chemelli et al.. 1999). Orexin (also known as hypocretin) is a newly discovered neuropeptide (De Lecea et al.. 1998; Sakurai et al., 1998) which is thought to have a role in feeding. metabolic control and possibly sleep regulation. Although orexinergic ce11 bodies are only located in the hypothalamus (De Lecea et al.. 19%). they have widespread monal projections (Peyron et al., 1998). There appear to be two main receptor types for the hypocretins: Hcrtr-1 and Hcrtr-2 ( Trivedi et al., 1998). The Hcrtr-2 is mainly found in cerebral cortex. nucleus accumbens, thalamus and subthalamic nucleus. Lin et al., (1999) have found that canine narcolepsy is caused by a disruption in the hypocretin (orexin) receptor 2 gene. Narcolepsy is a disabling CNS disorder that is characterized by excessive daytime sleepiness, abnormal REM sleep. cataplexy. sleep paralysis. and hallucinations (Guillerninault, 1989). Modafinil has recently been apgroved for treatment of narcolepsy in the United States. When mice were injected with modafinil and double immunostained for orexin and Fos (an indicator for neuronal activity), Chemelli et al., ( 1999) found that modafinil induced a 9-fold increase in the number of Fos-immunoreactive cells. Chemelli et al ., ( 1999) suggested this as evidence of modafinil's activation of orexin neurons.

8.3 Non-Cognitive Behavior

8.3.1 Open Field Spontaneous Activity Test The open Field is an activity test that is designed to measure explontory behavior. locomotion and other non-cognitive behaviors. It has been used to investigate the possible effects of pharmaceutical agents in a number of different species. as well as. the motor changes that may accompany aging. Age-dependent decrements in locomotor and exploratory behavior have been reported in cats (Levine et al.. 1987). rats (Goodnck, 1971, 1975: Masur et al.. 1980: Gage et al., 1984; Jucker et al., 1988: Pitsikas et al.. 1990) and mice (Elias et al.. 197% Elias and Redgate. l97% Lamberty and Gower. 1990. 199 1). In previous studies of aged canines. Head and Milgram (1991) have suggested that exploratory behavior was not age sensitive in dogs. In a later study, Head (1997) reported that there is a decrease in exploratory behavior with age but this is dependent upon rearing conditions and breed. as well as. age. Pet owners have also reponed a decrease in activity and affection in their geriatric dogs (Houpt and Beaver, 1981). In lesioned dos, a change in spontaneous behavior seems dependent on the area Iesioned. Prefrontal lesions result in hyperactivity (Allen. 1939; Bruthowski, 1964; Kononki and Lawicha, 1964) whereas Iesions in the Iateral pre-motor area result in depressed activity (Gerbner. 1971). Decrements in locomotor activity may not be a definite accompaniment to aging but for pets and aged individuals with age-associated locomotor problems, a stimulant without the undesirable side effects rnay improve their quality of life. 8.3.2 Non-Cognitive Behavior and Adrafinil One of the defining characteristics of adrafinil is its ability to increase behavioral activity without the stereotypies that accompany many other behavioral stimulants. Studies have shown that treatment with adrafinil has resulted in an increase in activity in mice. rats and monkeys (Duteil et al., 1979; Ramben et al.. 1984; Milhaud and Klein, 1985;DeIini-Stula and Hunn, 1990). Based on the findings of the present study, Siwak et al., (1999) perfomed two funher investigations on the effects of adrafinil on activity. In the first expenment. adrafinil was administered to older canines for 14 consecutive days and there was an increase in activiiy without any stereotypical behavior at dose levels between 70 and 40 mgkg. Adrafinil did not have any effect on other behavion such as sniffing. vocalization. grooming. jumping or rearing but there was a decrease in unnation at the highest dose. In the second study, Siwak et al., (1999) compared the effects of adnfinil to two other behavioral stimulants (nicergoline and propentofylline) and they found that only adrafinil caused an increase in activity. The study also investigated two different types of activity. the open field activity test and home-cage activity. The results showed that adnfinil caused an increase in the open field activity but the effect on home cap activity was much smaller. Siwak et al.. (1999) referred to this as adrafinil's ability to increase exploratory behavior in a novel environment. This was not seen in the more familiar home case environment.

8.4 Cognition

8.1.1 Cognition in the Aging Canine For approximately 10 years. Dr. Milgram's laboratory has been studying the dog as a mode1 of aging and dementia. Dunng this time we have investigated the age-associated declines of leming and memory and possible pharmaceutical agents that may slow the declines in cognition. We have found that aging in dogs is as complicated as aging in the human population and like us, dogs do not ail age at the same rate. When aged dogs are tested on a variety of cognitive tasks. a sub-population of the group will not show any sips of age-associated cognitive decline and will have performances similar to a young population of dogs. This is often referred to as successful aging. A second group of aged dogs show deficits on any task that they are tested on. These dogs will require more trials to leam simple discrimination tasks and will often be unable to learn more complex tasks. This sub-population is often referred to as unsuccessful aging. It is not known why some aged dogs show deficits in leaming and memory whereas others exhibit none of the signs of cognitive deterioration. Head ( 1997) suggested that the underlying neuropathology in aged dogs may be one possible reason for the individual differences in cognition. In a recent study investigating visual discrimination leaming and p-amyloid accumulation in aged dogs, Head et al.. (1998) found that there was an association bètween the cognitive task and the location and extent of B-amyloid pathology. The present investigation uses two types of visual discnmination problems. Of these two tasks. only the size discrimination has been previously tested in a group of aged dogs from our dog colony. Head et al.. (1998) reponed that the size discnmination task is age- sensitive. Old dogs made more erron and required more trials to solve the task than young dogs. This does not seem to be the case with the intensity discrimination task. Preliminary findings that were camed out after the completion of the present study have suggested that intensity discrimination is not age-sensitive. Old dogs and young dogs show a similar performance on the task. A phamaceutical agent that is able to enhance cognition would be very useful for the pet population. as well as, the human population. One of the complaints of pet owners is of their pet's inability to remember simple skills.

8.4.2 Cognition and Adrafinil There have not been any published studies using animal models to address the effects of adrafinil on leaming and memory. Clinical studies within the elderly human population have shown that adrafinil acis as an musa1 and vigilance-promoting agent. When elderl y patients were treated with adrafinil, the y showed irnproved performance on psychometric tests of attention. concentration and recall. The patients were also more energetic and displayed an improvement in mood (Israel et al.. 1989. Kohler and Lubin. 1990). Boyer et al., (1994) gave 548 subjects who complained of problems of attention. concentration, memory and orientation either adrafinil or a placebo control. Subjects treated wi th adrafinil exhibited significant improvement in dail y activities, attention. orientation and memory. In a study of hospitalized patients (over the age of 65) with deficits in wakefulness and vigilance. Defrance et al., (1991) found that adrafinil treatment was more effective in the younger patients and suggested this as justification for early treatment with adrafinil. None of the above mentioned studies have involved patients with dementia and subjects were excluded if they showed any signs of mental deterioration. Recently. Milgnm (personal communication) has investigated the effect of adrafinil on memory in a spatial delayed nonmatch-to-position task and has found that at higher dose levels, adrafinil impairs memory performance in aged dogs. When aged dogs were given oral doses of adrafinil over ZOmg/kg. they made more erron when required to remember the position of a previously seen object than when they were given a placebo control. Human studies have suggested that adrafinil causes an improvement in memory and the discrepancy may be a result of the dose levels nther than the effect of the dmg (Israel et al.. 1989: Kohler and Lubin, 1990; Boyer et al., 1991). In clinical studies. human subjects are often given doses that are Smg/kg or less and only one study by Kohler and Lubin ( 1990) has reported dose levels of IOrngkg. In the Kohler and Lubin (1990) study. the higher dose was separated into two separate treatments of 600mg in the moming and 300mg at noon. This suggests that at lower dose levels. adrafinil treatment results in an improvement in memory but at higher doses. there is an impairment in memory.

8.5 EEG

8.5.1 The Origin of the Electroencephalogram The spontaneous electrical activi ty of the brain. or the electroencephalogram, was first described by Canton in 1875. Canton detected this electrical activity with the use of electrodes on the skull or exposed brain of rabbits and monkeys. The use of the dog for studying EEG was fint undenaken by Beck in 1890. Beck used non-polarizable clay electrodes and a galvanometer to observe a current of variable strength from the cortical surface of dogs' brains. Beck noted that the current was present at dl times and did not coincide with respiration. pulse or the movcment of the animal. Although these were the first observations of electrical activity originating from the bnin, the real groundwork for Our current applications of human electroencephaiography came from Hans Berger. Berger was a Geman psychiatrist who believed the EEG would unlock the secrets of our mental processes and lead us to a better undentanding of the mind: it's normal functions in sleep and wakefulness and its denngements in mental disease (Gloor, 1969).

