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Pharmacological Challenges of an Animal Model of Self-Injurious Behavior

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PAGE 1

PHARMACOLOGICAL CHALLENGES OF AN ANIMAL MODEL OF SELFINJURIOUS BEHAVIOR By AMBER M. MUEHLMANN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Amber M. Muehlmann

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This document is dedicated to my parents, Richard and Susan Muehlmann, my brother, Aaron Muehlmann, and my loving family in Ga inesville, Florida, Nicholas, Shelby and Maximus Van Matre. Your love and support has allowed me to complete this work in only two years. Thank you.

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iv ACKNOWLEDGMENTS I would like to thank my committee member s, Dr. Mark Lewis, Dr. Andy Shapira and Dr. Timothy Vollmer, for their time, as well as Dr. George Casella for all of his help with the statistical analyses. I would especi ally like to thank my advisor, Dr. Darragh Devine, for his guidance throughout the development and completion of these projects. I also wish to thank my labmates, both past and present, who have helped tremendously with these experiments.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT....................................................................................................................... ..x CHAPTER 1 INTRODUCTION........................................................................................................1 2 METHODS.................................................................................................................12 Animals.......................................................................................................................1 2 Drugs.......................................................................................................................... .12 Experimental Procedures............................................................................................13 Drug Treatments-Experime nt 1: Risperidone.....................................................13 Drug Treatments-Experiment 2: Valproate.........................................................13 Drug Treatments-Experime nt 3: Nifedipine........................................................14 Drug Treatments-Experiment 4: Tramadol.........................................................14 Drug Treatments-Experiment 5: Memantine.......................................................14 Behavioral and Histological Asssays-All experiments...............................................14 Statistical Analyses.....................................................................................................16 3 RESULTS...................................................................................................................21 Experiment 1: Risperidone.........................................................................................21 Experiment 2: Valproate.............................................................................................28 Experiment 3: Nifedipine...........................................................................................35 Experiment 4: Tramadol.............................................................................................42 Experiment 5: Memantine..........................................................................................49 Inter-observer reliability.............................................................................................56 4 DISCUSSION.............................................................................................................57 LIST OF REFERENCES...................................................................................................69

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vi BIOGRAPHICAL SKETCH.............................................................................................79

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vii TABLE Table page 2.1 Injury score rating scale...........................................................................................20

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viii LIST OF FIGURES Figure page 3.1 Effects of risperidone on pemoline-induced self-injury...........................................23 3.2 Effects of risperidone on th e incidence of self-injury..............................................24 3.3 Effects of risperidone on the inducti on and maintenance of pemoline-induced self-injury.................................................................................................................24 3.4 Effects of risperidone on the duration of pemoline-induced self-injurious oral contact......................................................................................................................25 3.5 Effects of risperidone on the inducti on and maintenance of pemoline-induced self-injurious oral contact.........................................................................................25 3.6 Effects of risperidone on the grooming, inactivity and locomotion.........................26 3.7 Effects of risperidone on the health status of the rats...............................................27 3.8 Effects of valproate on pemoline-induced self-injury..............................................30 3.9 Effects of valproate on the incidence of self-injury.................................................31 3.10 Effects of valproate on the induction a nd maintenance of pemoline-induced selfinjury........................................................................................................................3 1 3.11 Effects of valproate on the duration of pemoline-induced self-injurious oral contact......................................................................................................................32 3.12 Effects of valproate on the induction a nd maintenance of pemoline-induced selfinjurious oral contact................................................................................................32 3.13 Effects of valproate on the gr ooming, inactivity and locomotion............................33 3.14 Effects of valproate on the health status of the rats..................................................34 3.15 Effects of nifedipine on pemoline-induced self-injury.............................................37 3.16 Effects of nifedipine on th e incidence of self-injury................................................38

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ix 3.17 Effects of nifedipine on the induction and maintenance of pemoline-induced self-injury.................................................................................................................38 3.18 Effects of nifedipine on the duration of pemoline-induced self-injurious oral contact......................................................................................................................39 3.19 Effects of nifedipine on the induction and maintenance of pemoline-induced self-injurious oral contact.........................................................................................39 3.20 Effects of nifedipine on the grooming, inactivity and locomotion...........................40 3.21 Effects of nifedipine on the health status of the rats................................................41 3.22 Effects of tramadol on pemoline-induced self-injury..............................................44 3.23 Effects of tramadol on the incidence of self-injury..................................................44 3.24 Effects of tramadol on the induction a nd maintenance of pemoline-induced selfinjury........................................................................................................................4 5 3.25 Effects of tramadol on the duration of pemoline-induced self-injurious oral contact......................................................................................................................45 3.26 Effects of tramadol on the induction a nd maintenance of pemoline-induced selfinjurious oral contact................................................................................................46 3.27 Effects of tramadol on the grooming, inactivity and locomotion.............................47 3.28 Effects of tramadol on the health status of the rats..................................................48 3.29 Effects of memantine on pemoline-induced self-injury...........................................51 3.30 Effects of memantine on the incidence of self-injury..............................................51 3.31 Effects of memantine on the induction and maintena nce of pemoline-induced self-injury.................................................................................................................52 3.32 Effects of memantine on the duration of pemoline-induced self-injurious oral contact......................................................................................................................52 3.33 Effects of memantine on the induction and maintena nce of pemoline-induced self-injurious oral contact.........................................................................................53 3.34 Effects of memantine on the grooming, inactivity and locomotion.........................54 3.35 Effects of memantine on the health status of the rats...............................................55

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x Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science PHARMACOLOGICAL CHALLENGES OF AN ANIMAL MODEL OF SELFINJURIOUS BEHAVIOR By Amber M. Muehlmann August 2005 Chair: Darragh P. Devine Major Department: Psychology Self-injurious behavior (SIB) is a devastating behavior di sorder that involves acts directed at a person’s own body and causes dama ge to skin and underlying tissues. These actions are often expressed in a stereotype d manner and include, but are not limited to, self-biting, head banging and se lf-punching. SIB is exhibite d by people with intellectual handicaps, particularly people with severe and profound intellectual impairment, and by people with several different congenital developmental disorders (e.g., Lesch-Nyhan syndrome, autism and Prader-Willi syndrome). Animal models of SIB have been developed using environmental restric tion, neurotoxins, and pharmacological manipulations. These models, in combination with post-mortem and in vivo clinical data, have provided evidence that monoaminergic di sregulation is an impor tant factor in the development and expression of SIB. Pemoline, an indirect monoamine agonist, produces stereotyped SIB in rats when administered at high doses. We have investigated the potential therapeutic effectiveness of five drugs (risperidone, valproate, nifedipine,

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xi tramadol and memantine) in this m odel of pemoline-induced SIB. These pharmacological challenges of the pemoline mode l were chosen in order to achieve four specific objectives. These objectives were to evaluate the predictive validity of the pemoline model, to test the generalizability of pharmacological interventions between several animal models of SIB, to investigat e the pharmacotherapeuti c potential of two of the drugs, and to further investigate the ne urobiological mechanisms that contribute to pemoline-induced SIB. Risperidone and valproate effectively decreased the occurrence of SIB in clinical trials, and they each decreased the occurrenc e of pemoline-induced SIB. Nifedipine blocked SIB in the 6-hydroxydopamine and Bay-K 8644 models, and it decreased the pemoline-induced SIB. We also investigated the pharmacotherapeutic potential of tramadol (a drug that atte nuates compulsive behaviors in obsessivecompulsive disorder and Tourette’s syndr ome) and memantine (a glutamate receptor antagonist that has shown promise in treatment of Alzheimer’s diseas e and other clinical disorders). These drugs did not significantly lessen the o ccurrence of pemoline-induced SIB. Each of these experiments also rev eals important new information regarding the neuronal changes that occur during chronic pemoline admini stration. These new findings will lead to future experiments on neurobiol ogical changes that produce SIB, and they may help to identify potential neurobiol ogical targets for new pharmacotherapies.

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1 CHAPTER 1 INTRODUCTION Self-injurious behavior (SIB) is a devast ating, maladaptive behavior disorder that is common in intellectually handicapped popul ations. The self-injurious actions are usually highly stereotypic ( Symons & Thompson, 1997 ), and they result in immediate or delayed damage to the skin or underlying tis sues. Self-injurers exhibit many different forms of SIB, including head banging ( Thompson & Caruso, 2002 ), self-biting ( Nyhan, 1968 ), skin-picking ( State et al., 1999 ), and self-punching ( Oliver et al., 1987 ); and although individual self-injurers usually exhibit stereotyped patterns of behavior that are directed at specific and gene rally invarian t body sites ( Bodfish et al., 1995 ), there is great diversity in the forms of self -injury within and across clin ical groups. These behaviors vary from mild self-injury producing bruises or calluses to severe self-injury leading to permanent tissue damage or tissue loss. In addition to the dire physical conse quences that self-injurers inflict upon themselves, these behaviors also limit social and cognitive development. SIB often results in exclusion from educational and soci alizing activities, and it interferes with all normal activities of daily living. SIB is hi ghly destructive for families and caregivers who live and work with self-injurer s, leading to increased stress ( Sarimski, 1997 ) and feelings of despair ( Bromley & Emerson, 1995 ). There are also significant costs to society (estimated at $3 billion in 1989), as self-injurers require a dditional resources in terms of specialized care a nd professional interventions ( NIH Consensus Development Conference Statement, 1989 ).

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2 SIB is positively correlated with the o ccurrence of non-injurious stereotypies and compulsions ( Bodfish et al., 1995 ) and has even been hypothesized as being a compulsive behavior in itself ( King, 1993 ). In fact, Powell and colleagues (1996) found that 46% of their self-injurious sample engage d in self-restraint in an apparent attempt to interrupt their self-injury – s uggesting that these individuals were resisting a compulsive need to self-injure. Unfortunately, these self-restraining behavi ors did not produce any decrease in the occurrence of SIB. SIB is also associated with stress, wher ein SIB increases in stressful situations (e.g., being around new people, being sick or when having restraints removed) ( Anderson & Ernst, 1994 ), and there is a disproportionately hi gh prevalence of SIB in disorders that involve abnormal amounts of distress ( Sovner & Fogelman, 1996 ; Lindauer et al., 1999 ). Estimates of the population prevalence of SIB range from 1.7% to 65.9% of the intellectually handicapped in general. Th ese estimates vary considerably because definitions of SIB are inconsistent; some studies include mild SIB whereas others only report the incidence of moderate to severe SIB (for review see Rojahn & Esbensen, 2002 ). Estimates also differ because indivi duals with severe or profound intellectual disabilities are more likely to self-injure than individuals with mild or moderate intellectual disabilities ( McClintock et al., 2003 ), and because individuals in institutions are more likely to self-injure than those who are not in institutions ( Eyman & Call, 1977 ). It is unclear, however, if th e greater severity of SIB in institutionalized populations is actually caused by the institutional environment, if it is because the more severely intellectually handicapped are more lik ely to live in an institution ( Eyman & Call, 1977 ), or if it is because the SIB is th e reason for institutionalization ( Eyman et al., 1972 ).

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3 SIB also presents as a ph enotypic trait of many specifi c congenital developmental disorders. Approximately 44% of individua ls with Cornelia de Lange syndrome selfinjure by head banging, self-s cratching and finger biting ( Berney et al., 1999 ). Among girls with Rett syndrome, 50% compulsively wring or bite their hands until there are lesions on the skin ( Sansom et al., 1993 ). Estimates of SIB in autism are more variable, as are all the characteri stics of autism. One study found mild SIB in 21.5%, moderate SIB in 17.1% and severe SIB in 14.6% of their autistic sample ( Baghdadli et al., 2003 ). Additionally, 80% of individuals with Prader-Willi syndrome will compulsively pick at their skin, leading to sores and infection ( Symons et al., 1999 ). SIB is almost always observed in Lesch-Nyhan syndrom e, but there have been rare cases where the expression of SIB has been delayed or non-existent ( Mitchell & McInnes 1984 ; Singh et al., 1986 ; Hatanaka et al., 1990 ; Adler & Wrabetz, 1996 ). The severity of their SIB is usually extreme and many individuals with Lesch-Nyha n syndrome exhibit self -injury that causes tissue loss and deformity of the hands and face. ( Nyhan, 1968 ; Anderson & Ernst, 1994 ). The biological basis of Lesch-Nyhan syndr ome is any single point mutation in the HPRT enzyme, which renders the enzyme completely inactive. The biological consequences of that deficiency that lead to SIB are not understood. There is disregulation in a variety of neurotransmitter systems, and most studies have indicated a prominent disregulation of dopamine. Studies of post-mortem brai n tissue have shown a significant loss of dopamine functioning in the nigrostriatal and mesolimbic dopamine terminals, as measured by decreases in dopamine content and functional activity of tyrosine hydroxylase and dopa decarboxylase ( Lloyd et al., 1981 ). Significant reductions in dopa decarboxylase activity and dopamine stor age have also been found in the caudate,

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4 putamen, frontal cortex and vent ral tegmental complex in an in vivo investigation using positron emission tomography (PET) imaging with a fluorodopa F 18 tracer ( Ernst et al., 1996 ). Using PET, Wong and colleagues (1996) found decreased binding of the radiolabeled dopamine transporter ligand, WIN-35,428, (50-63% reduction in the caudate and 64-75% in the putamen) in individuals with Lesch-Nyhan syndrome. This indicates a significant loss of dopaminergic nerve terminal s. There is also an upregulation of D1 and D2 receptors in the striatum ( Saito et al., 1999 ), suggesting post-synaptic supersensitivity for dopamine in the striata of individu als with Lesch-Nyhan syndrome. Reduced concentrations of the dopamine metabolite, homovanillic acid (HVA), have also been seen in cerebrospinal fluid ( Jankovic et al., 1988 ). Increased concentrations of serotonin and the serotonin metabolite, 5-hydroxyindoleacetic acid (5-H IAA), have also been found in the putamen ( Lloyd et al., 1981 ). There is evidence that decreased dopamine functioning may cause increases in serotonin levels in the brain ( Mrini et al., 1995 ). Marked decreases in caudate, putamen and cerebral volume in individuals with LeschNyhan syndrome have also been seen using magnetic resonance imaging ( Harris et al., 1998 ). Overall, these data indicate that there is profound disregulation of monoamine systems in the brains of Lesch-Nyhan syndrome, and that nigrostriatal and mesocorticolimbic dopamine neurotransmission ma y play a particularly important role in the etiology and expression of self-injury. Many drugs have been prescribed to help reduce the incidence of SIB in clinical populations. Unfortunately, no one medicati on, or class of medications, has proven effective for all patients. This suggests that these medications may not be able to correct the behavioral overlay (e.g., escape from a dema nding task) that is a ssociated with the