8.5.2 The Physiological Basis of the EEG The EEG is a technique that allows us to record the activity of many cortical neurons non-invasively through electrodes placed on the surface of the scalp. It allows us to gain information about sleep and wakefulness, epilepsy, coma and other ûrousal States. The EEG is composed of oscillating potentials denved from the scalp surface and originating from the electrical activity of the brain. It is the stimulation of postsynaptic potentials of neuronal ensembles rather than the spiking activity of individual neurons which is obtained while a subject is sleeping or sitting quietly. The frequencies of the oscillating potentials typically range from 1-30 Hz and are often divided into four dominant frequency bands: delta (14 Hz), theta (4-8 Hz), alpha (8-13 Hz), and beta (above 13 Hz). Delta and theta bands (slow waves) are often associated with sleep in hurnans and are often not found in alert subjects. Beta waves are norrnally associated with attentiveness and mental activity. Beta waves have also been associated with anxiety and apprehension and are often the result of medications such as benzodiazepene or barbiturates. The tem "gamma" rhythm was introduced in 1938 in order to designate frequencies above 30 Hz, usually between 30-45 Hz. Alpha waves (or Berger rhythms after the pioneenng work of Hans Berger) are usually associated with a state of relaxed wakefulness. In the past there has been interest in relating penonality and general behavior to alpha rhythm. Passive individuals have been regarded to be alpha-dominant whereas agressive penonnlities were considered to have minimal alpha activity. Whether or not this is the case, anxiety. agression and tension will cause a reduction in alpha waves in the EEG. 8.5.3 EEG and Aging Studies have shown that as an individual ages. there are accompanying changes to the EEG. With advancing age there is an increase in the lower frequencies of delta and theta and a decrease in alpha and slow beta activity. There is also an increase in the very fast betdgamma activity (van der Drift. 1977; Matejcek and Devos. 1976; Arrigo et al.. 1979; Obrist. 1979). The progression to slower rates can actually be seen when recordings are made from the same individual over tirne. There is considerable debate. however. regarding whether the changes in EEG with accornpanying age are a normal phenomenon of the aging process or are due to patholopical alterations frequentl y found in aged individuals. Hubbard et al.. ( 1976) found that in the absence of cerebral pathology. the EEG of individuals past the age of 60 may continue to show the normal healthy aspects of a mature adult. In some cases. normal adult EEG tracings may even be seen in individuals past the age of 80. In a study of IO individuals over the age of 100. Hubbard et al.. (1976) reported that there were no noteworthy changes in the EEG recordings of dl 10 subjects and Hubbard suggested that any changes in the EEG of elderly individuals were not caused by age. Giaquinto and NoIfe (1986) compared a group of elderly subjects (aged 60-80) with nomal subjects (aged 40-60) and found no significant difference in the EEG power spectrum of the two

ùoroups. Similar findings were also reported by Kazis et al., (1982). Sokoloff (1966) and Pollock et al., ( 1990) who failed to show a significant association between changes to the EEG and age. These studies suggest that the high proportion of abnormal EEG tracings in elderly individuals may reflect structural changes and possible cerebral pathology. Similar abnormal EEG tracings have also been reported in the EEG records of Alzheimer's patients and other types of dementias (Stigsby et al.. 1981: Coben et al.. 1983; Giaquinto and Nolfe, 1986; Coben et al., 1990: Maurer and Dierks, 1992: and Schreiter-Gasser et al., 1993)

8.5.4 EEG and Canines The first EEG recordings from dogs were performed by Beck in 1890. Since then, dogs have been used as test subjects in EEG studies of seizure activity. anesthetics, cerebral disorden and pharmaceuticals. The EEG of dogs has been reported to be similar to humans with a preponderance of power generally between 5-35 Hz and having smooth. rhythmic oscillations. There is a high frequency EEG pattern during periods of alenness or arousal and a slower EEG during periods of drowsiness and sleep (Bullock. 1974; Pettigrew and Daniels. 1973). One EEG feature that is shared by mature canines and humans is the prominent band of rhythmic activity in the alpha range of 8-13 Hz. The alpha rhythm of do,0s was reported by Lopes da Silva et a1..(1973: 1977, 1975) to be similar to the human alpha rhythm. It was recordable if the animal was in a quiet atmosphere having its eyes closed. Typically. a period of stable alpha rhythm had a duration of about 8-15 sec, after which it was replaced by higher frequencies when the animal opened its eyes or was aroused, or by lower frequencies as the animal became sleepy. Periods of drowsiness consisted of the dog having their eyes partially open or completely closed and requiring minimal stimulation or small disturbances in the environment to become readily aroused. During periods of drowsiness. the EEG rhythm consisted primarily of 6-8Hz. The development of these features can be seen at 5 months of age in the dog (Fox. 197 1) and there has been no known studies of the decline of EEG rhythm with increasing age in canines.

8.5.5 EEG and Adrafinil There have been very few studies that have examined the effect of adrafinil on EEG. One study by Saletu et al.. (1986) was a double-blind placebo-controlled study with 10 elderly subjects (mean = 66 yem). Subjects were given single oral doses of 200. 400 or 600 mg of modafinil, 900mg of adrafinil or a placebo with EEG testing occuning 0-8 hours after treatment. A computer-assisted spectral analysis of the EEG indicated that both adrafinil and rnodafinil had a significant CNS effect as compared to the placebo. There was a tendency

for a decrease in delta and theia frequency bands. as well as. û decrease in the very fast beta activity. There was also an increase in alpha activity and a trend towards an increase in slow beta activity. Saletu et al., (1986) suggested this as evidence for the vigilance- promoting effects of both adrafinil and modafinil. Two unpublished studies with adnfinil and EEG were also discussed by Ferner et al., (1983) as an example of exploratory data analysis. In the two double-blind placebo- controlled studies, 18 geriatric subjects were given either 600 or lZOOmg doses of adrafinil and EEG recordings were made 0-8 houn after treatment. Serum levels of adrafinil and its two metabolites (modafinil and CRL 40467) were measured after each EEG recording. Femer et al., (1983) found that subjects with the lowest serum concentration of adnfinil showed the least pronounced changes in EEG and suggested therefore, that any changes in EEG were due to the effects of adrafinil rather than it's two metaboli tes. Recently, Sebban et al.. (1999) have examined the changes in the EEG spectral power of rnodafinil in the prefrontal cortex of conscious rats and compared this to dmgs which act on dopaminergic or noradrenergic systems. Sebban et al.. (1999) have found that modafinil decreased the total EEG power over the frequencies 6-18 Hz and this effect was dose dependent. There was a decrease in power at 3. 5-6, 8 and 13 Hz and an increased power at 1-1 and 19-30 Hz. This was similar to other al-adrenergic agonists but it is the opposite of the effects of adrafinil on EEG that Saletu et a1..(1986) have reported previously. Funher investigation is required to detemine if adnfinil and modafinil behave differently on EEG power spectra and whether these dmgs have

different mechanisms of action. Most investigators assume that the dnigs are both al- adreneqic agonists but they are used within different populations (vigilance enhancement in the elderly with adrafinil and narcolepsy with modafinil) and there are few studies ihat have compared the two substances.

8.6 Present Investigation

The present investigation was undertaken to determine the effect of adrafinil on behavionl activity. cognition and EEG in an aging population of canines. The purpose was to determine whether adrafinil could act as a therapeutic agent in elderly dogs and to provide additional animal research for a new pharmacolo~cal agent wi th excellent promise. The spontaneous activity test was chosen to determine if adrafinil's effects on behavior were similar to the other behavioral stimulants that are currently being used or if adrafinil had a unique profile. Behavioral stimulants are often used in treating affective disorden. disorders of vigilance and disorders of sleep but there are a number of negative side-effects. such as hyperactivity, stereotypy and addiction. which makes them undesirable. The present investigation will determine if adrafinil causes an increase in behavioral activity in aging canines without the undesirable side-effects of other behavioral stimulants. The cognitive tasks were chosen because there is very little research that has been done to determine whether adrafini 1 can en hance cognition. In the nonscienti fic community. adrafinil is being used as a "smart-drug" to increase enegy. reduce fatigue and improve cognitive function. mental focus, concentration and memory, yet there is little scientific evidence available to support or refute whether adrafinil has this effect on cognition. There have been no animal studies perfonned to address the effect of adrafinil on learning and rnemory and only a limited number of clinical studies have been perfonned in France examining the effects of adrafinil on vigilance. The present investigation examines the effect of adrafinil on two types of discrimination learning tasks in aging canines. The final component of this study involved an examination of the effects of adrafinil on EEG. This component was included as a neurophysiological measure of cortical activity that could be compared to any behavioral and cognitive findings. It was also used to determine whether EEG analysis was a good indicator of dmg effects. There have been few studies on adrafinil and EEG. Most of the work has examined the effects of modafinil on sleep and narcolepsy. The present investigation examines the effect of adrafinil on resting EEG in aged canines. The purposes of the present investigations were therefore: 1 .) To determine whether adnfinil could act as a therapeutic agent in elderly dogs and to provide additional animal research for a new phmacological agent with excellent promise.

2.) To determine if adrafinil's effects on behavior were similar to the other behavioral stimulants that are currently being used or if adrafinil had a unique profile. 3.) To determine if adrafinil caused an increase in behavioral activity in aging canines without the undesirable side-effects of other behavioral stimulants.

4.) To determine adrafinil's effect on cognition based on the non-scientific community's use of the substance as a "smart-drug".

5.) To determine the effect of adrafinil on resting EEG in aged canines.

6.) To determine the effectiveness of EEG as a method of drug analysis and to compare it to mesures of cognition and behavioral function. 9. Experiment 1: Adrafinil as a Behavioral Activating Agent in the Aging Canine

9.1 Introduction

Adrafinil has been shown to act as a behavioral enhancing agent in monkeys. rats and mice (Duteil et al., 1979; Ramben et al., 1984; Milhaud and Klein, 1985: Delini-Stula and Hunn, 1990; Edgar et al., 1997). Unlike other behavioral activating agents. such as. amphetamine. adrafinil does not cause stereotypical responses. In the pet population. there is a demand for a therapeutic agent that will increase the behavioral activity of impaired old dogs without the negative side-effects of amphetamines. Based on these findings, the purpose of the present investiption is to test three di fferent dose levels of adrafini 1 to determine its be havioral acti vating characteristics in an aged beagle and to determine if adrafinil can counteract the behavioral declines that are often seen in aging dogs.

9.2 Materials and Methods

9.2.1 Subjects The subjects for the first expetiment were 9 beagle dogs (5 female and 4 male), nnging in age from 7 to 11 years and had been purchased from Hulan Sprague Dawley Breeding Fms. None of the animals had participated in any other studies prior to the start of the experiment. The dogs were housed in individual kennels and fed once daily at approxirnately 19:00 hours, after the completion of the behavioral testing. Water was available ad libitum. The characteristics of each animal are listed in Appendix A. 9.2.2 Behavioral Procedures After the dogs were acclimatized to the animal facility, they were tested for the effects of adrafinil on spontaneous behavior. The test procedures used for the spontaneous behavior task have been descnbed previously by Head and Milgram (1991). The dogs were piaced in a room measunng 3.66 x 3.66rn which contained a sink. cupboard and large glass window. The floor of the room was marked into squares measuring 61 x 61cm with black electrical tape to assist with localizing the dog's spatial position. The activity test was 10 minutes in duration and was observed by two experimenters through the window of the adjoining room. The dog's behavior in the room was recorded with a video camera and also with the use of a dedicated computer program. The computer program was designed to mesure locomotion. directed sniffing. urination. inactivity, grooming, rearing. vocalizing and jumping.

9.2.3 Experimental Design Each dog was randomly assigned to receive an oral dose of 10. 30 or 50mzAg of adrafinil. The dogs were then tested on spontaneous behavior at 2 hours, 4 hours. or 10 houn following treatment. There were also three control tests given at the same time points followin; the administration of a lactose placebo. Thus. each dog received 6 test sessions in total. There was a four-day interval between each of the tests and the order of testing was randomized by the sponsor. The test substance and the placebo control were prepared by Vétoquinol Spécialités Pharmaceutiques Vétérinaires B.P. 189-70204 Lure Cedex France and shipped to the experirnenter in such a way as to keep the study blind. The test substances and placebo controls were administered orally by a veterinariûn who was not involved in any of the behavioral testing. The substances were administered at the same time of day, on each of the test days, for al1 of the animals. The random assignment of each animal is listed in Appendix B. 9.2.4 Data Analysis A computer program was used to measure the total distance traveled by the animal. as well as. the frequency of unnation. jumping, rearing, grooming, sleeping, directed sniffing. vocalizations and periods of inactivity. AI1 of the statistical analyses were performed with the Statistica software package. The level of probability for significance was pcO.05. A three-factor mixed design analysis of variance (ANOVA) with repeated measures on two factors was used to determine the effect of the test substance on each of the behaviors. The factors included dose, time following dosing (2.4 and 10 hours) and treatment (drug venus placebo). The repeated measures factors were treatment and tirne following treatment while dose served as a within subject factor. A significance level of 0.05 was used. A computer printoot of the animal's total activity pattern was also obtained. These patterns provide a qualitative measure of the animal's behavior during the entire test session.

9.3 Results One of the dogs was not included in the statistical analysis because she would consistently run to a corner of the room and remain there for each of the entire test sessions. This dog continuously showed fear responses to both the mi mal caretakers and the expenmenters. Since this animal showed no sign of spontaneous behavior. she was not included in the final analysis. Of the eight behaviors measured none of the dogs engaged in groorning or jumping on any of the test sessions and therefore these behavion were not anal ysed.

9.3.1 Effect of Adrafinil on Locomotion When animals were treated with adrafinil. there was a statistically significant effect on locomotion. An increase in locomotion was seen as an effect of treatment (p= 0.0018) for each of the three doses of adrafinil. The animals exhibited a greater amount of movernent when they were lidministered adrafinil than when they were given a placebo control. Movement was measured as the total distance traveled by the animal. Figure 2 depicts the distance traveled by the animals at the 10, 30 and 50mg/kg dose of adrafinil. as well as. the distance traveled by the animals in the control tests at that dosage Ievel. There was also a statistically significant interaction between dose and treatment (p= 0.0353). Although there was an increase in movement for al1 three doses, the most dramatic increase in locomotion was seen at the highest dose level of 50rng/kg. This is also illustrated in Figure 3.

Adrafinil O Placebo

30 50 Dose Level (mgkg)

Figure 2: Total distance traveled after the administration of 10.30 or 50rndkg of adrafinil or a placebo control by aged beagles in Experiment 1. There is a statistically significant difference between adrafinii and the placebo control rit each of the dose levels. The interaction between dose and time following treatment approached statistical significance (p= 0.0635). The increase in locomotion was the greatest at 2 houn following administration of adrafinil and the lowest at 10 hours. At the 2-hour time period, there was only one dog that did not show any increase in locomotion and this dog was at the Iowest dosage level of 1Omgkg. At the 10 hour time period. locomotor activity was highest in the 50mglkg group and lowest in the 10mgBcg group. This is illustrated in Figure 3.

LIPlacebo

Hours Following Treatment

Figure 3: The cou1 distance traveled 2.4 or LO hours after the administration of adratinil or a placebo control by aged beagle dogs in Experiment 1. The interaction between dose and time following treatment approached statisticd significance for each of the tirne petiods. 9.3.2 Effect of Adrafinil on Urination Adrafinil had a statistically si gni ficant effect on urination. A decrease in unnation frequency was significantly affected by treatment (p= 0.0018) and time following treatment (p= 0.0405). There was also a significant interaction between dose and treatment (p= 0.0037) and between dose. treatment and time (p=0.0305). These results are primarily due to the decreased urination in the 50mzgkn<~group and are no longer apparent 10 houn later.

9.3.3 Effect of Adrafinil on Vocalization Adrafinil did not have a significant effect on vocalization. There was a tendency for a decrease in vocalizations in the SOmg/kg group. The treatment effect approached signi ficance (p= 0.069) and the interaction between treatment and time following dosing also approached statistical significance (p= 0.066).

9.3.4 Effect of Adrafinil on Activity Pattern Adrafinil did not affect the overall activity pattem of the dogs. The activity pattems were unique for each of the animals after the administration of adrafinil or the placebo control. There was an increase in totai activity but the animal's individual pattem within the testing room was not affected. Thus, if an animal spent the majority of the test time in the outer portion of the room. they continued to spend the majority of test time in that part of the room after the administration of adrafinil. The treatment of adrafinil did not result in a similar response pattem between any of the animals other than an increase in the total distance traveled. Two activity pattems of one of the animals (Iggy) ai the low dose of lOmgkg and the placebo control is illustrdted in Figure 4. Placebo

Figure 4: Activity patterns of one animal 2 hours after the administration of lOmg/kg of adrafinil or a placebo conuol. The dog showed an increase in activity after the administration of adrafinil but there was no change in the overall activity pattern. Dogs show different patterns of behavior in the testing room in cornparison to each other and adrafinil did not cause any type of stereotypical behavior (such as circling or pacing activity) at any of the doses. The activity patterns of one dog (Homer) has been provided in Figure 5. This dog was tested at the riomgkg dose of adrafinil and Figure 5 depicts the three activity pattems on adrafinil. at the 2,4 and 10 hour time following dosing, as well as, the three placebo control tests at the same time periods following dosing. Placebo

4 hrs

10 hrs 10 hrs

Figure 5: Activity patterns of one animal 2.4 or 10 hours foilowing the administration of SOrngkg of ridrafinil or a placebo control. There is an increase in acrivity rit each of the three time periods ridter the administration of ridrafinil in cornparison to the placebo control. The greatest increase crin be seen at the 2 hour time period. 9.4 Discussion The results of the present experiment indicate that adrafinil produces an increase in locomotor activity. This increase occun at al1 of the dose levels but has the greatest effect at the highest dose of 50mg/kg. The time following dosing was also an important factor on the effect of adnfinil with the greatest increase in locomotion occumnp 2 and 4 hours following administration. The enhancing effect of adrafinil was also long lasting and could be seen 10 houn following treatment. This finding is consistent with other studies on adrafinil and is similar to the results of a larger study that our Croup perfotmed with 32 beagle dogs in New Mexico (Siwak et al.. 2000). Adrafînil did not significantly affect sniffing. vocalization. inactivity. rearing or grooming. These results suggest that adrafinil does not have a stereotypical effect on behavior. Simon et a1..(1996) defined stereotyped behavior as the continuous repetition of one or a few actions. When dogs are given amphetamine. they show a change in their activity pattern and there is an increase in activities such as circling. pacing and sniffing (Randrup and Munhvad. 1975). This effect is not seen when dogs are given adrafinil. The activity patterns of each of the dogs did not show signs of circling or pacing and the generiil pattem of the individual dogs was maintained. Thus. if a dog spent most of the session in the outside perimeter of the test room. they continued to do so when they were given adrafinil. This suggests that adrafinil's mechanism of action as a behavioral stimulant differs from that of amphetamine. The results also indicate that adrafinil causes a decrease in urination. It is not known why adrafinil has this effect but one possibility may have been due to the design of the experiment. We did not perform Our fint activity test until 2 houn following treatment and the dogs were not closely monitored during this time. There is, therefore, the possibility of an increased urination during the first 2 hours following treatment and this would result in a decrease in urination during the test session. Plasma studies with adrafinil have shown peak plasma levels within one hour following on1 administration (Saletu et al.. 1986). Simon et al., (1995) have also found that rats exhibit a decrease in fluid intake following treatment with modafinil and this may dso account for the decrease in urination. In summary. a single dose of adrafinil causes an increase in locomotor activity and this increase cm last as long as 10 hours following treatment. The magnitude of the increase and the length of time it lasts is also dose dependent. Adrafinil does not induce the stereotypical behaviors that are seen with other stimulants but it may decrease urination frequency at higher dosage levels. Adrafinil's ability to increase behavionl activity without the stereotypies makes it an excellent behavionl enhancing agent for inactive, elderly dogs. 10. Experiment 2: Adrafinil as a Cognitive Enhancing Agent In Aging Beagle Dogs

10.1 Introduction

In the non-scientific community, adrafinil is being used as a "smart drug". The internet hüs a number of sites which are offering the substance for use in increasing aiertness and sensory vigilance. memory improvement and cognitive enhancement without the anxiety, agitation or the need for rebound sleep. Although adrafinil has been shown to increase vigilance in patients with Alzheimer's disease. as well as. in the aged population. it has not been tested for its cognitive enhancing abilities in ünimals. The purpose of the present investigation is to examine the effects of adrafinil on two tasks of discrimination learning in a population of aged beagles to determine if adrafinil can act as a cognitive enhancing substance and improve the learning ability of agd dogs.