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5 SIB. Clinical trials with typical neurol eptics such as haloperidol have produced inconsistent results. Some studies report successful a ttenuation of SIB ( Janowsky et al., 2005 ) and some studies report failures to decrease SIB ( Mace et al., 2001 ). Interestingly, Durand (1982) found that neither haloperidol nor mild punishment reduced a case of severe SIB, but a combination of haloperidol and behavioral intervention did significantly decrease the occurrence of SIB. This sugge sts that the effectiven ess of pharmacological treatment in human self-injur ers may be complicated by e nvironmental conditions that influence the expression of the behavior diso rder. Clinical trials with fluoxetine, a selective serotonin reuptake inhi bitor (SSRI), have also produced inconsistent results. In a blind, placebo-controlled experiment usi ng fluoxetine in children with Obsessive Compulsive Disorder, King and colleagues (1991) reported an emergence of both SIB and obsessive self-injurious ideations in six children, four of whom had to be hospitalized. Decreased SIB with fluoxetine treatment has also been reported (e.g., Ricketts et al., 1993 ), but many of those trials have not included placebo control or have not used blind observers to measure the depe ndent outcomes. In a ddition, naltrexone, an opioid antagonist, has produced conflicting results in its effectiveness to reduce SIB in clinical trials. There ar e reports that naltrexone produced increases in SIB ( Benjamin et al., 1995 ), decreases in SIB ( Symons et al., 2001 ) and no effects on SIB ( WillemsenSwinkels et al., 1995 ). Valproate, an indirect GABA agonist (that also has actions in other systems), has also reduced the incidence of SIB in small clinical trials with autistic ( Hollander et al., 2001 ) and intellectually handicapped individuals ( Kastner et al., 1993 ). Risperidone, an atypical neuroleptic that affects multiple monoaminergic systems, has had the most consistent effects across differe nt patient groups in reducing the amount of

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6 aggression, directed at both th e self and others, in children and adults with Lesch-Nyhan syndrome ( Allen & Rice, 1996 ), autism ( McCracken et al., 2002 ; Caicedo & Williams, 2002 ) and mental retardation ( Cohen et al., 1998 ). In summary, the results of these clinical trials have provided evidence of th e involvement of dopaminergic, serotonergic, opioid and GABAergic systems in clinical SIB, and these data further suggest that there are sub-groups of self-injur ers, who may respond differen tly to different kinds of pharmacotherapy. Animal models of SIB have also provid ed important information regarding the neurobiological basis of SIB. These an imal models include neonatal lesions, environmental deprivation and pharmacologi cal manipulations. In one model, 6hydroxydopamine (6-OHDA) is used to lesion st riatal dopamine neurons in neonatal rat pups. When these lesioned rats become adults they begin to self-injure after administration of either dire ct or indirect dopamine agoni sts (e.g., apomorphine, l-dopa), which affect multiple dopamine receptors ( Breese et al., 1984b ; Breese et al., 1984a ). Furthermore, agonists that are effective only at the D1 class of dopamine receptors (e.g., SKF 38393) will effectively induce SIB, whereas D2-selective agonists do not induce SIB in the 6-OHDA model ( Breese et al., 1985 ). Additionally, D1 antagonists block the SIB ( Breese et al., 1985 ). It has been hypothesized, therefore, that SIB in this model is due to a supersensitivity at the D1 receptors. This is consistent with evidence of a dopamine supersensitivity in individuals with Lesch-Nyhan syndrome ( Saito et al., 1999 ). Risperidone and nifedipine (a n L-type calcium channel bl ocker, which decreases the amount of dopamine released in the caudate ( Okita et al., 2000 ) have lowered the incidence of SIB in the 6-OHDA model ( Blake et al., 2004 ). In summary, the 6-OHDA

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7 model provides further evidence of dopamine’s important role in SIB. It is unclear if altered functioning of other neurotransmitter systems may also contribute to the induction of SIB in the 6-OHDA model. Early environmental and maternal depriv ation of non-human primates has been found to produce abnormal behaviors incl uding stereotyped locomotion, abnormal socialization and SIB ( Harlow & Harlow, 1962 ). The occurrence of whole-body stereotypies and the severity of SIB increase with an apomorphine challenge ( Lewis et al., 1990 ), which suggests dopamine supersensitivity. These changes in dopaminergic functioning are similar to those found in i ndividuals with Lesch-Nyhan syndrome and provide further evidence that dopamine disregul ation is an important contributor to SIB. There is also a significant decrease in th e density of immunoreact ivity for tyrosine hydoxylase, substance P and leucine-enkephalin in the striatum and related basal ganglia regions in rhesus monkeys with a history of social isolation ( Martin et al., 1991 ). This suggests that early environmen tal deprivation directly aff ects the development of the dopaminergic and peptidergic systems in the striatum. Once again, this resembles the striatal disorganization seen in Lesch-Nyhan syndrome ( Wong et al., 1996 ; Harris et al., 1998 ). A variety of different classe s of pharmacological manipulations have been used to induce self-injury in animals. Bay-K 8644, an L-type calcium ch annel agonist, causes dose-orderly expression of self-biting in mice ( Jinnah et al., 1999 ; Jinnah et al., 2003 ). Moreover, Bay-K 8644induced SIB is elim inated by administration of nifedipine ( Jinnah et al., 1999 ), which demonstrates that BayK 8644-induced SIB is specifically due to actions on the L-type cal cium channels. Injections of Bay-K 8644 directly into the

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8 striatum produce significant in creases in dopamine release in a dose dependent fashion ( Jinnah et al., 1999 ). Additionally, administration of fluoxetine ( Kasim et al., 2002 ) and amphetamine ( Kasim & Jinnah, 2003 ) (serotonin and dopamine agonists, respectively) each increase Bay-K 8644-induced SIB and ad ministration of drugs that antagonize serotonin ( Kasim et al., 2002 ) or dopamine decrease Bay-K 8644-induced SIB ( Kasim & Jinnah, 2003 ). These results demonstrate that Ba y-K 8644-induced SIB, like SIB seen in clinical populations, is associated with changes in dopaminergic and serotonergic neurotransmission. Caffeine, a non-selective adenosine recepto r antagonist, has also been reported to induce SIB when administered re peatedly at very high doses ( Miana et al., 1984 ). However, it was recently reported that the ca ffeine-induced SIB is not dose orderly, that the self-injury is extremely mild and only seen in a small percentage of the animals, and the required doses are highly toxic ( Kies & Devine, 2004 ). GBR-12909, an indirect dopami ne agonist that blocks the dopamine transporter and the uptake of dopamine into synaptic vesicles, produces doseand time-orderly induction of SIB in rats. GBR-12909-induced SIB is bl ocked by 6-OHDA lesions of nigrostriatal dopaminergic neurons, which suggests GBR-12909 produces SIB by altering presynaptic dopamine (for review see Sivam, 1996 ). This is consistent with evidence of altered presynaptic dopamine functioning in individuals with Lesch-Nyhan syndrome ( Lloyd et al., 1981 ). Pemoline, an indirect monoamine a gonist, has also been used as a pharmacological model of SIB ( Genovese et al., 1969 ; Mueller & Hsiao, 1980 ). Chronic administration of moderately high doses of pemoline produces dose-orderly self-injury in

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9 a large majority of rats in a few days time ( Kies & Devine, 2004 ). SIB in this model is usually directed towards the forepaws and a bdomen, but is occasionally directed at the hindpaws and tail ( Mueller & Hsiao, 1980 ; Kies & Devine, 2004 ). Pemoline-induced SIB is potentiated by impoverished envir onmental conditions during development ( Kies et al., 2002 ) and by stress exposure ( Kies et al., 2004 ). This is consistent with characteristics of clinical SIB, which is mo re prevalent in environmentally impoverished conditions, such as institutions than it is in community-based populations ( Eyman et al., 1972 ; Eyman & Call, 1977 ), and is commonly expressed in stressful situations ( Anderson & Ernst, 1994 ). An examination of brain structur es affected by pemoline administration, using an assay for cytochrome oxidase (t he end product of the mitochondrial electron transport chain and a marker of on-going neur onal activity) indicated that there is a significant pemoline-induced down-regulation of neuronal activity in the caudate nucleus, septum, bed nucleus of the stria terminalis hippocampus, periaqueductal grey and some hypothalamic nuclei ( Kies & Devine, 2003 ). These results suggest that the pemoline acts upon the dopaminergic nigrostriatal and me solimbic pathways, and that there is significant indirect impact on a variety of lim bic structures that ar e known to participate in processing of emoti onally-relevant stimuli ( Herman et al., 1996 Herman & Cullinan 1997 ). Disregulation of the nigrostriatal dopami ne pathway is strongly implicated in clinical populations in which SIB is manifested and negative affect and SIB are highly correlated in some individuals with intell ectual handicaps (as prev iously discussed) ( Lindauer et al., 1999 ). In addition to dopaminergic ac tions, there is evidence that other neurotransmitter systems are involved in pemoline-induced SIB. Specifically, King and colleagues (1995) have found that pemoline-indu ced SIB is attenuated by MK-801

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10 administration, which suggests glutamatergic in volvement. Additionally, paroxetine (an SSRI) significantly potentiated pemoline-induced SIB, suggesting a role of serotonin in the pemoline model ( Turner et al., 1999 ). Based on the results from these investiga tions of SIB using the pemoline model, we have begun to further characterize the m odel in rats by pharmacologically challenging the induction of SIB with five specific drugs. Those drugs are risp eridone (Risperdal), valproate (Depakote), nifedipine, tramadol (Ultram) and memantine (Namenda). We have examined the predictive validity of the pemoline model by investigating the effectiveness of two drugs that have been cl inically useful in re ducing SIB, risperidone and valproate. Additionally, we have examined the eff ectiveness of nifedipine, an L-type calcium channel antagonist, to lessen pemolineinduced SIB. Nifedipine has been used to lower the incidence of self-injury in the 6-OHDA model and to decrease Bay-K 8644induced SIB (as previously discussed). The pur pose of this investigation is to evaluate the generalizability between an imal models as this may help to reveal whether common neurobiological mechanisms contribute to th e induction and expression of SIB in the various animal models. Commonality in th ese models may be useful in further investigating the neurobiological basis of SIB. In all of these drug challenges, we are also considering the specific biological mechanisms that are acted upon by the drug challenges in order to further evaluate th e neurobiological mechanisms that underlie pemoline-induced SIB. We have also evaluated the pharmacotherap eutic potential of two drugs that have not previously been assessed in clinical populations, using the pemoline model. Since

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11 SIB is a behavior disorder that appears hi ghly stereotypic and compulsive in clinical populations ( Bodfish et al., 1995 ; King, 1993 ) and in the pemoline model, we investigated the effectivene ss of tramadol to reduce the incidence of pemoline-induced SIB. Tramadol is a low affin ity mu-opioid receptor agonist ( Dhasmana et al., 1989 ), which also blocks reuptake of serotonin ( Driessen & Reimann, 1992 ) and norepinephrine ( Driessen et al., 1993 ). It has been shown to reduce the amount of compulsive behaviors in individuals with Obsessive Compulsive Disorder (OCD) ( Goldsmith et al., 1999 ) and to reduce the amount of mo tor tics in individuals w ith Tourette’s syndrome ( Shapira et al., 1997 ). Additionally, we evaluated the effectiv eness of memantine, a non-competitive NMDA receptor antagonist, to lessen SIB in the pemoline model. MK-801, another noncompetitive NMDA receptor antagonist with high affinity, blocks SIB in the pemoline model ( King et al., 1995 ). MK-801, however, cannot be used as a clinical pharmacotherapy because of its psychotomimetic side effects ( Koek et al., 1988 ). Memantine lacks these side effects because it has a lower affinity for the NMDA receptor, and was recently approved by the FDA for use in Alzheimer’s patients ( Molineuvo et al., 2005 ). In light of the effects of MK-801, we hypothesized that memantine could be clinically effective for treatment of clinical SIB.