10.2 Materials and Methods

10.2.1 Su bjects The subjects for Expenment 2 were 8 beagle dogs (5 femüle and 3 male) ranging in age from 7 to 11 yean and purchased from Harlan Sprague Dawley Breeding Fms. Seven of the dogs hdbeen used in Expenment 1. The eighth dog was purchased prior to the start of Experiment 3 and had no previous experience with the test substance. The housing and feeding schedule were the same as Experiment 1. A list of the dogs used for Experiment 2 can be seen in Appendix A.

10.2.2 Test Apparatus The testing apparatus was a wooden box 0.609 n 1.15 n 1.08m in size with vertical steel bars at the front. The box was based on the canine adaptation of the Wisconsin Genenl Test Apparatus (Fox. 197 1) and has been described previously by Milgram et al.. (1994). The heights of the vertical bars were adjustable and had three equal size openings that could be adjusted for each individual dog and allowed access to a sliding Plexiglas tray. The tray had three equally spaced food wells at the front. The experimenter was separated from the animal by a one-way mirror with a small hinged door at the bottom. Raising the door enabled the experimenter to present the tny to the animal. Dedicated software was used for al1 randomization procedures involved in determining the location of the stimulus and reward. A diagram of the test box has been provided in Figure 6.

Figure 6: The Canine Adaptation of the W isçonsin Testing Apparatus.

Openings for do? to respond

Hinged door Cornputer for randomization procedures. SIiding tray to present objects. 10.2.3 Re-Training Procedures Pnor to the start of the behavioral testing for Experiment 2, the dogs received pre- training sessions in which they learned to obtain food from the wells in the Plexiglas tray and move objects to obtain a food reward. Following the pre-training, the dogs were tested on visual object discrimination and reversa1 tasks. The dogs were presented with a round yellow lid and a blue block. with one of the objects being a positive stimulus and the other the negative stimulus. A food reward was always beneath the positive stimulus. The food reward for both the pre-training sessions and the behavioral testing was Hill's@ Pet Food dog biscuits.

10.2.4 Behavioral Testing Following the completion of the pre-training and the object discrimination. the animais were tested for the effect of adnfinil on two different discrimination learning problems. a size discrimination and a blacklwhite intensity discrimination task. For the size discrimination task. the dogs were presented with two objects that differed in size only. The small object consisted of a single red wooden block and the larger object consisted of two of the red wooden blocks glued together. For the intensity discrimination task, the objects differed in colour only, one object was white and the other object was black. The task each dog would start on was quasi-nndomized so that half of the dogs would start on the size discrimination task first and the other half would start on the intensity discrimination. Two equall y difficult tasks were required so that the animals could be tested on both adrafinil, as well as, the placebo control. The choice of the tasks was based on the performance of a separate group of dogs in a pilot study used to determine the difficulty of the tasks. The results of the pilot study indicated that the tasks were similar in difficulty, requiring the same number of trials to reach a pre-determined aiterion level of performance. On the fint day of testing, each dog was aven 10 trials with food located beneath each of the objects. This was used to establish object preferences. When a preference existed. on the subsequent testing days, the animal's non-preferred object was associated with the food reward. A nndom procedure was used to select the positive object for animals that showed no object preference. After the day of preference testing, the dogs were required to rnove the block that was the positive stimulus to receive the food reward. The dogs were considered to have made an error when they came into contact with the negative, unbaited stimulus. Only one correction trial was pemitted each day. The test session consisted of 10 trials per day with an inter-trial interval of 30 seconds. Test sessions were repeated until the animal achieved a criterion level of performance that included: (1) obtaining either 9 correct responses out of 10 in a single test session or 8 correct out of 10 on two successive sessions and; (2) performing at an accuracy of at least 70% over three successive additional sessions following achievement of the criterion level. The mimals were given a total of JO test sessions (400 trials) to achieve cri tenon.

10.2.5 Experimental Design Each of the dogs was given either an oral dose of IOmu& of adrafinil or a lactose placebo two hours before behavioral testins. A cross over design was used whereby half of the dogs were tested on the size discrimination task and the other half were tested on the intensity discrimination task. Of these iwo groups. half of each were tested with adrafinil and the other half the placebo control. After the anirnals completed the first task, they were given a two-week washout period and then they were switched to the opposite task and treatrnent condition. Thus. animals tested on the size discrimination under adrafinil were now tested on the intensity discrimination with the placebo control. The test substance and the placebo control were prepared by Vétoquinol Spécialités Pharmaceutiques Vétérinaires B .P. 189-7020J Lure Cedex France and shipped to the experimenter in such a way as to keep the study blind. The test substances and placebo controls were administered orally by a veterinarian who was not involved in any of the cognitive testing. The substances were administered at the sarne time of day. on each of the test days, for al1 of the animals. The randomization of each animal was done by the sponsor and has been provided in Appendix C.

This crossover design resulted in 4 groups: Group 1: was tested first on size discrimination under adnfinil and then on intensity discrimination under placebo control. Group 2: was tested first for size discrimination under the placebo control and then on intensity discrimination under adrafinil.

Group 3: was tested first on intensity discrimination under adrafinil and then on the size discrimination under the placebo control.

Group 4: was tested first on intensity discrimination under the placebo control and then on size discrimination under adrafinil.

10.2.6 Data Anaiysis All statistical analysis used the Statistica software Package. Two performance measures were used in the statistical analysis. errors to criterion and trials to criterion. The errors measure was the total number of incorrect responses made up to and including the cri terion session. The trials measure was the total number of trials taken to reach criterion. The level of probability for significance was pc0.05. After comparing the animals' performance on the two tasks. it was determined that there was a statistically significant difference between the total errors to criterion (t= 0.0503 two tailed test) and in total trials to criterion (t= 0.03 13, two tailed test). This is iilustrated in Figure 7. This difference in scores indicated that the tasks were not equivalent in difficulty and therefore the distributions of the two tasks were normalized and each animal was assigned a standard score. The standard scores were calculated by subtracting the animal's score from the mean of the distribution and then dividing by the standard deviation, Intensity Discrimination Size Discrimination

Figure 7: Mean number of trials and mors during the acquisition of the intensity and size discrimination tasks. A two-tailed test indicated that there wrts a statistically significant difference between the two tasks. Error bars indicate standard errors.

A three-way analysis of variance (ANOVA) was used to test for the effects of adrafinil on acquisition. The main conditions were treatment (adrafinil versus placebo), treatment order (dmg first or placebo fint), and task order (size discnmination first or intensity discnmination first). Treatment with adrafinil had a statistically significant effect on learning. A decrease in the number of errors required to reach criterion (p= 0.0372) and a decrease in the number of trials required to reach criterion (p= 0.015886) were seen when the animal was treated with adrafinil. The depiction in Figure 8 is the mean number of errors and trials required to reach cnterion after the administration of adrafinil or a placebo control. Figure 8 shows that the effects reflect more rapid leaming after treatment with adrafinil than after treatment with a placebo. The results also indicate that there was no effect of order of drug administration so it did not matter whether the animal was treated fint with adrafinil or the placebo. The effect of task order was very closely approaching significance for both errors required to reach criterion (p= 0.0502) and trials (0.0525).

Adrafinil 0Placebo

Errors

------Figure 8: Mean errors and trials required to reach criterion on two discrimination tasks foliowing the administration of adrafinil or ci placebo control. When the dogs were administered adrrifinil. they made fewer errors and required fewer trials than when they were ridministered the placebo control.1 Figure 9 illustrates that the animals showed a stronger effect in improved learning under the administration of adrafinil when they were tested on the size discrimination task fini.

Adrafinil 0Placebo

Size First Intensity First

Figure 9: The effect of task order was statistically significant for both the number of trials and errors required to rexh criterion. The rtnimds showed a suonger effect in improved leming following the administration of adratinil when they wcre tested on the size discrimination tsk first. An examination of one's animal's data indicates that adrafinil may have had an effect on response motivation. This dog showed penods where it failed to respond within the allotted time period when treated with the placebo control but the animal's motivation consistent1y improved when treated with adrafinil. This is illustrated in Figure IO.

1 1 I r I I I O 5 10 15 a0 25 33 Test se!ssiul

Figure IO: Response pattern of one dog following the administration of adrrifinil or a placebo control. The animal showed a suonger motivation to respond after the administration of adratinil. When the animal was given a placebo conuoi. there were fewer triah completed for a majority of the test sessions. 10.4 Discussion

The present results indicate that repeated daily administration of adrafinil can facilitate leaming in canines. When the dogs were given adrafinil. they made fewer erron and required fewer trials to reach criterion on two different types of discrimination leaming tasks. Saletu et al.. (1986) found that when hurnan subjects are given adrafinil. they show an increase in attention, concentration and memory in a number of psychornetric tests. These effects could account for the effectiveness of adrafinil on discrimination leaming in this study. The results have also show that the effects of adrafinil depended on the task and the order in which the tasks were given. Animals that were tested first on the size discrimination task showed the greatest effect of adrafinil treatment. The size discrimination task was a more difficult task and therefore any cognitive enhancing effects of adrafinil may have resulted in those dogs leaming the task much more rapidly than dogs on a placebo control. Thus. the effect of the dru; on the more difficult iask will be larger than the effect of the drug on an easier task. In a previous study by Our lab. we have shown that aged dogs have difficulty leaming a size discrimination task (Head et al.. 1998). We also have preliminary evidence suggesting a much smüller age effect on intensity discrimination learning. Thus. if dogs have little difficulty leaming an intensity discrimination task. adr~finilmay have less of an effect. The effectiveness of prior training of one task on subsequent training on a second task rnay also account for the differences between the drug effect on each task. When animals were tested on the intensity discrimination task fint. they showed a faster acquisition of the size discrimination task. Since both tasks are discrimination leaming problerns. acquisition of one discrimination problem may have facilitated the subsequent acquisition of the second problem. Adrafinil's mechanisrn of action has yet to be determined. Most of the evidence indicates that adrafinil serves as an u-l adrenergic agonist (Rambert et al., 1986) and this potential action on brain adrenergic systems could account for the cognitive enhancing effects of the dnig in aged subjects. Studies have shown that noradrenergic agonists can enhance both leming and memory (Arnsten, 1997) and there is extensive literature indicating the role of norepinephrine in learning and memory. Siruio et al., (1991) has shown that noradrenergic systems show a particular sensitivity to age-related degeneration (Vijayashanhan and Brody, 1979) and therefore, adnfinil may be a suitable cornpound for aging pets. as well as, aging individuals. Finally, the results of one dog indicated that adrafinil also affects arousal and motivation. This animal failed to respond consistently when tested dunng the pre-training phase, the placebo control phase and also dunng subsequent testing after the completion of the study. When the animal was given adrafinil. the responding was more consistent and reliable. In a separate study by Siwak et al.. (3000) adnfinil has been shown to increase exploratory behavior in dogs and this may be further evidence of the arousal or motivational effects of the drug. The present study provided the first evidence of adrafinil's ability to enhance cognition in non-human subjects. These findings are consistent with the human literature and suggest the requirement of further siudies to gain a better understanding of the effectiveness of adrafinil on cognition in aged pets. aged individuals and possibly the improvement of cognitive dysfunction associated with dementia. 11. Experiment 3: The Effects of Adrafinil on EEG Activity in an Aged Canine: A Pilot Study.

11.1 Introduction There have been very few studies performed to determine the effects of admfinil on EEG. Although adrafinil and modafinil are currently being used as arousal agents in a number of therapeutic environments. their effects on the brain have yet to be determined. The clinical potency of a vigilance-enhancing agent may be its ability to directly alter bnin function and its accessibility to the CNS. An indication of modifications to the CNS may be evident in any electroencephalographic effects. as well as, motor, sensory or cognitive alterations. Wikler (1954) sugpested that regardless of the nature or mechanism of action of the dmg administered. shifts in the pattern of the electroencephalogram in the direction of desynchronization occurred in association of alertness. anxiety. illusions or tremors, and in the direction of synchronization with euphoria. relaxation and drowsiness. One way to define arousd states is by the pattern of electrical activity created by the cortical neurons. In awake states. many neurons are firing but not in a coordinated fashion. The EEG shows lower amplitude, higher frequency waves. This lack of coordination can be referred to as desynchronization and it is produced by ascending signals from the reticular formation (Fisch. 1991). In the aeeper stages of sleep. the frequency of the waves slows down and the amplitude is increased. The waves are depolarizing in a coordinated and more regular fashion and the Iarger the amplitude, the more synchronized the firing of the cortical neurons. The results of Experiment 1 and 7 suggest that adrafinil has a behaviorally activating effect. as well as, a cognitive enhancing effect on aged beagles. There is limited researc h on the effects of adrafinil and modafinil on EEG and the research that is available is conflicting. Saletu et al.. (1986)stated that adrafinil and modafinil caused a decrease in the lower frequencies. an increase in the mid-frequencies and a decrease in the high frequencies. Saletu et al.. (1986) provided this as evidence for the vigilance-promoting effects of both adrafinil and modafinil. Conflicting evidence, however. was provided by Sebban et al., (1999) in their study with modafinil. Sebban et al., (1999) stated that modafinil caused an increase in the low and high frequencies and a decrease in the mid- frequencies, the exact opposite of the study by Saletu et al., (1986). Based on this limited and conflicting evidence, as well as, the findings of our behavioral and cognitive tasks, we investigated the effect of three differerent doses of adrafinil on the resting EEG of an aged beagle. The purpose of the present experirnent was to determine some of the physiological components of adnfinil and its effects on EEG to ascertain whether adrafinil acts as an enhancing agent in the CNS and if its effects are similar to other u- adrenergic agonists. 11.2 Materials and Methods

11.2.1 Subjects The subject for Experiment 3 was an 11 year old female beagle who had participated in Experiment 1. The housing and feeding were the same as it had been for Expenrnent 1.

11.2.2 Electrode Implantation Procedures In EEG recordings. the electrodes are placed on the scalp and the tracings are the summated changes of the underlying cortex. The EEG recorded with scalp electrodes differ from the tracings with electrodes placed directiy on the underlying cortex (electrocortiograrn or ECoG) or from those implanted deeper into the cortex (depth EEG or DEEG). The scalp EEG is generally of lower amplitude due to the increase in electrical impedance and reduction in current fiow caused by the thickness of the skull. Electrodes implanted directly into the cortex may also produce tracings from a more localized area as opposed to the scalp EEG which is generated from a larger surface area. In the present experirnent. depth electrodes were used because of the large muscles located across the dog's skull. The purpose of this muscle is to aide in chewing and its presence on the skull results in artifacts in the scalp EEG tracings. The term EEG has been used to represent the depth EEG tracings recorded from the implanted elecirodes. Pnor to the surgical procedures. the dog was sedated with an intra-muscular injection of aceprornazine (O.jrn~A/kg) and atropine sulfate (0.05mzAg). An N was inserted into the dog's forearm and 25m~Agof thiopental was injected, the dog was intubated and the head was shaved. The dog was then moved to the surgical suite and maintained under general inhalation anesthetic of halothane and nitrous oxide. A stereotaxic frame was used to maintain the stability of the dog's head and to determine the placement of the electrodes. The dog's scalp was incised and the muscle was sepanted to reveal the skull. Two holes measuring 0.8 cm in diameter were trephined through the skull on either side of the midline over the parietal /occipital cortex. Six bipolar electrodes were attached to a connector and implanted to a depth of approxirnately 2mm into the cerebnl cortex just right of the midline. The electrodes consisted of 3 insulated stainless steel wires that were twisted together and had their tips cut so that they were 0.5-1.Omm apan. Dental acrylic was applied to the top of the skull to keep the connectors in place. Six stainless steel screws were tapped into the skull and a wire was wrapped around them and attached to the connector to act as a ground electrode.

11 J.3 Experimental Design: Prior to the start of the actual EEG recording. the dog spent a number of days being acclimatized to the testing facility. We used a hammock for the dos to rest in during testing. The harnmock was padded and had four holes for the dogs limbs to protrude through. During the acclimatization period. the time the dog spent resting quietly in the hammock was gradua11 y increased. The experimenter also spent part of this time handlin; the dog. After the acclimatization petiod. the dog undenvent surgery to have the electrodes implanted. The dog was given a recovery period of 10 days with the surgical site being cleaned every second day. Following the 10-day recovery penod, the dog wüs re- acclimatized to the testing facility. During this time. the connector in the dog's head was attached to the recording apparatus as part of the acclimatization period. For the dmg-testing penod. the dog was given on oral dose of 10mkg/kgof adrafinil for three consecutive days with EEG recordings being done 1 hour before dosing and every half hour after dosing for a period of 5 hours. The EEG samples were 10 minutes in duntion. The dog was then tested for 3 consecutive days at 20m~Agdose followed by 3 days at a 40m& dose of adnfinil. There was a 4-day washout period between dosage levels when the dog was not tested and did not receive adrafinil. There was dso no set baseline period. EEG recordings that were made pnor to the start of the drug testing were used as baseline control. Al1 of the EEG recordings were made at the same time of day throughout the dmg-testing penod.