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12 CHAPTER 2 METHODS Animals Male Long Evans (LE) rats weighi ng 225-275 grams were housed in an AAALAC-approved, climate controlled vivarium. The rats were maintained on a 12hour light/dark schedule with lights on at 7 am. Standard laboratory rat chow (Lab Diet 5001) and tap water were available ad libitum The rats were pair-housed in standard polycarbonate cages (43 x 21.5 x 25.5 cm) during 5-7 days of acclimation to the housing facility. After the acclimation period the rats were singly-housed in similar polycarbonate cages. All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and all procedures were pre-approved by the Institutional Animal Care and Use Committee at the University of Florida. Drugs Pemoline (Spectrum Chemicals, New Bruns wick, New Jersey) was suspended at a concentration of 50 mg/ml in warm peanut oi l (held at approximately 36 Celsius), with constant stirring. Risperidone was purchased from Sigma-Aldrich Co. (St. Louis, Missouri) and was suspended in a so lution of 0.45% (w/v) hydroxypropyl-betacyclodextrin. The risperidone was suspe nded at concentrations of 0, 0.1, 0.5 and 1.0 mg/ml. Sodium valproate was purchased fr om Sigma-Aldrich Co. and was suspended in a solution of 0.04% (w/v) Na2EDTA. The valproate was susp ended at concentrations of 0, 50, 100 and 200 mg/ml and was adjusted to a neutral pH of 7.4. Nifedipine was

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13 purchased from Sigma-Aldrich Co. and was su spended in a solution consisting of 40% propylene glycol (v/v), 10% ethanol (v/v), 15% benzyl alcohol (v/v), 5% sodium benzoate (w/v) and approximately 35% distille d water (v/v). Nife dipine was suspended at concentrations of 0, 1.5, 5 and 15 mg/ml. Tramadol hydrochloride was purchased from Sigma-Aldrich Co. and was dissolved in ster ile saline at concentrations of 0, 1.0, 10 and 100 mg/ml. Memantine was purchased from Sigma-Aldrich Co. and was dissolved in sterile saline at concentra tions of 0, 3, 10 and 30 mg/ml. Experimental Procedures Drug Treatments-Experiment 1: Risperidone Twenty-three male LE rats (Charles River Laboratories, Raleigh, NC) were weighed and injected with pemoline (200 mg/kg s.c.) at approximately 8:00 a.m. on each of five consecutive days. These injections were administered at the nape of the neck and either flank on a rotating basis, using 21 gauge ne edles. The rats were also injected twice daily with risperidone (0, 0.1, 0.5 or 1.0 mg/kg, i .p.) on each of the five days (n = 5-6 per group), using 26-gauge needles. The risper idone injections were administered at approximately 8:00 am (immediately after the pemoline injection) and approximately 6:00 pm. Drug Treatments-Experiment 2: Valproate Thirty-six male LE rats (Charles River La bs) received daily pemoline injections at 200 mg/kg and twice-daily injections of valp roate (0, 50, 100 or 200 mg/kg, i.p.) for five days (n = 9 rats per group), following the same procedures as in Experiment 1.

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14 Drug Treatments-Experiment 3: Nifedipine Twenty-three male LE rats (Charles River Labs) received daily pemoline injections at 200 mg/kg and tw ice-daily injections of nife dipine (0, 3, 10 or 30 mg/kg, s.c.) for five days (n = 6 rats per group), fo llowing the same procedures as in Experiment 1. Drug Treatments-Experiment 4: Tramadol Seventy-two male LE rats (Harlan Inc., Indianapolis, Indiana) received daily pemoline injections at 200 mg/kg and twice-da ily injections of tramadol (0, 0.1, 1.0 or 10 mg/kg, s.c.) for five days (n = 18 rats per group), following the same procedures as in Experiment 1. Drug Treatments-Experi ment 5: Memantine Twenty-two male LE rats (Charles Rive r Labs) received daily pemoline injections at 200 mg/kg and twice-daily injections of me mantine (0, 3, 10 or 30 mg/kg, i.p.) for five days (n = 5-6 rats per group). The injection pr ocedures were similar to the procedures in Experiment 1 except that memantine was admi nistered 30 minutes before pemoline each day, and then again at approximately 6:00 p.m. Behavioral and Histological Assays All Experiments The rats were visually inspected each tim e they were injected (i.e. twice per day for five days), and the inspections were videotaped. These inspections were also performed on the morning of the sixth day, but no injections were administered on the sixth day. The rats were held in front of a video camera and the head, forepaws, hindpaws, ventrum and tail were displayed. An injury score (see Table 1 ) was assigned to describe the presence (or absence) and sever ity of all injuries. An observer blind to the

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15 drug treatment independently re-scored the injuries from the vi deotapes, and interobserver reliability was assessed. After the morning injections of pemolin e and challenge drug (i.e. risperidone, valproate, nifedipine, tramadol or memantin e) each rat was placed back into its home cage. A locomotor monitor (San Diego Inst ruments, San Diego, CA) was then raised around each cage in order to measure the locomo tor activating (or inhi biting) effects of the pemoline and challenge drug. Each locomo tor monitor had four LED sensors spaced along the length of the cage. A locomoto r count was recorded each time the rat interrupted a photocell sensor, and then no furt her counts were recorded from that sensor until the rat interrupted another photocell se nsor. Accordingly, each locomotor count represented actual movement across the cage (approximately 3.5 inches) rather than repetitive interruption of any single phot ocell sensor. The rats’ locomotion was monitored for two hours after the injections each morning. A variety of behaviors were recorded us ing night-vision cameras, where a camera was focused on the cage of each rat each night. Five-minute time samples were recorded once per hour for eight hours each night. Thes e recordings were scored for duration of self-injurious oral contact, duration of gr ooming, duration of inactiv ity and the amount of locomotion. Self-injurious oral contact was de fined as all oral contact that stayed fixed on any one body part for longer than two second s. Grooming was defi ned as oral contact with any part of the body that continued to move from site to site on the body (e.g., oral contact with the paws, then moving up each arm and continuing to the ventrum, in which the contact was not sustained in any spot on the body for longer than two seconds). Inactivity was defined as complete lack of movement except respiratory movements.

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16 Locomotion was counted by sectioning the cage into three equal parts (along the length of the cage) and tallying the number of times the rat entered into a different section without returning to the secti on that he occupied immediat ely prior to that movement. On the morning of the sixth day each ra t was inspected and assigned an injury score. Immediately after this inspection the rat was rapidly decapitated. Each rat’s brain was removed and rapidly frozen in 2-methylbut ane at -40C and later stored at -80C. These brains are being retained for histoc hemical analyses to compare a variety of potential neurochemical differences between self-injurious and noninjurious rats that were treated with the pemoline and challenge drugs. These analyses are not included in this thesis. The thymus a nd adrenal glands were also removed, frozen on dry ice and stored at -80C. These glands were later we ighed in order to determine the health status of the rats. Statistical Analyses The induction of self-injury was determin ed by graphing the injury scores of each rat across the six days of the experiment. The onset of SIB was defined for each experiment at the time the first rat in that ex periment exhibited self -injury (i.e. received an injury score of 1 or more). A linear re gression line was then plotted from the time of onset to the time when injury scores began to asymptote, and the slope of that line was determined for each rat. Between groups diffe rences in the slopes of the regression lines were then compared using a one-way anal ysis of variance (ANOVA). All significant effects were further analyzed with Fisher’s Least Significant Diffe rence (LSD) post-tests for each experiment.

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17 The maintenance of the self-injury was compared between the groups by taking the mean of the injury scores that were recorded for each ra t during the final days of the experiment (i.e. all days after the asymptot e was reached). On those days, the injury scores leveled off or began to decline in most groups. A one-way ANOVA was used to compare the mean injury scores for the rats in each treatment group in each experiment. All significant effects were further an alyzed with Fisher’s LSD post-tests. The percentage of time that the rats e xhibited self-injurious oral contact (data scored from the 40 minutes of videotaped r ecordings for each night) was used to quantify the behavioral expression of self-injury. Th e induction of self-injur ious oral contact was assessed from the first night until the night that the most self-injurious group reached the peak mean duration of self-injurious oral co ntact. The duration of self-injurious oral contact data were then transf ormed by calculating the square root of the percentage of time spent with self-injurious oral contact to equalize the variability between the treatment groups (see below). A linear regres sion line was then plotted from the first night to the night when the duration of self-injurious or al contact peaked, using the square root transformed scores, and the slope of that line was determined for each rat. Between groups differences in the slopes of the regression lines were then compared using an ANOVA for each experiment. All signifi cant effects were further analyzed with LSD post-tests. The maintenance of the self-injurious oral contact was compared by taking the mean of the transformed percentage of oral contact data for the final nights of the experiment, when the percent of self-injurious oral contact duration started to decline in the groups that were treated with pemoline a nd vehicle. The mean of the transformed

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18 duration of self-injurious oral contact was determined for each rat. These data were compared for each treatment group using a one-way ANOVA. All significant effects were further analyzed with LSD post-tests. The dose of pemoline (200 mg/kg/day) that was used in these experiments induces self-injury in approximately 75% of th e rats. Consequently, there was substantial variability of the duration of self-injurious oral contact within each treatment group. In fact, in each experiment we observed some rats with self-injurious oral contact for 100% of the time sampled and other rats, in the same treatment group, with no self-injurious oral contact whatsoever (see Results). Acco rdingly, square root tr ansformations of the duration of self-injurious or al contact were used to c ontrol for this variability ( Ott & Longnecker, 2001 ). The power of the statistical analys es of injury scores and oral contact duration was also diminished by the fact that the range of doses fo r the challenge drugs included doses that were ineffective at re ducing the different measures of pemolineinduced SIB. In these cases, the statis tical outcome was conf ounded by the ineffective dose(s). We therefore set the a cceptable alpha error level at 0.1 for all analyses of injury scores and oral contact durations. The duration of grooming behavior, in activity, and the amounts of overnight locomotion and post-injection locomotion (m easured by the locomotor monitors) were compared for each group using 4x5 (group x day) repeated measures ANOVAs. All significant effects were further analyzed with LSD post-tests. Body weights were analyzed by two-way (4 groups x 5 days) repeated measures ANOVA. Glandular weight s were compared using a one-way ANOVA for each experiment. All significant effects were further analyzed with LSD post-tests.

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19 Some rats were euthanized before the e nd of the experiment because they had an injury score of 4 (open lesion or amputated di git). In these cases, the missing data were replaced by repeating the final score that was attained for each dependent measure through the end of the experiment. This strate gy was used to avoid the potential that the group means would underestimate the injury scores and self-injurious oral contact scores, and to avoid the potential that the gro up means would overor under-estimate the locomotor, inactivity, and grooming scores wh en the most severe self-injurers were removed from any group. The prevalence of pemoline-induced SIB is dose-dependent ( Kies and Devine, 2004 ), and we chose to work with a dose (200 mg /kg/day) that would induce SIB in some but not all of the rats. Accordingly, the variab ility in expression of SIB is high in all the groups of rats, including the c ontrol (pemoline + vehicle) gro ups. Furthermore, the doses of challenge drugs included ineffective doses in most of the experiments. These factors decreased the power of our ANOV As to identify effective trea tments. In light of these statistical issues, treatment-associated decrease s in injury scores and self-injurious oral contact scores were treated as statistically re liable when the p-values were less than 0.10. Between-groups differences in all other depende nt measures were treat ed as statistically reliable when the p-values were less than 0.05.

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20 Table 2.1. Injury score ra ting scale (adapted from Turner et al., 1999 ). Score Classification Description 0 no self-injury 1 very mild self-injury denuded skin, edema or erythema; involves small area 2 mild self-injury denuded skin, edema or erythema; involves medium area or multiple small sites 3 moderate self-injury denuded skin, edema or erythema; involves large area or multiple medium sites 4 severe self-injury open lesion, amputated digit. Requires euthanasia.

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21 CHAPTER 3 RESULTS Experiment 1: Risperidone In the experiment with risperidone, ther e were no signs of self-injury until the second night during the pemoline treatment. On the morning of day 3, mild injury was observed in one rat in the group that was trea ted with pemoline plus vehicle, and in one rat that was treated with pemoline plus the medium dose (0.5 mg/kg) of risperidone. The injury scores of the vehicle-treated rats reached an asymptote around the morning of day 4 (see Fig. 3.1 ). During the experiment, two rats were assigned an injury score of 4, and both were in the vehicle-treated group. One was put down on day 4; the other reached an injury score of 4 on day 6 and was terminated with the other rats. By the final morning of the experiment, 5 of the 6 vehicle-treated rats exhibited injury scores of 1 or higher (i.e. had self-i nduced tissue damage). Fewer rats in each of the risperidone-treated groups exhibited injury scores of 1 or higher, and this effect was dose-orderly. The prevalence of positive in jury scores throughout the experiment is depicted in Fig. 3.2 The rats that were treated with risperidone exhibited lo wer injury scores than the vehicle-treated rats did, and th is effect was dose-orderly ( Fig. 3.1 ). The induction of selfinjury (i.e. the slope of the in jury scores starting with the onset on day 3 to the asymptote on day 4) occurred at a significan tly lower rate in all the ri speridone-treated groups, than in the vehicle-treated group (F(3, 22) = 7.340, p< 0.01; Fig. 3.1 and Fig. 3.3a ). The mean injury scores during the maintenance phase (i.e the asymptotic expression from day 4 to

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22 day 6) were significantly lower in the risperidone-treated ra ts than they were in the vehicle–treated rats, and this e ffect was roughly dose-orderly (F(3,22) = 5.276, p< 0.01; Fig. 3.1 and Fig. 3.3b ). The duration of self-injurious oral c ontact was also significantly less in the risperidone-treated rats than it wa s in the vehicletreated rats ( Fig. 3.4 ). The induction of self-injurious oral contact (taken from night 1 until the peak on night 3) was significantly lower in the risperidone-t reated rats than in th e vehicle-treated rats (F(3, 22) = 4.380, p< 0.10; Fig. 3.4 and Fig. 3.5a ). The maintenance of the self -injurious oral contact (taken from night 3 until night 5) appeared to be lo wer in the risperidone-treated rats, but this effect did not reach st atistical significance (F(3,22) = 2.336, p> 0.10; Fig. 3.4 and Fig. 3.5b ). Risperidone did not significantly aff ect the duration of time spent grooming (F(12,76) = 0.7929, p> 0.05; Fig. 3.6a ). Although risperidone did significantly increase the duration of inactiv ity overnight (F(3,76) =3.393, p< 0.05; Fig. 3.6b ), there were no significant time or group by time interaction eff ects. The amount of locomotion recorded on videotapes overnight decreased significan tly across the days of the experiment (F(4,76) =10.67, p< 0.05; Fig. 3.6c ), but there were no significant between-groups differences, and there were no group by time interaction effect s. The amount of post-injection locomotion (counts taken from the photocell monitors each morning) was not significantly affected by risperidone treatment (F(12,76) = 0.9274, p> 0.05; Fig. 3.6d ). All the groups exhibited weight loss for the first four days, followed by a slight weight gain. There was a significant inte raction between group and time for body weight (F(15,95) =1.947, p< 0.05; Fig 3.7a ), wherein the risperidone-tr eated rats exhibited less

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23 weight loss than the vehicle-treated rats di d. Risperidone treatment, however, did not appear to impact the health of the rats. There were no significant between-groups differences in thymus (F(3,22) =2.630, p> 0.05; Fig. 3.7b ), or adrenal wei ghts (left adrenal: F(3,22) =.4862, p> 0.05; right adrenal: F(3,22) =.5737, p> 0.05; Fig. 3.7 c,d ). Fig. 3.1. Effects of risperidone on pemo line-induced self-injury: Risperidone dose-dependently delayed the onset of se lf-injury, with the highest dose of risperidone blocking the pemoline-induced self-injury until day 4. Risperidone also dose-dependently attenuated the severi ty of pemoline-induced self-injury, as measured by the injury scores. All values expressed are group means S.E.M. 0 1 2 3 4 5 6 0 1 2 3 40 mg/kg 0.1 mg/kg 0.5 mg/kg 1.0 mg/kg dayinjury score

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24 0 0.1 0.5 1.0 0.0 0.5 1.0 1.5 2.0 2.5* *dose of risperidone (mg/kg)induction of tissue damage(slope of regression lines of injury scores) 0 0.1 0.5 1.0 0 1 2 3* *dose of risperidone (mg/kg)maintenance of tissue damage(mean injury scores)A B 0 1 2 3 4 5 6 0 25 50 75 1000 mg/kg 0.1 mg/kg 0.5 mg/kg 1.0 mg/kg daypercentage of rats that exhibited injury Fig. 3.3. Effects of risper idone on the induction and maintenance of pemolineinduced self-injury: (a) Risperidone dos e-dependently reduced the induction of pemoline-induced self-injury. (b) Risperi done also dose-dependently reduced the maintenance of pemoline-induced self-injury. All values expressed are group means S.E.M. Significant differences be tween the pemoline plus vehicle and pemoline plus risperidone treated groups (L SD) are depicted with asterisks, where indicates p < 0.10. Fig. 3.2. Effects of risperi done on the incidence of self -injury: Risperidone dosedependently lowered the incide nce of self-injury, as meas ured by the percentage of rats that exhibited injury on day 6 of the experiment.