11.2.4 EEG Collection During the collection of EEG recording, the dog was placed in the suppon hammock with her legs hanging freely through the four leg holes. A sponge cushion was placed at the front of the hammock for the dog to rest her head. The hammock was situated in a radio-frequency shielded recording room and the cables leading to the EEG amplifier headstage were attached to the connector in the dog's head. The impedance of each of the electrodes was measured prior to the start of EEG collection and the lights of the recording room were turned off. EEG recordings were collected for durations of 10 minutes every half an hour for a total of 5 hours after treatment. The digitizing rate of each analog EEG channel was 256 sümples /sec. If the dog began to move its head ioo much or if there were any electrical interference. the recording was discontinued until the animal was quiet and the interference was removed. If this continued for a number of recording samples throughout the day. the data collection was discontinued for the remainder of the day.

11.2.5 Data Analysis Al1 of the EEG samples were analyzed with the Labview software package. Analysis consisted of applying the Fast Fourier Transform (FFï) to determine the frequency content of each of the samples. The FFT is a mathematical manipulation that converts the EEG from a time domain to a frequency dornain. It is based on the fact that any signal can be described as a combination of sine and cosine waves of various phases, frequencies and amplitudes. The FFT generates a number of numerical coefficients that represent the sine and cosine waves of the original signal. To view the signal in terms of the intensity of the frequencies, the Fourier coefficients are squared and this results in the power spectrum (Fisch. 1991). The power spectrum of the present experiment was subdivided into the following frequencies: 1) Delta: 1-4 Hz 2) Theta: 4-8 Hz 3) Low Alpha: 840.5 Hz 4) High Alpha: 10.5- 13 Hz 5) Low Beta: 13-18 Hz 6) Mid Beta: 18-24 Hz 7) High Beta: 73-30 Hz

11.3 Resul ts

11.3.1 Chronic Effect of Adrafinil on Resting EEG Treatment with adrafinil had an enhancing effect on cortical EEG. Since there was no set baseline period. the EEG recordings that were made prior to the stan of the drug testing were used as baseline control. In cornpaison to recordings made when the dog was administered adrafinil. the total power of the control data was rnuch lower than the total power of the recordings at the 10. 20 or 40mgkg dose levels. This was seen across al1 of the seven frequencies. Figure 1 I depicts the total power for each frequency for a sample of one day of control data and one day of recording at the 10mgkg dose as an example of the change in power after treatment with adrafinil. The total power of each frequency was L 540 times greater in the lOmg/kg dose than it was for the control recordings. Sirnilar findings were also seen at the 20 and 4Omgkg doses. One of the defining characteristics of the control data of this animal is the predominance of delta and theta activity and the near absence of any other frequency. Although delta and theta activity is increased after the administration of adnfinil. the other frequencies show a greater increase in power in cornparison to their initial baseline power. The chronic effect of adrafinil appears to be an enhancement of the frequency composition of the resting EEG in cornparison to the frequency composition of the baseline control.

+Control .O+ . 1Orng/kg

Frequency (Hz)

Figure 11: Total power ris a function of Frequency for EEG recordings of conuol data and following the administration of 1Omgkg of ridrafinil. There is an increrrse in the power for each of the frequencies rifter the administration of adratinil, with the greritest increrise in the delta frequency. 11.3.2 Acute Effect of Adrafinil on Individual Frequencies The acute effects of the drug were determined by the change in the power spectrum of the animal's EEG pnor to. and following the administration of adrafinil on each individual day of recording. The frequency composition of the EEG would return to pre-dmg levels after approximately 5 houn after administration of the dng. and would be at pre-drug levels at the start of the next recording session. Pre-dnig levels were considered to be the sarnples that were collected one hour pnor to the administration of adrafinil, Although there was an overall enhancement in the chronic EEG after treatrnent with adrafinil. the effect of the drug differed for each individual frequency at the ricute level. When the dog was treated with adrafinil. there was a dramatic decrease in the power of delta and a less pronounced decrease in theta. There was an increase in the mid-frequencies of low alpha. high alpha and low beta. High alpha and low beta had a large increase in power whiie low alpha had a smaller overall increase in power. For the higher frequencies of mid beta and high beta. there was a slight reduction in mid beta and a large decrease in high beta after the administration of adrafinil. This change in frequencies was similar at ail three dose levels of adrafinil with the greatest effect being at the 70mg/kg dose. When the dog was administered the LOmglkg dose of adrafinil. there was a change in the EEG tracings beginning at approximately one half hour after ireatment and lastin; approximately 4-5 hours after treatment. Changes in al1 seven frequencies began at the same time and lasted for the same duration. This was also seen for the 20 and the 40m~@kgdoses. A sample of the changes that occurred over the time course of one day is illustrated in Figure 12. The figure depicts the change in delta activity and high alpha activity. There is a reduction in the total power of delta and an increase in the power of high alpha beginning at one haif hour after treatment and continuing until 4 houn after treatment. After the 4-hour time period. the total powen of delta and high alpha returned to pre-treatment conditions. Delta

Time Following Dosing (hours)

Time Following Dosing (hours) Figure 11: Changes in the power of delta and high alpha following the administration of 10rngkp of adrafinil. There was a strong decrease in power for delta and an increase in power for high alpha. The changes in the frequencies began approximately one haIf hour after treatment and Iristed approximately four hours after treatment. A sample of the power spectrums of one channel has dso been provided as an exarnple of the change in power for each of the frequencies over the course of one day. This is illustrated in Figure 13. The figure depicts the power specuum at the time periods of prior to treatment, 30 minutes. 1 hour, 2 hours and 4 hours after treatment. At the 30- minute time period, there is a decrease in the low frequencies (between 2 and 8 Hz) and an increase in the middle and high frequencies (between 14 and 30 Hz). This continues up to the four hour time period when there is a reduction of the mid-frequencies and an increase in the lower frequencies. The increase in the high frequencies of mid beta (18-14 Hz) and high beta (74-30 Hz) beginning 30 minutes after treatment that are depicted in Figure 13. were not common to al1 of the channels recorded. The trend is generally for a decrease in the higher frequencies of rnid and high betû, as opposed to the increase that is seen here. The power spectrums depicted in Figure 13 were a sample of one day of recording from one channel and are a good demonstration of the shift in power away from the lower frequencies of delta and theta. Prier to adrafinil

30 mins atier adratinil

1 hour after adrafinil

2 hours after adrafinil

4 hours riftet adrafinii

Figure 13: Power spectrums prior to the administration of adrafinil and approximately 30 minutes. 1 hour. 2 hours and 4 hours after the administration of 10mg/kg of adrafinit. The power spectrums show the shift in power from the low frequencies. prior to receiving the drue, to the higher frequencies after 30 minutes. This pattern continues up to 4 hours and then returns to pre-dnig conditions. b -46- 11.3.3 Comparison of 10,20 and 40 mgkg of Adrafinil

As indicated previously, al1 three of the doses showed a similar trend of a decrease in the low and high frequencies. as well as. an increase in the mid frequencies of alpha and low beta. The IOmglkg dose had the greatest effect, and the 10 and JOmplkg doses were very similar in magnitude to each other. When the dose levels were compared for total power, the 20mgkg dose also showed the greatest increase in total power and was often twice as high as that seen for the 10 or JOrngkg doses. Total power consists of recordings obtained from one channel throughout the entire day for each of the seven frequencies. Figure 11 illustrates the average total power for each dose level for the three days of recordings. 20mgkg

Dose (mglkg)

Figure 14: Average total power of EEG recordings from one channel following the administration of LO. 20 or JOmgkg of adratinil. The animal showed the greatest increase in total power ;ir the ZOmgkg dose. Error bars indicate standard deviation. 11.4 Discussion

The present results indicate that administration of adrifinil cm enhance the resting EEG of aged beagles. When the dog was given adrafmil, the total power of the resting EEG was dramatically increased by 15-40 times that of the control recordings. A number of studies have suggested that as we age, there is a decrease in our resting EEG, as well as, changes in its frequency composition (van der Drift. 1973: Matejcek and Devos. 1976: Amgo et al., 1979: Obrist. 1979). Other studies have sugpested that the changes that are often seen in the EEG recordings of aged individuals are not due to aging but the pathological alterations that are often found in elderly subjects (Hubbard et al., 1976: Kazis et al.. 1981: Giaquinto and Nolfe. 1986; Pollock et al.. 1990). Whether the changes in the EEG of some elderly individuals are due to the process of aging or pathology. the results in the present experiment suggest that adrafinil has a reversal effect on those EEG patterns. The baseline EEG of the one animal that was tested for this experiment showed a predominance of the low frequencies of delta and theta and the near absence of any of the other frequencies pnor to the administration of adrifinil. It is unknown whether this finding is similar to the EEG tracings of other aged beagles because there have been no known studies of the changes that accompany aging in the dog. It is important to mention. however, that after the completion of the EEG studies. goss anatomical investigations revealed a large lesion in the media1 frontal lobe of the dog's brain. The lesion did not appear to have been caused by the invasiveness of the experimental protocol but was a long-term disorder pnor to the purchasing of the animal. This animal may therefore represent a form of dementia or what we have previously referred to as an unsuccessful ager. The resuIts also indicated that there was a difference between the chronic and acute effects of adrafinil on the frequency composition of the resting EEG. When this animal began baseline recordings, the EEG consisted mainly of delta and a small amount of theta activity. There was little to no evidence of the remaining five frequencies. After the animal was consistently brought to the testing facility, the power of each of the frequencies increased. This increase in power was even more dramatic after the administration of adrafinil and there was no indication of a return to the levels that had been previously seen at the baseline recording. This may be evidence of adrafinil's effect on dementia but further studies are required to detemine if this is the case. This increase in total power has not been reported in the literature regarding the cffect of adrafinil on EEG and is the opposite effect of what would be expected if adrafinil were an al-adrenergic agonist. The ai-adrenergic agonists usually cause a decrease in total power rather than an increase (Sebban et al.. 1999). The acute effects of the drug were determined by the change in the power spectrum of the animal prior to, and following the administration of adrafinil on each individual day of recording. The results of the acute studies showed that adrafinil had a possible vigilance-promoting effect on the EEG of aged animals by decreasing the lower frequencies and the high frequencies of beta. and increasing the mid- frequencies of alpha and low beta. Similar findings were reported by Saletu et al.. (1986) in a study of the effects of adrafinil and rnodafinil on EEG in elderly patients. Saletu et al.. (1986) suggested this was evidence for the vigilance-enhancing effect of the drug and its ability to reverse the changes in the EEG with accornpanying age. Finally. the results provide evidence of the thenpeutic benefits of adrïfinil in aged non-human subjects with the suggestion of a possible treatment for the aged pet population. 12. Experiment 4: The Neurophysiological Modulation of Cortical Function by Adrafinil

12.1 Introduction Based on the results of Experiment 3, a second EEG expenment was canied out with two additional aged beagles. The focus of the second EEG experirnent was to use only the 70rngkg dose of adrafinil because it had been determined to be the optimum dose level in Experiment 3. The purpose of the present experiment was to determine if the same effect could be obtained in two additional aged canines or if the results of the pilot experiment were speci fic to one individual animal.

12.2 Materials and Methods

12.2.1 Subjects The subjects for this expenment were two old beagle dogs (1 male and 1 female) over the age of 10 years who had participated in Experirnent 1 and 3 prior to the start of this study. These dogs were selected at random from the larger proup of dogs that had been used for Experiment 1 and 2. The housing and feeding schedule were the same as it had been for the other expenments.

12.2.2 Electrode implant Procedures The surgical procedures for the implanting of the electrodes were very similar to what we had done for Experiment 3. The dog was sedûted with an iM injection of acepromazine and atropine sulfate and an IV was inserted into the forearm for a 25rng/kg thiopental injection. The dog was intubated. the head was shaved and the animal was then moved to the surgical suite and placed on a general inhalation anesthetic of halothane and nitrous oxide. The dog's head was then placed in a stereotaxic frme to maintain stability and to detennine the placement of the electrodes. The scalp was incised and the muscle separated to reveal the skull. Two holes measuring 0.8mm in diarneter were trephined through the skull on either side of the midline over the parietal/occipital cortex. Four bipolar electrodes were attached to two equal size connectors and implanted to a depth of approximately 2mm into the cerebral cortex on either side of the rnidline. The electrodes consisted of 2 insulated stainless steel wires which were twisted together and their tips cut so that they were 0.5-1.Omm apart. Dental acrylic was applied to the top of the skull to keep the connectors in place. Six stainless steel screws were tapped into the skull with a wire wrapped around them and attached to the connecton to act as a ground electrode. A screw overlying the primary auditory cortex was used as a reference c lec trode.

12.2.3 Experimental Design: Prior to the start of the EEG recording, the dogs were brought to the test facility daily for an acclimatization period. During this tirne. the dogs were placed in the hammock in the recording room and handled by the experimenter. The purpose of the acclimatization period was to get the dogs used to the hammock. the testing facility and the experimenter. After a week of acclimatization. the dog undenvent surgical pmcedures to implant the electrodes and this was followed by a 10-day recovery period. Dunng the recovery period. the animal's head was cleaned every second day and monitored for signs of infection. The recovery period was followed by a 3-day re-acclirnatization period. At this time. the animal was placed in the hammock in the recording room, the surgical site was cleaned and the dog was connected to the recording apparatus. The purpose of the re- acclimatization period was to prepare the dog for the expenmental conditions during the EEG collection. The collection of the EEG consisted OF three phases. A baseline phase, a drug phase and a washout phase. Each of the phases were 8 days in duration with EEG collection occumng on days 1, 3, 5 and 8. The baseline phase was performed first and was used to determine the initial EEG components in the absence of the test substance. For the dmg phase, the dog was given an oral dose of 20m_&g of adrafinil for 8 consecutive days. with EEG recording occumn_pon days 1, 3, 5 and 8 only. The washout phase was the last eight days of the study. During this time the EEG recordings were made to determine whether the EEG would return to baseline rneasures and the amount of time it would take to do so. The EEG recordings were collected approximately 1 hour prior to dosing and every half an hour after dosing for a penod of 5 houn. The EEG sarnples were 5 minutes in duration. During the baseline and the washout periods, the animal was given a placebo control and the EEG was collected in the sarne manner as during the dmg phase. There were no additional days between each of the phases.

12.2.4 EEG Collection: The collection of the EEG was identical to that described in Experiment 3. The dog was placed in the hammock with the legs hanginp freeiy through the le; holes. The head could be rested on a sponge cushion that was provided at the front of the hammock. The hammock was placed in a radio-frequency shielded recordinp room and the cables leading to the EEG amplifier headstage were attached to the two connectors in the dog's head. The impedance of the electrodes was measured prior to the start of the EEG collection. A 5-minute EEG sample was collected every half-hour for a period of 5 hours after treatment. The digitizing rate of each analog EEG channel was 356 samples/sec. If the dog began to move its head too much or if there was any electrical interference. the recording was discontinued. The EEG recordings were performed at the same time of day for each of the animals during the baseline. drug and wash-out conditions.

12.2.5 Data Analysis: The analysis of the EEG samples was performed with the Labview software program and was identical to the analysis in Experiment 3. The FFT analysis was performed on each of the EEG recordings and the resulting power spectrum was divided into the 7 frequency bands which were provided in Expenment 3. 12.3.1 Resting EEG The baseline resting EEG of the two dogs in the current experiment were different in frequency composition thnn the dog that had previously been tested in Experiment 3. Unlike Bonnie, the current two dogs showed high activity at each of the frequencies for their baseline recordings. There was also a lack of predorninant delta and theta bands and there was approximately equal power across al1 seven frequencies. In cornparison to each other, there was a similarity in the shape of the power spectrum but the male dog (Denny) showed slightly higher power across al1 frequencies than was seen for the female dog (Eve).

12.3.2 Effect of Adrafinil on Resting EEG The results of the present experiment provide further evidence of the enhancing properties of adrafinil. Treütment with adrafinil had a slightly different effect on the current two animals than had been discussed in Experiment 3. There was a decrease in total power for both dogs following the administration of îOmg/kg of adrafinil than there was for the placebo control. The tothl power for both the baseline recordings and the washout recordings were higher than the total power after the administration of 2Om~Ag of adrafinil. This is the opposite of what was described for Experiment 3. Figure 15 depicts the average total power of one channel for the baseline. dng and washout test sessions of both dogs. The figure illustrates the difference in average total power with less power for the dmg sessions than for the baseline or washout sessions. Similar findings were seen at each of the eight channels that were used to record EEG from the dogs. Eve

Baseline Washout

Denny

Baseline D% Washout

Figure 15: Average total power of EEG recordings for baseline. drue and washout sessions recorded from one channel for both dogs in Experiment 4. There was a reduction in total power after the administration of 20mgkg of adrafinil. This reduction in power returned CO baseline levels during the wash-out session. When the dogs were administered 20rnglkg of adrafinil, there was a change in the EEG tracings beginning approximately I to 1.5 houn after treatment and lasting approximately 4-5 hours after treatment. Changes in al1 seven frequencies began at the same time and lasted for the same duration. The changes in frequency power began at either 1 or 1.5 houn after treatment for both dogs but the duration of the effect was different for the two dogs. The changes in the frequencies of one dog (Denny) had not retumed to pre-treatment levels at the end of the test session on any of the dru, treatment days. The frequencies had retumed to pre-treatment levels. however. by the tirne of the next test session two days later.

12.3.3 Effect of Adrafinil on Individual Frequencies Treatment with adrafinil resulted in a decrease in the low frequencies (delta and theta). as well as. a decrease in the high frequencies (mid and high beta). There was generally no change in the middle frequencies of low alpha. high alpha and low beta. The decrease in upper and lower frequencies results in the decrease in the total power that was discussed previously. These findings also slightly differ from the results descnbed in Experiment 3. The mid-frequencies (low and high alpha and low beta) for Expenment 3 were increased after the administration of adrafinil but there was no effect of adrafinil on the mid- frequencies for the animals in the current experiment. The other frequencies showed a similar response to adrafinil as was reported previously in Experiment 3. A sample of the changes that occurred over the rime course of one day is iilustrated in Figure 16. The figure depicts the activity of delta and high alpha for both Eve and Denny. There is a reduction in the power of delta and high alpha which Stans approximately 30 minutes after treatment and continues for approximately 3.5 hours after treatment for Eve. The power of delta does not return to pre-treatment conditions for Denny after 5 houn of treatment but the levels of delta activity did retumed to pre-treatment conditions by the next test session. There was no change in the activity of high alpha for Eve or Denny after the administration of adrafinil. -2 - 1 O 1 2 3 4 5 6 Time Following Dosing (hours) High Alpha

Time Following Dosing (hours)