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25 Fig. 3.4. Effects of risperi done on the duration of pemolin e-induced self-injurious oral contact: Risperidone re duced the overall percent dura tion of self-injurious oral contact. Self-injurious oral contact p eaked on day 4 for most groups. All values ex p ressed are g rou p means S.E.M. Fig. 3.5. Effects of risper idone on the induction and maintenance of pemolineinduced self-injurious oral contact: (a ) Risperidone dose-dependently decreased the induction of pemoline-i nduced self-injurious oral contact. (b) Although it appeared that risperidone also reduced the maintenan ce of pemoline-induced selfinjurious oral contact in a dose-orderly manner, this effect was not statistically significant. All values expressed are group means S.E.M. Significant differences between pemoline plus vehicle and pemo line plus risperidone treated groups (LSD) are depicted with asterisks, where indicates p < 0.10. 0 0.1 0.5 1.0 0 5 10 15 20 25 30 35* *dose of risperidone (mg/kg)induction of self-injurious oral contact(slope of regression lines) 0 0.1 0.5 1.0 0 10 20 30 40 50 60 70dose of risperidone (mg/kg)maintenance of self-injurious oral contact(mean scores)A B 0 1 2 3 4 5 0 20 40 60 80 1000 mg/kg 0.1 mg/kg 0.5 mg/kg 1.0 mg/kg nightself-injurious oral contact(% duration)

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26 Fig. 3.6. Effects of risperidone on groo ming, inactivity and locomotion: (a) Risperidone did not significantly affect time spent grooming. (b) Risperidone-treated rats did exhibit more inactivity as compared to vehicle-treated rats. Significant differences between vehicleand risperidone-treated ra ts (LSD) are depicted as follows: p< 0.05 for comparisons between ri speridone at 1.0 mg/kg and vehicle. Risperidone had no significant effect on locomotion, either (c) overnight or (d) after injections. All values expressed are group means S.E.M. 0 1 2 3 4 5 0 50 100 150 200 2500 mg/kg 0.1 mg/kg 0.5 mg/kg 1.0 mg/kg nightgrooming (sec.) 0 1 2 3 4 5 0 250 500 750 1000 12500 mg/kg 0.1 mg/kg 0.5 mg/kg 1.0 mg/kg nightinactivity (sec.) 0 1 2 3 4 5 0 25 50 75 1000 mg/kg 0.1 mg/kg 0.5 mg/kg 1.0 mg/kg nightovernight locomotion(scores from videotape analysis) 0 1 2 3 4 5 0 250 500 750 10000 mg/kg 0.1 mg/kg 0.5 mg/kg 1.0 mg/kg daypost-injection locomotion(photocell crossings)AB C D

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27 Fig. 3.7. Effects of risperidone on the health status of the ra ts: (a) there was a significant interaction between time and group for body weig ht. Pemoline plus vehicle treated rats lost more weight than pemoline plus risperi done treated rats did. Significant differences between vehicleand risperidone-treated ra ts (LSD) are depicted as follows: p< 0.05 for comparisons between risperi done at 1.0 mg/kg and vehicle; p< 0.05 for comparisons between risperi done at 0.5 mg/kg and vehicle; # p< 0.05 for comparisons between risperidone at 0.1 mg/ kg and vehicle. Risperidone did not significantly affect the weights of the (b) thymus glands or (c,d) adrenal glands. All values expressed are group means S.E.M. 0 1 2 3 4 5 6 0 50 1000 mg/kg 0.1 mg/kg 0.5 mg/kg 1.0 mg/kg 200 225 250 275# # *# *daybody weight (g) 0 0.1 0.5 1.0 0 100 200dose of risperidone (mg/kg)thymus weight(mg/100g body weight) 0 0.1 0.5 1.0 0 5 10 15dose of risperidone (mg/kg)left adrenal weight(mg/100g body weight) 0 0.1 0.5 1.0 0.0 2.5 5.0 7.5 10.0 12.5dose of risperidone (mg/kg)right adrenal weight (mg/100g body weight)A B C D

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28 Experiment 2: Valproate In the experiment with valproate, there were no signs of self-injury during the first day or night of pemoline treatment. The onset of self-injury occurr ed on day 2, with a rat in the pemoline plus vehicle group exhibiting m ild injury. The onset of self-injury was further delayed in all the valp roate treated groups. The in jury scores of the vehicletreated rats reached an asymptot e around the morning of day 5 (see Fig. 3.8 ). Five rats were assigned an injury score of 4, two in the vehicle-treated group (one euthanized on day 4, the other on day 5), two rats in the lowe st dose (50 mg/kg) of valproate group (one euthanized on day 4, the other on day 5) a nd one in the medium dose (100 mg/kg) of valproate group (euthanized on day 4). By the final morning of the experiment, 8 of the 9 vehicle-treated rats exhibited injury scores of 1 or higher (i.e. had self -induced tissue damage). Fewer rats in the groups treated with the middle and high doses of valproate exhibited injury scores of 1 or higher, and this effect was dose-orderly. The prevalence of positive injury scores throughout the experiment is depicted in Fig. 3.9 The rats that were treated with valproate exhibited lower injury scores than the vehicle-treated rats did, and this effect wa s primarily observed at the highest dose of valproate ( Fig. 3.8 ). The induction of self-injury (i.e the slope of the injury scores starting with the onset on day 2 to the as ymptote on day 5) occurred at a significantly lower rate in the valproate-treated gro ups, than in the vehicle-treated groups (F(3,35) =2.593, p< 0.10; Fig. 3.8 and 3.10a ), and this effect was prim arily observed at the highest dose of valproate. The mean injury scor es during the maintenance phase (i.e. the asymptotic expression from day 5 to day 6) were significantly lower in the valproate-

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29 treated rats than they were in the vehicle-treated rats (F(3,35) =2.420, p< 0.10; Fig. 3.10b ), and this effect was seen primarily in the highest dose of valproate. In contrast to the effects of valproate on actual tissue da mage (i.e. injury scores), valproate administration did not significantly a ffect the duration of self-injurious oral contact ( Fig. 3.11 ). The induction of self-injurious oral contact (taken from night 1 until the peak on night 4) was not significan tly affected by valproate treatment (F(3,35) =.8013, p> 0.10; Fig. 3.12a ). The maintenance of self-injurious oral contact (taken from night 4 until night 5) was also not significantly reduced by valproate (F(3,35) =.3864, p> 0.10; Fig. 3.12b ). Valproate administration did not signifi cantly affect any of the other behaviors that were measured during the experime nt. Although the time spent grooming (F4,128) =5.378, p> 0.05; Fig. 3.13a ), or inactive (F(4,128) =3.784, p> 0.05; Fig. 3.13b ), and the amount of locomotion recorded on videotapes overnight (F(4,128) =20.19, p> 0.05; Fig. 3.13c ) decreased significantly across the days of the experiment, there were no significant between-groups differences a nd there were no group by time in teraction effects. Postinjection locomotion, recorded with the phot ocell monitors immediately after pemoline and valproate injections each morning, wa s not significantly affected by valproate treatment (F(3,35) =1.991, p> 0.05; Fig. 3.13d). All groups exhibited weight loss for th e first four days, followed by a slight weight gain. This weight loss was signi ficant across days of the experiment (F(5,160) =10.79, p< 0.05; Fig. 3.14a ). There were no significant between-groups differences or group by time interaction effects. Thymus weights were found to be significantly different between groups (F(3,35)=5.841, p< 0.05; Fig. 3.14b ), but no significant

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30 differences were found between the valproat e-treated groups and the vehicle-treated group. There were also no significant betw een-groups differences on adrenal weights (left: F(3,35) =.6223, p> 0.05; right: F(3,35) =.8762, p> 0.05; Fig. 3.14c,d ). Fig. 3.8. Effects of valproate on pemoline-i nduced self-injury: Valproate delayed the onset of self-injury. The two highes t doses of valproate (100 and 200 mg/kg) attenuated the severity of pemoline-induced self-injury, as measured by the injury scores. All values expressed are group means S.E.M. 0 1 2 3 4 5 6 0 1 2 3 40 mg/kg 50 mg/kg 100 mg/kg 200 mg/kg dayinjury score

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31 Fig. 3.10. Effects of valproate on the induction and maintenance of pemolineinduced self-injury: (a) Valproate reduced the induction of pemoline-induced selfinjury. (b) Valproate also reduced th e maintenance of pemoline-induced selfinjury. All values expressed are group means S.E.M. Significant differences between the pemoline plus vehicle and pemoline plus valproate treated groups (LSD) are depicted with asterisks, where indicates p< 0.10. 0 50 100 200 0.00 0.25 0.50 0.75 1.00 1.25*dose of valproate (mg/kg)induction of tissue damage(slope of regression lines of injury scores) 0 50 100 200 0 1 2 3*dose of valproate (mg/kg)maintenance of tissue damage(mean injury scores)A B Fig. 3.9. Effects of valproate on the incide nce of self-injury: The middle and high doses of valproate lowered the inciden ce of self-injury, as measured by the percentage of rats that exhibited injury on day 6 of the experiment. 0 1 2 3 4 5 6 0 25 50 75 1000 mg/kg 50 mg/kg 100 mg/kg 200 mg/kg daypercentage of rats that exhibited injury

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32 Fig. 3.11. Effects of valproate on the durat ion of pemoline-induced self-injurious oral contact: Valproate did not signifi cantly reduce the overall percent duration of self-injurious oral contact. Self-injuri ous oral contact peaked on night 4 for all groups. All values expressed are group means S.E.M. 0 50 100 200 0 1 2 3dose of valproate (mg/kg)induction of self-injurious oral contact(slope of regression lines) 0 50 100 200 0 1 2 3 4 5 6 7 8dose of valproate (mg/kg)maintenance of self-injurious oral contact(mean scores)A B Fig. 3.12. Effects of valproate on the induc tion and maintenance of pemoline-induced self-injurious oral contact: (a) Valproat e did not significantly affect induction of pemoline-induced self-injurious oral contac t. (b) Valproate also did not significantly affect the maintenance of pemoline-induced se lf-injurious oral contact. All values expressed are group means S.E.M. 1 2 3 4 5 0 20 40 60 80 1000 mg/kg 50 mg/kg 100 mg/kg 200 mg/kg nightself-injurious oral contact (% duration)

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33 Fig. 3.13. Effects of valproate on groomi ng, inactivity and locomotion: Valproate did not significantly affect (a) time spen t grooming or (b) time spent inactive. Valproate also had no significant effect on locomotion, either (c ) overnight or (d) after injections. All values expressed are group means S.E.M. 0 1 2 3 4 5 0 50 100 150 200 250 300 3500 mg/kg 50 mg/kg 100 mg/kg 200 mg/kg nightgrooming (sec.) 0 1 2 3 4 5 0 100 200 300 400 500 600 7000 mg/kg 50 mg/kg 100 mg/kg 200 mg/kg nightinactivity (sec.) 0 1 2 3 4 5 0 10 20 30 40 50 60 700 mg/kg 50 mg/kg 100 mg/kg 200 mg/kg nightovernight locomotion (scores from videotape analysis) 0 1 2 3 4 5 0 500 1000 1500 20000 mg/kg 50 mg/kg 100 mg/kg 200 mg/kg daypost-injection locomotion(photocell crossings)A B C D

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34 Fig. 3.14. Effects of valproate on the health stat us of the rats: (a) There was a significant effect of time on body weight, wherein all groups lost weight during the first four days of the experiment and then exhibi ted a slight weight gain. (b) Valproate did significantly affect the weights of the thymus glands, however, no significant differences were found between the valproate-treated groups and th e vehicle-treated group. (c,d) Valproate did not significantly affect the weights of the ad renal glands. All values expressed are group means S.E.M. 0 1 2 3 4 5 6 0 50 1000 mg/kg 50 mg/kg 100 mg/kg 200 250 300200 mg/kg Daybody weight (g) 0 50 100 200 0 100 200dose of valproate (mg/kg)thymus weight(mg/100g body weight) 0 50 100 200 0.0 2.5 5.0 7.5 10.0 12.5dose of valproate (mg/kg)left adrenal gland weight(mg/100g body weight) 0 50 100 200 0.0 2.5 5.0 7.5 10.0 12.5dose of valproate (mg/kg)right adrenal gland weight(mg/100g body weight)A B C D