- - Figure 16: Changes in the power of delta and high alpha for Eve and Denny folIowing the administration of 20mg/kg of adratinil. There wcis a decrease in power for delta approximately 30 minutes after treatment for both animais and it lasted approximately 3.5 hours after treatrnent for Eve. The power of delta did not return tn nrtwrentme=ntcnnrlitinnc fnr nennv Th~rpwnc nn pffert nf arlrnfinil nn hioh alnha fnr FVPnr n~nnv 12.4 Discussion A cornputer-assisted power spectmm analysis indicated that for these two anirnals. adrafinil behaved iike other al-adrenergic agonists in its effect on EEG. There was a gnenl decrease in the total power. and this decrease was greater during the treatment phase than it was for either the baseline or washout periods. The overall tendency was a decrease in the power of the frequencies. with the greatest decrease in delta and theta activity, as well as, decreases in mid and high beta activity. There was no change for low and high alpha and low beta. This is similar to the findings of Sebban et al., ( 1999) but differed from the previous study, as well as. other reported studies of the effects of adnfinil on EEG (Saletu et al.. 1986). There was no increase in the middle frequencies of alpha and low beta. which were reported by Saletu et al.. and this may be due to the high baseline activity already present at those frequencies or it may be due to the differences in impairment among the animals. We have found that the effect of adrafinil varied ümong the dogs and seemed to be dependent on the frequency composition of the baseline EEG. Thus, dogs that had an initial EEG composition which was predominantly low frequency showed a greater increase in alpha and beta activity than dogs that had high alpha and beta activity at baseline recordings. The difference in the effect of the drug on resting EEG was discussed in the previous experiment. Adrafinil initially caused an enhancement of each of the frequencies when the dog's baseline consisted predominantly of delta activity. When the EEG activity reached levels indicating a more aroused or alen state. the effect of adrafinil wûs a reversa1 of the changes in EEG that often accompany aging. For the animals that possibly show no signs of aging or dementia. the effects of adrafinil on EEG is similar to the effects of ui-adrenergic agonists on EEG. As a dog ages, typically we see an increase in the low frequencies (delta and theta). a decrease in the mid-frequencies (especially alpha activity) and an increase in the very high frequencies of betn. Adrafinil seems to counter-act this effect. This finding was also seen in human EEG and adrafinil (Saletu et al.. 1986). Our dog work differs from the human EEG studies with adrafinil in the fact that we have observed adrafinil to have a stronger and slightly different effect on the dogs that were more impaired. The hurnan EEG studies groups elderly subjects into one category and does not look at individual differences of the drug. Our dog work has traditionally grouped anirnals into impaired versus unimpaired categones based on performance on cognitive tasks. Although it is unknown whether these dogs cm be divided into two separate groups. the differences in resting EEG and the effect of adrafinil seems to suggest that this is the case. This may be related to the effects that adrafinil has on dementias but further study is required. 13. General Discussion and Conclusions

The results from the behavioral, cognitive and EEG studies are al1 consistent with each other in suggesting the vigilance-enhancing properties of adrafinil. The dogs that showed the greatest decline in cognitive function and resting EEG analysis also showed the greatest improvements with treatment. Dogs that were less cognitively impaired or whose baseline frequency composition was more active showed improvement after treatment with adrafinil but the effects were not as dramatic as they were for the impaired animals. This may be the result of adrafinil's effect on unsuccessful agers or animals that show possible signs of decline and dementia. It may also be due to the greater amount of improvement that can be obtained from animals with a poorer performance. Animals that had little difficulty solving the cognitive tasks. for example. may not show a greater performance after the administration of adrafinil simply because they are already performing at a maximum level. The effects of the dmg on EEG also differed based on the frequency composition of the baseline recordings. When an animal's EEG consisted of delta and theta activity. there was a stronger effect of enhancement than for animals with an EEG composition that suggested a more aroused or alert state. The effect of adrafinil on behavior was consistent among each of the animals, with the maximum effect of the dmg being dependent on dose level and time following treatmrnt rather than the baseline inactivity of the dogs. This rnay be due to the fact that although there can be decreases in activity with accompanying age. it is not a defining feature of the aging process. The mechanism of action of adrafinil is currently unknown. Although there have been sevenl hypotheses suggested, most of the available evidence indicates that adrafinil is an ul-adreneqic agonist. Evidence has shown that the adrenergic system and adrenersic agonists are involved in improved cognitive function, increased behavionl activity and a decrease in EEG power (Amsten and Golman-Rakic. 1990: Arnsten and Contant, 1991: Cai et al.. 1993: Sebban et al.. 1999). The activation of ul-adrenoceptors promotes learning and working memory, vigilance and behavioral activation (Sirvio and MacDonald. 1999) and this evidence is similar to the findings expressed in the previous experiments. The preseni studies found that adnfinil improved leaming and increased behavionl activity in a population of aged dogs, as well as. decreased the EEG power spectrum of two of the three dogs tested. These studies therefore, provide more evidence for adrafinil's adrenergic mechanism of action. There is other evidence that adrafinil affects other neurotransmitters such as glutamate, GABA and dopamine. The effects on these neurotransmitters, as well as. norepinephrine may be the cause of the animals' response to the drug. The action of adrafinil may be due to its complex in vivo phmacological profile and adrafinil rnay be having its behavioral. cognitive and neurophysiological effects by a cascade of actions that are both directly and indirectly operating. The increase in noradrenaline. dopamine and glutamate and the decrease in GABA may be causing an arousal or alening response through several of the brain areas involved in the sleep waking cycle and the arousal systems. It is unknown why adrafinil has such a varied effect on the different neurotransmitten but the recent discovery of the orexin neurons in the lateral hypothalamus. their widespread axonal projections throughout the brain. and its possible role in sleep regulation may provide an answer to the mystery. Funher studies are required but adnfinil may be acting on the orexin neurons. which in tum are affecting other neurotransmitters involved in sleep regulation and arousal. The real importance of the EEG and its relation to the behavioral and cognitive studies became available after the completion of al1 of the experirnents. The organization of the experiments was developed pnor to our deeper understanding of adrafinil and its effecis on behavior, cognition and neurophysiology. The EEG information that we obtained in the final experiments could have been useful prior to the stan of the cognitive and behavionl studies. The results of Expenment 1 indicated that adrafinil caused an increase in activity 10 hours after treatment. Had the drug been active for a shoner duration. we may have been unaware of its effects due to the behavioral tests being conducted at no less than 7 hours after administration of the drug. The EEG recordings offered information regarding the time when adnfinil first hd an effect, the duration of the effect and infomation on the optimum period for cognitive and behavionl testing. The EEG recordings also offered information regarding the response of the animals to adrafinil. This information could be used as a possible screening test to determine a population of animals that would be best suited for administration of the dmg. EEG iecordings are currently being used as a possible method for detecting the presence of AIzheimer's disease and other dementias (Pentilla et al., 1985; Soininen et al., 1982, 1989, and 1991). EEG recordings may therefore offer a means of distinguishing cognitively impaired dogs from a population of aged dogs prior to performing vast amounts of cognitive tasks which are both time consuming and expensive. Overall, the results of al1 four experiments indicate that adnfinil offers wide promise as a therapeutic agent in treating aged pets and aged individuals with cognitive or behavionl declines that are consistent wirh aging, Alzheimer's disease or other types of dernentias. Adrafinil is currently being used as a rreatment for Alzheimer's patients in France and has been shown to be safe and effective. REFERENCES

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Animals Used in Study

ANIMAL NAME DATEOF SEX WIGHT EXPERIMENT ID BIRTH (KG)

KZX-5 Andy 1 1/85 Male 13 -40 1 WVK-7 Bonny 6/87 Female 10.50 1.3 WW-7 Clyde 11/87 Male 14.60 1,2 SSB-8 Denn y 01/88 Maie 12.80 1,2,4 DXI-8 Eve 05/8 8 Fernale 14.60 1,2,4 OCU-8 Flash 10/88 13.80 C Female 1 1.2 ZAE-9 Genny 1 03/89 FernaIe 13.20 1.3 Q.JD-O Homer 02/90 Male 13.00 1.3 HBE-O Igg y 03/90 Femde 13.00 1 ,? B IE-9 Jewels ?/90 Femaie 12.80 -3 Randomization of Animals Used in Experiment 1

ANIMALID NAME SEX TEST TEST TEST TEST TEST TEST DOSE 1 2 3 4 5 6 LEVEL KZX-5 Andy MaIe Pl0 P2 A4 P4 Al0 A2 30nig/kg WVK-7 Bonnie Female P3 P2 Pl0 Al0 A4 A2 50rnjAg W UC-7 Clyde Male P4 Pl0 A2 A4 P2 A10 l0rnoAg SSB-8 Denny Male Pl0 P4 Ai0 A2 P2 A4 30rn:Aj DXI-8 , Eve Female P2 PL0 A2 Al0 P4 A4 1Orn~Ag ZAE-9 Genny Female A10 A2 Pl0 P2 P4 A4 30mg/kkg OCU-8 Flash Female A2 P4 A4 Al0 Pl0 P2 50rno& OJD-O Homer Male A? A4 P2 1 P4 PL0 A10 50rn.dkg

a P indicates placebo. a A indicates adrafinil. 2.4 and 10 indicate hours after treatment. APPENDIX C

Randomization of Animals in Experiment 2

1 ANIMAL ID 1 NAME 1 SEX 1 DRUG ORDER 1 TEST ORDER 1 Clyde 1 Male 1 Placebo First 1 Size First SSB-8 Denny Male Placebo First 1 Intensity First DXI-8 Eve Female Placebo First Intensity First OCU-8 Flash Female Adrafini l First Intensi ty First ZAE-9 Genn y Female Placebo First Size First QJD-O Homer Male Adrafinil Fin? Intensity Fint HBE-O Ig gy Female Adrafinil First Size First B IE-9 Jewels Female Adrafinil First Size First