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35 Experiment 3: Nifedipine In the experiment with nifedipine, th ere were no signs of self-injury until the second day during the pemoline treatment. On the afternoon of da y 2, mild injury was observed in one rat that was treated with pe moline plus the medium dose (10 mg/kg) of nifedipine. The injury scores of the vehicle-treated rats reached an asymptote around the morning of day 5 ( Fig. 3.15 ). During the experiment, thirteen rats were assigned an injury score of 4 and were euthanized before the end of the experiment. The rats in the vehicle-treated group exhibited ex tremely severe self-injury. In fact, all the rats in the vehicle-treated group received an injury score of 4 and were terminated early (one on day 3, four on day 4 and one on day 5). Three rats in the group treated with pemoline plus the low dose (3 mg/kg) of nifedipi ne reached an injury score of 4 (one on day 4 and two on day 6). One rat from the group treated with pemoline plus the middle dose (10 mg/kg) of nifedipine reached an injury score of 4 on da y 4 and three rats in the group treated with pemoline plus the high dose (30 mg/kg) of ni fedipine group were eu thanized early (one on day 3 and two on day 4). By the final morning of the experiment, al l rats exhibited injury scores of 1 or higher (i.e. had self-induced tissue damage) in the group treated with pemoline plus vehicle and the group treated with pemoline plus the low dose (3 mg/ kg) of nifedipine. All but one rat in each of the groups that we re treated with pemoline plus the middle (10 mg/kg) and high (30 mg/kg) doses of nifedipine also exhibi ted injury scores of 1 or higher. The prevalence of positive injury scor es throughout the experiment is depicted in Fig. 3.16 The rats that were treated with nifedipine exhibited lower injury scores than the vehicle-treated rats did ( Fig. 3.15 ). The induction of self-i njury (i.e. the slope of the

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36 injury scores starting with the onset on day 2 to the asymptote on day 4) occurred at a significantly lower rate with all the nifedipine-treated gr oups, than in the vehicle-treated group (F(3,22) =2.626, p< 0.10; Fig. 3.15 and Fig. 3.17a ). The mean injury scores during the maintenance phase (i.e. the asymptotic expression from day 4 to day 6) were significantly lower in the nifedipi ne-treated rats than they were in the vehicle-treated rats (F(3,22) =3.041, p< 0.10; Fig. 3.15 and Fig. 3.17b ). In contrast to the effects of nifedipine on actual tissue da mage (i.e. injury scores), nifedipine administration did not significantly affect the dura tion of self-injurious oral contact ( Fig. 3.18 ). The induction of self-injurious oral contact (taken from night 1 until the peak on night 3) was not significan tly affected by nifedipine treatment (F(3,21) =0.4573, p> 0.10; Fig. 3.18 and Fig. 3.19a ). The maintenance of self-injurious oral contact (taken from night 3 until night 5) wa s also not significantly reduced by nifedipine (F (3,22) =0.7444, p> 0.10; Fig. 3.18 and Fig. 19b ). Nifedipine did not signif icantly affect any of the other behaviors that were measured during the experiment. Time spent grooming (F(4,72) =4.676, p< 0.05; Fig. 3.20a ), inactive (F(4,72) =6.579, p< 0.05; Fig. 3. 20b ) and the amount of locomotion recorded on videot apes overnight (F(4,72) =23.94, p< 0.05; Fig. 3.20c ) or recorded with the photocell monitors immediatel y after pemoline and nifedipine injections each morning (F(4,76) =10.79, p< 0.05; Fig. 3. 20d ) all changed significantly across the days of the experiment, but no significant group by time interaction effects were found. All groups exhibited weight loss during the experiment. This weight loss was significant across days of the experiment (F(5,95) =86.06, p< 0.05; Fig. 3.21a ). There were no significant between-groups differences or group by time interaction effects.

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37 Nifedipine treatment significan tly affected thymus weights (F(3,21) =6.917, p< 0.05; Fig. 3.21b ), with the thymus glands of nifedipine -treated animals weighing significantly less than the glands of the vehicl e-treated animals did. Adrenal weights, however, were not different between groups (left: F(3,21) =.4816, p> 0.05; right: F(3,21) =1.095, p> 0.05; Fig. 3.21c,d ). Fig. 3.15. Effects of nifedipine on pemolineinduced self-injury: Nifedipine dose dependently attenuated the severity of pemoline-induced self-injury, as measured by the injury scores. Nifedipine did not delay the onset of pe moline-induced selfinjury. All values expressed are group means S.E.M. 0 1 2 3 4 5 6 0 1 2 3 40 mg/kg 3 mg/kg 10 mg/kg 30 mg/kg dayinjury score

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38 Fig. 3.16. Effects of nifedipine on the incide nce of self-injury: The highest doses of nifedipine lowered the incidence of se lf-injury, as measured by the percentage of rats that exhibited injury on day 6 of the experiment. Fig. 3.17. Effects of nifedipine on the induction and maintenance of pemolineinduced self-injury: (a) Ni fedipine reduced the inducti on of pemoline-induced selfinjury. (b) Nifedipine also decreased th e maintenance of pemoline-induced selfinjury. All values expressed are group means S.E.M. (Significant differences between pemoline plus vehicle and pemoline plus nifedipine treated groups (LSD) are depicted with asterisks, where indicates p< 0.10). 0 1 2 3 4 5 6 0 25 50 75 1000 mg/kg 3 mg/kg 10 mg/kg 30 mg/kg daypercentage of rats that exhibited injury 0 3 10 30 0 1 2 3* *dose of nifedipine (mg/kg)induction of tissue damage(slope of regression lines of injury scores) 0 3 10 30 0 1 2 3 4* *dose of nifedipine (mg/kg)maintenance of tissue damage(mean injury scores)A B

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39 Fig. 3.18. Effects of nifedipine on the durat ion of pemoline-induced self-injurious oral contact: Nifedipine di d not significantly reduce the overall percent duration of self-injurious oral contact. Vehicle-tr eated rats exhibited self-injurious oral contact for 100% of the night, beginning on night 3. All valu es expressed are group means S.E.M. Fig. 3.19. Effects of nifedipine on the induction and maintenance of pemolineinduced self-injurious oral contact: (a) Ni fedipine did not sign ificantly affect the induction of pemoline-induced self-injurious oral contact. (b) Nifedipine also did not significantly affect the maintenance of pemoline-induced self-injurious oral contact. All values expressed are group means S.E.M. 0 3 10 30 0 25 50dose of nifedipine (mg/kg)induction of self-injurious oral contact(slope of regression lines) 0 3 10 30 0 50 100dose of nifedipine (mg/kg)maintenance of self-injurious oral contact(slope of regression lines)A B 0 1 2 3 4 5 0 25 50 75 1000 mg/kg 3 mg/kg 10 mg/kg nightself-injurious oral contact(% duration)30 mg/kg

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40 0 1 2 3 4 5 0 100 2000 mg/kg 3 mg/kg 10 mg/kg 30 mg/kg nightgrooming (sec.) 0 1 2 3 4 5 0 100 200 300 400 500 600 7000 mg/kg 3 mg/kg 10 mg/kg 30 mg/kg nightinactivity (sec.) 0 1 2 3 4 5 0 25 50 750 mg/kg 3 mg/kg 10 mg/kg 30 mg/kg nightovernight locomotion(scores from videotape analysis) 0 1 2 3 4 5 6 0 250 500 750 1000 12500 mg/kg 3 mg/kg 10 mg/kg 30 mg/kg daypost-injection locomotion(photocell crossings) Fig. 3.20. Effects of nifedipine on grooming, in activity and locomotion: Nifedipine did not significantly affect (a) tim e spent grooming or (b) time spent inactive. Nifedipine also had no significant effect on locomotion, eith er (c) overnight or (d) after injections. All values expressed are group means S.E.M.

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41 Fig. 3. 21. Effects of nifedipine on the heal th status of the rats: (a) There was a significant effect of time on body weight, wher ein all groups lost we ight during the six days of the experiment. Nifedipine si gnificantly affected the (b) thymus gland weights. Significant thymus i nvolution was seen in all rats in the nifedipine-treatment groups. Nifedipine, however, had no significant effect on (c ,d) adrenal gland weights. All values expressed are group means S.E.M. (Significant differences between pemoline plus vehicle and pemoline plus nife dipine treated groups (LSD) are depicted with asterisks, where indicates p< 0.05). 0 1 2 3 4 5 6 0 50 1000 mg/kg 3 mg/kg 10 mg/kg 200 250 30030 mg/kg daybody weight (g) 0 3 10 30 0 50 100 150dose of nifedipine (mg/kg)* *thymus weight(mg)/ 100g body weight) 0 3 10 30 0 10 20dose of nifedipine (mg/kg)left adrenal gland weight (mg/100g body weight) 0 3 10 30 0 5 10 15dose of nifedipine (mg/kg)right adrenal gland weight(mg/100g body weight)

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42 Experiment 4: Tramadol In the experiment with tramadol, ther e were no signs of self-injury until the second day of pemoline treatment. On day 2, mild injury was observe d in one rat in the group treated with pemoline plus vehicle, and one rat that was treate d with pemoline plus the medium dose (1.0 mg/kg) of tramadol. The injury scores of the vehicle-treated rats reached an asymptote around the morning of day 4 ( Fig. 3.22 ). During the experiment, five rats were assigned an injury score of 4. Two rats in the ve hicle-treated group (one euthanized on day 2, the other on day 3), one ra t in the group treated with the lowest dose (0.1 mg/kg) of tramadol (euthanized on day 6), one rat in the group treated with the medium dose (1.0 mg/kg) of tramadol (eut hanized on day 4) and one rat in the group treated with the highest dose (10 mg/kg) of tramadol (euthanized on day 3) were assigned an injury score of 4. By the final morning of the experiment 12 of 18 vehicle-treated rats exhibited injury scores of 1 or higher (i.e. had self-i nduced tissue damage). More rats in each of the tramadol-treated groups exhibited positive injury scores than in the vehicle-treated group. The prevalence of positive injury scores throughout the experiment is depicted in Fig. 3.23 The rats that were treated with tramadol did not exhibit lower injury scores than did the vehicle-treated rats ( Fig. 3.22 ). The induction of self-i njury (i.e. the slope of the injury scores starting with the onset on day 2 to the asymptote on day 4) was not significantly affected by tramadol treatment (F(3,71) =1.532, p> 0.10; Fig. 3.22 and Fig. 3.24a ). The mean injury scores during th e maintenance phase (i.e. the asymptotic expression from day 4 to day 6) was also not significantly affected by tramadol treatment (F(3,71) =.7915, p> 0.10; Fig. 3.22 and Fig. 3.24b ).

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43 The duration of self-injurious oral contact was also not significantly altered in the tramadol-treated rats ( Fig. 3.25 ). The induction of self-injuri ous oral contact (taken from night 1 until the peak on night 3) was not si gnificantly affected by tramadol treatment (F(3, 42) = 1.942, p> 0.10; Fig. 3.25 and Fig. 3.26a ). The maintenance of self-injurious oral contact (taken from night 3 until ni ght 5) was also not significantly reduced by tramadol (F(3,42) =0.8608, p> 0.10; Fig. 3.25 and Fig. 3.26b ). Tramadol did not significantly affect the other behaviors that were measured during the experiment. The time spent grooming (F(4,164) =9.596, p< 0.05; Fig. 3.27a ), inactive (F(4,164) =5.113, p< 0.05; Fig. 3.27b ) and the amount of locomotion recorded on the videotapes overnight (F(4,164) = 7.454, p< 0.05; Fig. 3.27c ) or recorded with the photocell monitors immediatel y after pemoline and tramadol injections each morning (F(4,272) = 3.050, p< 0.05; Fig. 3.27d ) were significantly changed across days of the experiment. However, there were no signifi cant between-groups differences or group by time interaction effects for any of these behaviors. All groups exhibited weight loss during th e first four days of the experiment, followed by a slight weight gain. This wei ght loss was significant across days of the experiment (F(5,340) =22.90, p< 0.05; Fig. 3.28a ). No between-groups differences or interaction effects were significant. Tram adol treatment, however, did not appear to impact the health of the rats. There were no significant between-groups differences in thymus (F(3, 71) = 0.7867, p> 0.05; Fig. 3.28b ), or adrenal weights (left: F(3, 71) = 0.5434, p> 0.05; right: F(3, 71) = 1.016, p> 0.05; Fig. 3.28c,d ).

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44 Fig. 3.22. Effects of tramadol on pemolineinduced self-injury: Tramadol did not affect pemoline-induced self-injury, as meas ured by the injury scores. All values expressed are group means S.E.M. Fig. 3.23. Effects of tramadol on the incide nce of self-injury: Tramadol did not significantly affect the incidence of self-i njury, as measured by the percentage of rats that exhibited self-injur y on day 6 of the experiment. 0 1 2 3 4 5 6 0 1 2 3 40 mg/kg 0.1 mg/kg 1.0 mg/kg dayinjury score10 mg/kg 0 1 2 3 4 5 6 0 25 50 75 1000 mg/kg 0.1 mg/kg 1.0 mg/kg 10 mg/kg daypercentage of rats that exhibited self-injury

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45 Fig. 3.24. Effects of tramadol on induction and maintenance of pemolineinduced self-injury: Tramadol did not significantly affect the induction of pemoline-induced self-injury. (b) Tramadol also did not significantly affect the maintenance of pemoline-induced self-injury. All values expressed are group means S.E.M. Fig. 3.25. Effects of tramadol on the durat ion of pemoline-induced self-injurious oral contact: Tramadol did not significan tly affect the overall percent duration of pemoline-induced self-injurious oral contac t. Self-injurious oral contact peaked on day 3 for most groups. All va lues expressed are group means S.E.M. 0 0.1 1.0 10 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8dose of tramadol (mg/kg)induction of tissue damage (slope of regression lines of injury scores) 00.11.010 0.0 0.5 1.0 1.5 2.0 2.5dose of tramadol (mg/kg)maintenance of tissue damage (mean injury scores)A B 0 0.1 1.0 10 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8dose of tramadol (mg/kg)induction of tissue damage (slope of regression lines of injury scores) 00.11.010 0.0 0.5 1.0 1.5 2.0 2.5dose of tramadol (mg/kg)maintenance of tissue damage (mean injury scores)A B 0 1 2 3 4 5 0 25 50 75 1000 mg/kg 0.1 mg/kg 1.0 mg/kg nightself-injurious oral contact(% duration)10 mg/kg

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46 Fig. 3.26. Effects of tramadol on the i nduction and maintenance of pemolineinduced self-injurious oral contact: (a) Tr amadol did not significantly affect the induction of pemoline-induced self-injurious oral contact. (b) Tramadol also did not significantly affect the maintenance of pemoline-induced self-injurious oral contact. All values expressed are group means S.E.M. 0 0.1 1.0 10 0 10 20 30 40dose of tramadol (mg/kg)induction of self-injurious oral contact(slope of regression lines) 0 0.1 1.0 10 0 10 20 30 40 50 60 70dose of tramadol (mg/kg)maintenance of self-injurious oral contact(mean scores)A B

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47 Fig. 3.27. Effects of tramadol on grooming, in activity and locomotion: (a) Tramadol did not significantly affect time spent groom ing. (b) Tramadol di d significan tly alter the amount of time spent inactive. Howe ver, no tramadol-treated group differed significantly from the vehicle-treated group. Tramadol had no significant effect on locomotion, either (c) overnight or (d) after injections. A ll values expressed are group means S.E.M. 0 1 2 3 4 5 0 100 200 300 400 500 600 7000 mg/kg 0.1 mg/kg 1.0 mg/kg 10 mg/kg nightgrooming (sec.) 0 1 2 3 4 5 0 1000 20000 mg/kg 0.1 mg/kg 1.0 mg/kg 10 mg/kg nightinactivity (sec.) 0 1 2 3 4 5 0 25 50 75 1000 mg/kg 0.1 mg/kg 1.0 mg/kg 10 mg/kg nightovernight locomotion (scores from videotape analysis) 0 1 2 3 4 5 0 1000 20000 mg/kg 0.1 mg/kg 1.0 mg/kg 10 mg/kg daypost-injection locomotion (photocell crossings)A B CD

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48 Fig. 3.28. Effects of tramadol on the health status of the ra ts: (a) There was a significant effect of time on body weight, wh erein all groups lost weight during the first four days of the experiment. Trama dol did not significantly affect the weights of the (b) thymus glands or (c,d) adrena l glands. All values expressed are group means S.E.M. 0 1 2 3 4 5 6 0 50 1000 mg/kg 0.1 mg/kg 1.0 mg/kg 10 mg/kg 200 225 250 275daybody weight (g) 00.11.010 0 100 200dose of tramadol (mg/kg)thymus weight(mg/100g body weight) 0 0.1 1.0 10 0.0 2.5 5.0 7.5 10.0 12.5dose of tramadol (mg/kg)left adrenal gland weight(mg/100g body weight) 0 0.1 1.0 10 0.0 2.5 5.0 7.5 10.0 12.5dose of tramadol (mg/kg)right adrenal gland weight(mg/100g body weight)A B C D

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49 Experiment 5: Memantine In the experiment with memantine, th ere were no signs of self-injury until the second night during the pemoline treatment. On the morning of day 3, moderate injury was observed in one rat in the group treated with pemoline plus vehicle. The injury scores of the vehicle-treated rats reac hed asymptote around the afternoon of day 4 ( Fig. 3.29 ). During the experiment, five rats were assigned an injury score of 4, one in the vehicle-treated group (euthanize d on day 3), two rats in the group treated with the lowest dose (3 mg/kg) of memantine (one euthanized on day 3, the other on day 4) and two rats in the group treated with the medium dose (10 mg/kg) of memantine (one euthanized on day 4, the other on day 6). By the final morning of the experiment, 5 of the 6 vehicle-treated rats exhibited injury scores of 1 or higher (i.e. had self-i nduced tissue damage). All rats in the groups treated with the lowest and medium doses (3 and 10 mg/kg) of memantine exhibited injury scores of 1 or higher, and 4 out of th e 6 rats in the group tr eated with the highest dose (30 mg/kg) of memantine exhibited injury scores of 1 or higher. The prevalence of positive injury scores throughout the experiment is depicted in Fig. 3.30 The rats that were treated with memantine did not exhibit lower injury scores than the vehicle-treated rats did. The induction of self-injury (i.e. the slope of the injury scores starting with the onset on day 3 to th e asymptote on day 4) was not significantly affected by memantine treatment (F(3,21) = 1.042, p> 0.10; Fig. 3.29 and Fig. 3.31a ). The mean injury scores during the maintenance phase (i.e. the asymptotic expression from day 4 to day 6) was also not significa ntly affected by memantine treatment (F(3,21) = 0.4803, p> 0.10; Fig. 3.29 and Fig. 3.31b ).

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50 The duration of self-injurious oral contact was also not significantly affected in the memantine-treated rats, as compared to the oral contact dura tions in the vehicletreated rats ( Fig. 3.32 ). The induction of self-injurious oral contact (taken from night 1 until the asymptote on night 4) was not signi ficantly affected by memantine treatment (F(3,21) = 0.5208, p> 0.10; Fig. 3.32 and Fig. 3.33a ). The maintenance of the selfinjurious oral contact (taken from night 4 until night 5) was also not significantly affected by memantine (F(3,21) = 0.1132, p> 0.10; Fig. 3.32 and Fig. 3.33b ). Although there were significant cha nges in the duration of grooming (F(4,72) = 5.442, p< 0.05; Fig. 3.34a ) and inactivity (F(4,72) = 4.869, p< 0.05; Fig. 3.34b ) across the days of the experiment, there were no significant between-groups differences or interaction effects with memantine treat ment. There were significant group by day interactions for both overnight locomotion (taken from the overnight videotapes) (F(12,72) = 2.948, p< 0.05; Fig. 3.34c )and post-injection locomotion (counts taken from the locomotor monitors) (F(12,72) = 7.753, p< 0.05; Fig. 3.34d ), wherein memantine-treated rats exhibited greater locomotion than did vehicle-treated rats. All groups exhibited weight loss for th e first four days, followed by a slight weight gain. There was a significant inte raction between group and time for body weight (F(30,180)=2.243, p< 0.05; Fig. 3.35a ), wherein the memantine-tr eated rats exhibited more weight loss than the vehicle-treated rats did. Thymus weights were found to be significantly different between groups (F(3,21) =3.657, p< 0.05; Fig. 3.35b ), but no significant differences were found between th e weights in the memantine-treated and vehicle-treated groups. Adre nal weights were not significantly affected by memantine

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51 0 1 2 3 4 5 6 0 1 2 3 40 mg/kg 3 mg/kg 10 mg/kg dayinjury score30 mg/kg 0 1 2 3 4 5 6 0 25 50 75 1000 mg/kg 3 mg/kg 10 mg/kg daypercentage of rat that exhibited injury30 mg/kg administration (left adrenal: F(3,21) =0.5132, p> 0.05; right adrenal: p> 0.05, F(3,21) = 2.857, p> 0.05; Fig. 3.35c,d ). Fig. 3.29. Effects of memantine on pemo line-induced self-injury: Memantine did not affect pemoline-induced self-injur y, as measured by th e injury scores. All values expressed are group means S.E.M. Fig. 3.30. Effects of memantine on the incide nce of self-injury: Memantine did not significantly affect the incidence of self-i njury, as measured by the percentage of rats that exhibited self-injur y on day 6 of the experiment.

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52 0 3 10 30 0.0 0.5 1.0 1.5 2.0 2.5doses of memantine (mg/kg)induction of tissue damage(slope of regression lines of injury scores) 0 3 10 30 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5doses of memantine (mg/kg)maintenance of tissue damage(mean injury scores)A B 0 1 2 3 4 5 0 25 50 75 1000 mg/kg 3 mg/kg 10 mg/kg 30 mg/kg nightself-injurious oral contact(% duration) Fig. 3.31. Effects of memantine on inducti on and maintenance of pemoline-induced self-injury: Memantine did not significan tly alter either (a) induction or (b) maintenance of pemoline-induced self-injur y. All values expressed are group means S.E.M. Fig. 3.32. Effects of memantine on the durati on of pemoline-induced self-injurious oral contact: Memantine did not signi ficantly affect pemoline-induced selfinjurious oral contact. Self-injurious or al contact peaked on day 4 for most groups. All values expressed are group means S.E.M.

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53 Fig. 3.33. Effects of memantine on the i nduction and maintenance of pemolineinduced self-injurious oral contact: Memantine did not significantly affect either (a) induction or (b) maintena nce of pemoline-induced self-i njurious oral contact. All values expressed are group means S.E.M. 0 3 10 30 0 5 10 15 20 25 30 35dose of memantine (mg/kg)induction of self-injurious oral contact(slope of regression lines) 0 3 10 30 0 25 50 75dose of memantine (mg/kg)maintenance of self-injurious oral contact(mean scores)AB

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54 Fig. 3.34. Effects of memantine on gr ooming, inactivity and locomotion: Memantine did not significantly affect (a ) time spent grooming or (b) time spent inactive. Memantine-treated rats exhi bited significantly greater counts of locomotion, both (c) overnight and (d) pos t-injection. Signif icant differences between vehicleand memantine-treated rats (LSD) are depicted as follows: p< 0.05 for comparisons between meman tine at 30 mg/kg and vehicle; p< 0.05 for comparisons between memantine at 10 mg/kg and vehicle; # p< 0.05 for comparisons between memantine at 3 mg/kg and vehicle. All values expressed are group means S.E.M. 0 1 2 3 4 5 50 150 2500 mg/kg 3 mg/kg 10 mg/kg 30 mg/kg nightgrooming (sec.) 0 1 2 3 4 5 0 100 200 300 400 500 600 7000 mg/kg 10 mg/kg 30 mg/kg 3 mg/kg nightinactivity (sec.) 0 1 2 3 4 5 0 100 2000 mg/kg 3 mg/kg 10 mg/kg 30 mg/kg *nightovernight locomotion(scores from videotape analysis) 0 1 2 3 4 5 0 1000 2000 3000 4000 50000 mg/kg 3 mg/kg 10 mg/kg 30 mg/kg *# *daypost-injection locomotion(photocell crossings)A B C D

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55 Fig. 3.35. Effects of memantine on the health status of the rats: (a) There was a significant interaction between time and group for body weight. The rats in the pemoline plus memantine groups lost more weight than the rats in the pemoline plus vehicle group did. Si gnificant differences between vehicleand memantinetreated rats (LSD) are depicted as foll ows: p< 0.05 for comparisons between memantine at 30 mg/kg and vehicle; p< 0.05 for comparisons between memantine at 10 mg/kg and vehicle; # p< 0.05 for comparisons between memantine at 3 mg/kg and vehicle. (b ) There were significant between-groups differences in the weights of the thymus glands, however, no significant differences were found between the mema ntine-treated groups and the vehicletreated group. Memantine had no affect ad renal gland weight (c,d). All values expressed are group means S.E.M. 0 1 2 3 4 5 6 0 50 100Pem + veh Pem + 3 Mem Pem + 10 Mem 200 225 250 275 300Pem + 30 Mem * * # daybody weight (g) 0 3 10 30 0 100 200dose of memantine (mg/kg)thymus weight (mg/100g body weight) 0 3 10 30 0 5 10 15dose of memantine (mg/kg)left adrenal gland weight (mg/100g body weight) 0 3 10 30 0 5 10 15dose of memantine (mg/kg)right adrenal gland weight (mg/100g body weight)A B C D

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56 Inter-observer reliability Inter-observer reliability for injury scores across the five experiments was as follows. In 93% of cases, the two observers’ sc ores matched exactly. In 6% of cases, the scores were mismatched by one point on the 5-point scale (e.g., one observer assigned a score of 2, and the other observer assigned a sc ore of 3). In less than 1% of cases, the scores were mismatched by 2 points, and th e scores were never mismatched by 3 points or more.

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57 CHAPTER 4 DISCUSSION The results of the current study replicat e previous findings that approximately 75% of the rats exhibit SIB when treated w ith pemoline at 200 mg/kg/day, and most of the self-injury was targeted at the forepaws and ventrum ( Kies and Devine, 2004 ). In four of the five current experiments, appr oximately 75-80% of the rats exhibited tissue injury when injected daily with 200 mg/kg pe moline (i.e. those that were injected with pemoline plus vehicle). The only exception was the nifedipine experiment in which 100% of the rats self-injured when treated with pemoline and the vehicle (see discussion below). The fact that some of the rats in the groups that were treated with pemoline and vehicle did not self-injure in four of the five experime nts, suggests that there are individual differences in vul nerability to develop pemolin e-induced SIB. Individual differences in vulnerability to develop pemo line-induced SIB resemble the expression of SIB in clinical populations. Ev en in disorders with a high prevalence of SIB, there are individuals who do not self-injur e. Individual differences in the vulnerability to develop pemoline-induced SIB may provide a useful to ol for investigating the neurobiological differences between rats that exhibit pe moline-induced SIB and those that do not. In these experiments we used multiple measures to characterize pemoline-induced SIB. These included injury scores that detail the severity and the pr evalence of injury in each treatment group. We also evaluated a meas ure of the behavioral expression of SIB, using the duration of self-injurious oral contact on the body, and we used the injury scores and the oral contact scores to ev aluate the rate of onset and the ongoing

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58 maintenance of the pemoline-induced self-i njury. An analysis of the behavioral expression, or duration, of SI B has generally not been used (for an exception, see King et al., 1995 ). It has even been proposed that the behavioral expression of pemoline-induced SIB could not be quantified because of its resemblance to normal grooming ( Mueller & Hsiao, 1980 ). In fact, we found that there was general concordance between the measures of self-injury, a nd that the measure of prol onged oral contact reliably discriminates between grooming and SIB. A dditionally, we found that the quantification of the behavioral expressi on of SIB highlighted important information about the pemoline-induced SIB; information that the in jury scores alone were not sensitive enough to decipher. The oral contact data from th e overnight videos indicated that there was pemoline-induced self-biting behavior before there were any signs of injury. The quantification of SIB also indi cated that the behavioral e xpression of SIB actually peaks and then declines during the five nights of the experiment in most of the groups of rats that were treated with pemoline plus vehicle. The injury scores remained at asymptotic levels around this time and so they did not ac curately reflect this eventual decrease in self-biting behavior. The reason for the de cline in behavioral expression of pemolineinduced SIB is not known. Perhaps the decline in self-injurious oral contact results from tolerance to the injury-inducing effect of pemo line. This decline could also be a response to the pain of injuring tissue that has been traumatized. This interesting finding will require further investigation. On the other ha nd, the analysis of the self-injurious oral contact was not very sensitive to the severi ty of biting. In two of the experiments (valproate and nifedipine), th e rats that were treated with the drug challenges exhibited lowered injury scores, but no si gnificant effect on oral cont act scores. Apparently, these

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59 rats engaged in prolonged oral contact, with less severe self-biting so that they exhibited lower amounts of tissue damage than the vehicl e-treated rats did. Overall, these multiple dependent variables each measure different aspects of pemoline-induced SIB and allow for a more thorough characteri zation of the self-injury. The drugs that were evaluated in thes e experiments were designed to provide specific information about the pemoline model of SIB, and about the potential effects of drug challenges in this model. In particular the drug challenges were designed to assess the predictive validity of the model (i.e. risper idone and valproate), th e generalizability of the pemoline model in relation to other animal models of SIB (i.e. risperidone and nifedipine), and pharmacotherapies that may ha ve clinical potentia l (i.e. tramadol and memantine). The results of these pharmaco logical studies generated support for the predictive validity and generalizability of th e pemoline model. They did not yield any promising leads for previously untested pha rmacotherapy, but thes e studies revealed interesting information about the pote ntial use of the model to uncover the neurobiological basis of SIB. The fact that the rats expressed lowe r amounts of SIB if they were treated with some of the cha llenge drugs (risperidone, valproate, and nifedipine) suggests that the ne urochemical mechanisms that are directly or indirectly addressed by these drugs may be important me diators of the inducti on and expression of SIB, and these mechanisms may be importa nt targets for future development of pharmacotherapies. The fact that risperidone attenuates pe moline-induced SIB suggests that the model has predictive validity. Risperidone decr eased SIB in both clinical samples ( Allen & Rice, 1996 ; Cohen et al., 1998 ; McCracken et al., 2002 ; Caicedo &Williams, 2002 ) and

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60 in this animal model. Additionally, risper idone has lessened the occurrence of SIB in another animal model ( Allen et al., 1998 ). These results provide evidence for the generalizability between the different animal models of SIB. This generalizability will allow for neurobiological analyses that can hi ghlight the common substrates that lead to SIB in these models. The results from the risperidone experiment also provide further evidence that disregulated monoaminergic neurotransmission is important for the etiology and maintenance of SIB. Pemoline is a monoamine agonist ( Molina & Orsinghen, 1981 ) and risperidone is a monoamine antagonist ( Leysen et al., 1988 ). Thus, the opposing actions of these drugs, as they induce SIB and block SIB, suggests that disregulated monoaminergic systems cause SIB in the pemoline model. This is consistent with the evidence of disregulated monoaminergic neurotransmission in clinical populations that exhibit SIB ( Lloyd et al., 1981 ; Ernst et al., 1996 ; Wong et al., 1996 ). The inconsistency between the effects of valproate on injury sc ores and its effects on duration of self-injurious or al contact suggests that the e ffects of valproate were more subtle than were the effects of risperidone. These results show that valproat e lessened the severity of the pemoline-induced self-injury, as measured by the injury scores, but did not reduce the expression of stereotyped oral be haviors, as quantified from the overnight videotaped samples. The significant valproat e-induced decrease in the severity of the tissue injury further indicates that the pemo line model has predictive validity since this finding is consistent with decreases in SIB in autistic and intellectually handicapped patients treated with valproate ( Kastner et al., 1993 ; Hollander et al., 2001 ). Valproate increases extracellular GABA concentrations by blocking the degrad ation of GABA by GABA transaminase ( Loscher, 1993 ). It also blocks volta ge-gated sodium channels

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61 ( McLean & McDonald, 1986 ), calcium channels ( Kito et al., 1994 ) and protein kinase C ( Chen et al., 1994 ). Accordingly, it is not clear whether valproate reduced pemolineinduced self-injury through GABAergic mech anisms, ion channels actions or through alterations in cell signaling pa thways. But, it did not appe ar that the injury-reducing effects of valproate were due to general se dation because the duration of self-injurious oral contact, grooming behaviors, inactivity and amount of locomotion were not different between the groups. The fact that nifedipine attenuated pemo line-induced self-injury, and appeared to produce an overall inhibition of the self-injurious oral cont act (although this did not reach statistical significance), concurs with the findi ngs that nifedipine lowers the SIB in the 6OHDA ( Blake et al., 2004 ) and Bay-K 8644 ( Jinnah et al., 1999 ) models. This indicates that there is generalizability between these di fferent animal models of SIB. This could indicate that there are commonalities in the ne urobiological actions that initiate SIB in each of these animal models. Identification of these common mechanisms could help to characterize the neurobiological conditions th at are necessary and sufficient to induce SIB. One obvious factor is dopamine disr egulation, and this investigation using nifedipine contributes furthe r evidence that dopamine is im portant in the expression of SIB. L-type calcium channels are found predominantly on the presynaptic neurons ( Okita et al., 2000 ) in the striatum and cortex ( Hirota and Lambert, 1997 ) and are associated with increased release of dopa mine from the caudate when activated ( Okita et al., 2000 ). Additionally, bloc king these channels decreases th e rate of action potentials of midbrain dopaminergic neurons ( Mercuri et al., 1994 ).

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62 One potential confounding variable in th is study is that the vehicle for the nifedipine contained et hanol. Ethanol was not used in any of the other experiments in this or previous studies, and the effect of ethanol on pemoline-induced SIB is not known. The rats that just received pemoline plus ve hicle exhibited more se vere self-injury than the rats did in any other experiment, even though all the groups of ra ts were treated with pemoline at 200 mg/kg/day. All the rats in the pemoline plus vehicle group were euthanized before the end of the experiment because they reached an injury score of 4. Accordingly, it is possible that this anomal ous outcome may have actually resulted from an interaction between the ethanol and the pemo line in this experiment. This possibility will require further study. Nevertheless, nife dipine significantly reduced the overall SIB, demonstrating the effectiveness of this drug challenge. Significant thymus involution was observed in all the nifedipine-treated groups. This is consistent with reports that nifedi pine administration caus es thymic apoptosis ( Balakumaran et al., 1996 ). Accordingly, it does not appe ar that nifedipi ne could have any clinical potential for treatm ent of SIB, but further studies with this drug may help to reveal neurobiological mechanisms that under lie the induction and maintenance of SIB in the various animal models in which it is effective. Although tramadol has been reported to decrease compulsive behaviors in individuals with OCD a nd Tourette’s syndrome ( Goldsmith et al., 1999 ; Shapira et al., 1997 ) it did not significantly a ffect any of our dependent measures of SIB in the pemoline model. One potential interpretation of these results is that pemoline-induced SIB is not a compulsive behavior. This is contradictory to our casual observations, where we have noticed that the rats are extremely di fficult to distract after they have initiated

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63 self-biting behavior and that even if the injury site is out of reach for the rat (i.e. when the paw is being shown to the camera during the visual inspections) th e rat will begin to nibble on other items, such as the examiners glove or lab coat. Anothe r possibility is that tramadol does not disrupt compulsive behavi or patterns with enough strength to combat the compulsive nature of pemoline-induced SIB. The two studies that describe the effectiveness of tramadol to combat com pulsive behaviors in OCD and Tourettes syndrome are open-labeled and they included small sample sizes. Thus, the clinical efficacy of tramadol is not yet well establishe d. Moreover, it is possible that the dosage (0, 0.1, 1.0, 10 mg/kg) or dosing re gimen (b.i.d.) of tramadol th at we used in this study may not have been aggressive enough. We chose doses below 25 mg/kg/day because higher doses have been shown to indu ce abnormal chewing movements, vigorous grooming and spasms ( Matthiesen et al., 1998 ). Accordingly, it seems unlikely that higher doses of tramadol would be effective in the pemoline model of SIB. The potential that compulsions play an important role in pemoline-induced SIB merits further evaluation. The fact that memantine did not aff ect any of our dependent measures of pemoline-induced SIB is disappointing from a clinical perspective. Although the pemoline model is not a definitive screening t ool for the efficacy of therapeutic drugs, the results from the memantine study suggest th at it may not be an effective drug for reducing SIB in clinical populations. This negative outcome is somewhat surprising since memantine and MK-801 are both non-competitive NMDA receptor antagonists ( Wong et al., 1986 ; Bormann, 1989) and MK-801 blocked pemoline-induced SIB ( King et al., 1995 ). This difference in findings coul d be explained by the difference in

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64 experimental procedures. King and colleague s investigated interactions between MK801 and pemoline only acutely, as MK-801 and pe moline were administered only once in their study. In the present study the effects of blocking the NMDA r eceptor were studied with repeated administration of memantine (b.i.d.) and pemoline (q.d.) for five days. Although there is no specific re ason to suspect that these di fferent dosing regimens would produce differing outcomes, it is possible that higher doses or more frequent administration of memantine may have had so me effect. However, memantine has been found to produce some learning deficits and ataxia when admini stered at 20 mg/kg ( Hesselink et al., 1999 ) and in our study we used doses up to 30 mg/kg b.i.d. Accordingly, higher doses of memantine ma y not be appropriate in this model. Another potential reason fo r the effectiveness of MK801 and the ineffectiveness of memantine is that the actions of these drugs at the NMDA receptor differ substantially from each other. MK-801 exhibits high a ffinity binding to activated NMDA receptors, and becomes trapped in the ionophore where it cannot be displaced. Accordingly, MK801 blocks the physiological acti ons that result from transi ent release of glutamate (e.g., learning and memory) and it also blocks path ological actions that result from prolonged glutamate stimulation (e.g., apoptotic cascad es in neurodegenerative diseases). Memantine, on the other hand, exhibits low affinity binding to the ionophore of the activated NMDA receptors. As such, the binding is transient. Accordingly, memantine is neuroprotective in conditions where th ere is prolonged glutamate stimulation (e.g., neurodegenerative disorders such as Alzhei mer’s disease) because it decreases the calcium influx during the prolonged stimulati on. However, under basal conditions, when the postsynaptic NMDA receptor is generally quiescent, memantine is not bound to the

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65 receptors, which allows transient binding of glutamate and the opening of the ion channel to occur. If memantine does bind, the binding is rapidly reversible, and so the next time normal transient glutamate stimulation occurs the process is repeated. Accordingly, memantine is only a very low potency antagon ist against normal physio logical actions of glutamate at NMDA receptors (for review see Sonkusare et al., 2005 ). In fact, a comparative analysis reveals that MK-801 e xhibits very high potency blockade of NMDA receptor-dependent hippocampal l ong term potentiation (LTP), whereas memantine has only very low potency and does not affect LTP ( Frankiewicz et al., 1996 ). The discrepancy between the effects of MK-801 ( King et al., 1995 ) and the lack of effects of memantine (present results) c oupled with the observati on that there is a lag between the onset of treatment and the onset of SIB when the rats are treated with a moderately high dose of pemoline ( Kies and Devine, 2004 ; present results), raises the interesting possibility that glutamate-mediat ed neuroadaptations may play an important role in pemoline-induced SIB. In this case, the MK-801 was effective because it blocked the ability of pemoline to initiate these adaptations, and memantine was ineffective because it did not block the neuroadaptations An analysis of the systems in which pemoline induces neuroadaptation through gl utamate-mediated actions may reveal neuropathological disregulation in the pemo line-treated rats that exhibit SIB. Identification and characterization of disregulat ed systems that differentiate self-injurious from non-injurious rats may provide intere sting leads to examine neuropathological substrates that underlie SI B in clinical populations. In the valproate, nifedipine and trama dol experiments, the drug challenges had no significant impact on any of the measures of grooming, inactivity or locomotion

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66 (overnight videotapes and phot ocell counts). In the risper idone experiment, the duration of inactivity on the last night of the experiment was significan tly greater in rats treated with the highest dose of risperidone, as comp ared to vehicle-treated rats. It does not appear that risperidone was ex erting its effect through sedati ve actions, however, because duration of grooming and amount of locomotion were not different between risperidoneand vehicle-treated groups. During the first two days of the memantine experiment, the memantine-treated rats exhibited greater loco motor counts, both overnight and after the morning injections, compared to that of the vehicle-treated rats. Beginning on day 3, the locomotor counts of the memantine-treated ra ts no longer differed from that of the vehicle-treated rats. The differences in locomotor counts between memantineand vehicle-treated rats are mostly inconseque ntial because memantine had no significant effect on pemoline-induced self-injury. The rats remained reasonably health y throughout the experiments. Pemoline caused some weight loss, which began to le vel off or improve on day 3 or 4 in most experiments. This may be due to the psyc homotor stimulating effects of pemoline or altered feeding behaviors during pemoline treatment. Chromodaccryorrhea (porphyrin containing secretions around the eyes; Payne, 1994) was noticed in some of the rats. These secretions have been associated with stress ( Harkness & Ridgway, 1980 ; Ross, 1994 ; Chen et al., 1997 ) and with toxic actions of a variety of drugs ( Moser, 1991 ; Sauer et al., 1995 ; Pegg et al., 1996 ; Graziano et al., 1996 ; Sauer et al., 1997 ). The presence of chromodaccryorrhea replicates a previous finding when rats were treated daily with 200 mg/kg pemoline ( Kies & Devine, 2004 ), but as in the previo us study, this was a minor

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67 effect that occurred in a very small number of the rats, and did not appear to be associated with the induction or maintenance of SIB. In summary, these projects provide ev idence of the predictive validity of the pemoline model to mimic the SIB seen in clinical populations as risperidone and valproate lessened pemoline-induced SIB. They also provide more evidence of monoamine involvement in SIB because risperid one and nifedipine e ach reduced the selfinjury in the pemoline model. Results from these experiments also indicate that common neurobiological substrates may underlie the i nduction of SIB in multiple animal models, as evidenced by the fact that nifedipine cons istently blocks SIB in these various animal models. Also, the current evidence suggests that glutamate-mediat ed neuroadaptations may be involved in producing pemoline-i nduced SIB since memantine did not affect pemoline-induced SIB. This interesti ng possibility merits further attention. Unfortunately, the tramadol experiment sugge sts (within the limits that the model may predict clinical efficacy) that this treatm ent does not hold therapeutic promise. In accordance with the observations of th ese experiments, the predictive validity and pre-clinical screen ing potential of the pemoline model should be investigated further using drugs that are effective in decreasing SIB in different clinical populations. In light of the fact that the monoamine agonist pemoline only produced SIB after days of treatment, and that a monoamine antagonist bl ocked the effect, the dynamic regulation of neurotransmission in dopaminergic, serotone rgic, and noradrenergic systems should be examined in relation to the onset and expres sion of the SIB. Add itionally, the role of specific glutamate-mediated neuroadaptations should also be investigated. Challenging the pemoline model of SIB with drugs that inhibit the protein ki nases that mediate

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68 neuroadaptations, such as a protein kinase B and protein kinase C, will begin to elucidate the changing mechanisms that produce pemolin e-induced SIB. Additionally, challenging the pemoline model with a protein kinase C inhibitor and with another GABA agonist, topiramate, will also help decipher whether valproate reduced the pemoline-induced selfinjury through its actions on GABA or its actions on the kinase. These future experiments will improve our knowledge about the neurobiological mechanisms that are producing and maintaining this devastating behavior disord er and will lead to the elucidation of potential target s for new pharmacotherapies.

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79 BIOGRAPHICAL SKETCH Amber Marie Muehlmann graduated cum laude from San Diego State University with a Bachelor of Arts in May 2002. She began her graduate education in August 2003 working towards her Master of Science degree in the behavioral neuroscience program in the Psychology Department at the University of Florida.


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PHARMACOLOGICAL CHALLENGES OF AN ANIMAL MODEL OF SELF-
INJURIOUS BEHAVIOR















By

AMBER M. MUEHLMANN


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Amber M. Muehlmann














This document is dedicated to my parents, Richard and Susan Muehlmann, my brother,
Aaron Muehlmann, and my loving family in Gainesville, Florida, Nicholas, Shelby and
Maximus Van Matre. Your love and support has allowed me to complete this work in
only two years. Thank you.















ACKNOWLEDGMENTS

I would like to thank my committee members, Dr. Mark Lewis, Dr. Andy Shapira

and Dr. Timothy Vollmer, for their time, as well as Dr. George Casella for all of his help

with the statistical analyses. I would especially like to thank my advisor, Dr. Darragh

Devine, for his guidance throughout the development and completion of these projects. I

also wish to thank my labmates, both past and present, who have helped tremendously

with these experiments.

















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES ....................................................... ............ .............. .. vii

LIST OF FIGURES .............. .......................................................... viii

A B STR A C T ................................................. ..................................... .. x

CHAPTER

1 INTRODUCTION ............... .................................................... 1

2 METHODS ...................................................................... ........... 12

A n im a ls ................................................................................................................. 1 2
D ru g s .....................................................................1 2
Experim mental Procedures ........................................................................... 13
Drug Treatments-Experiment 1: Risperidone ........................... ............... 13
Drug Treatments-Experiment 2: Valproate ............. ........................................13
Drug Treatments-Experiment 3: Nifedipine.................... .............................14
Drug Treatments-Experiment 4: Tramadol ............................... ................ 14
Drug Treatments-Experiment 5: Memantine ..................................................14
Behavioral and Histological Asssays-All experiments.............................................14
Statistical A n aly ses ................................................................. ........ ....... .. 16

3 R E S U L T S .............................................................................2 1

E xperim ent 1: R isperidone .............................................................. .....................2 1
Experim ent 2: V alproate.................................................. ............................... 28
Experim ent 3: N ifedipine .......................... ........ ........................................ 35
E xperim ent 4 : T ram adol ..................................................................... ..................42
E xperim ent 5: M em antine ...................................................................................... .... 49
Inter-observer reliability .......................... ........... ........ ............ 56

4 D ISC U S SIO N ............................................................................... 57

L IST O F R EFE R E N C E S ............................................................................ .............. 69



v









B IO G R A PH IC A L SK E TCH ...................................................................... ..................79
















TABLE

Table pge

2 .1 Inju ry score rating scale ........................................ .............................................2 0
















LIST OF FIGURES


Figure pge

3.1 Effects of risperidone on pemoline-induced self-injury ..........................................23

3.2 Effects of risperidone on the incidence of self-injury ................... ................24

3.3 Effects of risperidone on the induction and maintenance of pemoline-induced
se lf-inju ry ......................................................................... 2 4

3.4 Effects of risperidone on the duration of pemoline-induced self-injurious oral
co n tact ..............................................................................2 5

3.5 Effects of risperidone on the induction and maintenance of pemoline-induced
self-injurious oral contact............................................... .............................. 25

3.6 Effects of risperidone on the grooming, inactivity and locomotion........................26

3.7 Effects of risperidone on the health status of the rats..................... ..................27

3.8 Effects of valproate on pemoline-induced self-injury................... ............... 30

3.9 Effects of valproate on the incidence of self-injury .............................................. 31

3.10 Effects of valproate on the induction and maintenance of pemoline-induced self-
inju ry .............................................................................. 3 1

3.11 Effects of valproate on the duration of pemoline-induced self-injurious oral
co n tact .............................................................................. 3 2

3.12 Effects of valproate on the induction and maintenance of pemoline-induced self-
injurious oral contact ......................................................... ............... ... 32

3.13 Effects of valproate on the grooming, inactivity and locomotion............................33

3.14 Effects of valproate on the health status of the rats...............................................34

3.15 Effects of nifedipine on pemoline-induced self-injury ...................................37

3.16 Effects of nifedipine on the incidence of self-injury............................ .............38









3.17 Effects of nifedipine on the induction and maintenance of pemoline-induced
se lf-inju ry ......................................................................... 3 8

3.18 Effects of nifedipine on the duration of pemoline-induced self-injurious oral
co n tact .............................................................................. 3 9

3.19 Effects of nifedipine on the induction and maintenance of pemoline-induced
self-injurious oral contact............................................... .............................. 39

3.20 Effects of nifedipine on the grooming, inactivity and locomotion...........................40

3.21 Effects of nifedipine on the health status of the rats ............................. .............41

3.22 Effects of tramadol on pemoline-induced self-injury ................................. 44

3.23 Effects of tramadol on the incidence of self-injury....................... ...............44

3.24 Effects of tramadol on the induction and maintenance of pemoline-induced self-
inju ry ...............................................................................4 5

3.25 Effects of tramadol on the duration of pemoline-induced self-injurious oral
co n tact ..............................................................................4 5

3.26 Effects of tramadol on the induction and maintenance of pemoline-induced self-
injurious oral contact .................. ................. ......... .. ....... ................. 46

3.27 Effects of tramadol on the grooming, inactivity and locomotion.............................47

3.28 Effects of tramadol on the health status of the rats ......... .............. ............... 48

3.29 Effects of memantine on pemoline-induced self-injury .........................................51

3.30 Effects of memantine on the incidence of self-injury ........................................ 51

3.31 Effects of memantine on the induction and maintenance of pemoline-induced
se lf-inju ry ......................................................................... 5 2

3.32 Effects of memantine on the duration of pemoline-induced self-injurious oral
co n tact .............................................................................. 5 2

3.33 Effects of memantine on the induction and maintenance of pemoline-induced
self-injurious oral contact............................................... .............................. 53

3.34 Effects of memantine on the grooming, inactivity and locomotion.........................54

3.35 Effects of memantine on the health status of the rats...............................................55















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

PHARMACOLOGICAL CHALLENGES OF AN ANIMAL MODEL OF SELF-
INJURIOUS BEHAVIOR

By

Amber M. Muehlmann

August 2005

Chair: Darragh P. Devine
Major Department: Psychology

Self-injurious behavior (SIB) is a devastating behavior disorder that involves acts

directed at a person's own body and causes damage to skin and underlying tissues. These

actions are often expressed in a stereotyped manner and include, but are not limited to,

self-biting, head banging and self-punching. SIB is exhibited by people with intellectual

handicaps, particularly people with severe and profound intellectual impairment, and by

people with several different congenital developmental disorders (e.g., Lesch-Nyhan

syndrome, autism and Prader-Willi syndrome). Animal models of SIB have been

developed using environmental restriction, neurotoxins, and pharmacological

manipulations. These models, in combination with post-mortem and in vivo clinical data,

have provided evidence that monoaminergic disregulation is an important factor in the

development and expression of SIB. Pemoline, an indirect monoamine agonist, produces

stereotyped SIB in rats when administered at high doses. We have investigated the

potential therapeutic effectiveness of five drugs (risperidone, valproate, nifedipine,









tramadol and memantine) in this model of pemoline-induced SIB. These

pharmacological challenges of the pemoline model were chosen in order to achieve four

specific objectives. These objectives were to evaluate the predictive validity of the

pemoline model, to test the generalizability of pharmacological interventions between

several animal models of SIB, to investigate the pharmacotherapeutic potential of two of

the drugs, and to further investigate the neurobiological mechanisms that contribute to

pemoline-induced SIB. Risperidone and valproate effectively decreased the occurrence

of SIB in clinical trials, and they each decreased the occurrence of pemoline-induced

SIB. Nifedipine blocked SIB in the 6-hydroxydopamine and Bay-K 8644 models, and it

decreased the pemoline-induced SIB. We also investigated the pharmacotherapeutic

potential of tramadol (a drug that attenuates compulsive behaviors in obsessive-

compulsive disorder and Tourette's syndrome) and memantine (a glutamate receptor

antagonist that has shown promise in treatment of Alzheimer's disease and other clinical

disorders). These drugs did not significantly lessen the occurrence of pemoline-induced

SIB. Each of these experiments also reveals important new information regarding the

neuronal changes that occur during chronic pemoline administration. These new findings

will lead to future experiments on neurobiological changes that produce SIB, and they

may help to identify potential neurobiological targets for new pharmacotherapies.














CHAPTER 1
INTRODUCTION

Self-injurious behavior (SIB) is a devastating, maladaptive behavior disorder that

is common in intellectually handicapped populations. The self-injurious actions are

usually highly stereotypic (Symons & Thompson, 1997), and they result in immediate or

delayed damage to the skin or underlying tissues. Self-injurers exhibit many different

forms of SIB, including head banging (Thompson & Caruso, 2002), self-biting (Nyhan,

1968), skin-picking (State et al., 1999), and self-punching (Oliver et al., 1987); and

although individual self-injurers usually exhibit stereotyped patterns of behavior that are

directed at specific and generally invariant body sites (Bodfish et al., 1995), there is great

diversity in the forms of self-injury within and across clinical groups. These behaviors

vary from mild self-injury producing bruises or calluses to severe self-injury leading to

permanent tissue damage or tissue loss.

In addition to the dire physical consequences that self-injurers inflict upon

themselves, these behaviors also limit social and cognitive development. SIB often

results in exclusion from educational and socializing activities, and it interferes with all

normal activities of daily living. SIB is highly destructive for families and caregivers

who live and work with self-injurers, leading to increased stress (Sarimski, 1997) and

feelings of despair (Bromley & Emerson, 1995). There are also significant costs to

society (estimated at $3 billion in 1989), as self-injurers require additional resources in

terms of specialized care and professional interventions (NIH Consensus Development

Conference Statement, 1989).









SIB is positively correlated with the occurrence of non-injurious stereotypes and

compulsions (Bodfish et al., 1995) and has even been hypothesized as being a

compulsive behavior in itself (King, 1993). In fact, Powell and colleagues (1996) found

that 46% of their self-injurious sample engaged in self-restraint in an apparent attempt to

interrupt their self-injury suggesting that these individuals were resisting a compulsive

need to self-injure. Unfortunately, these self-restraining behaviors did not produce any

decrease in the occurrence of SIB.

SIB is also associated with stress, wherein SIB increases in stressful situations

(e.g., being around new people, being sick or when having restraints removed) (Anderson

& Ernst, 1994), and there is a disproportionately high prevalence of SIB in disorders that

involve abnormal amounts of distress (Sovner & Fogelman, 1996; Lindauer et al., 1999).

Estimates of the population prevalence of SIB range from 1.7% to 65.9% of the

intellectually handicapped in general. These estimates vary considerably because

definitions of SIB are inconsistent; some studies include mild SIB whereas others only

report the incidence of moderate to severe SIB (for review see Rojahn & Esbensen,

2002). Estimates also differ because individuals with severe or profound intellectual

disabilities are more likely to self-injure than individuals with mild or moderate

intellectual disabilities (McClintock et al., 2003), and because individuals in institutions

are more likely to self-injure than those who are not in institutions (Eyman & Call, 1977).

It is unclear, however, if the greater severity of SIB in institutionalized populations is

actually caused by the institutional environment, if it is because the more severely

intellectually handicapped are more likely to live in an institution (Eyman & Call, 1977),

or if it is because the SIB is the reason for institutionalization (Eyman et al., 1972).









SIB also presents as a phenotypic trait of many specific congenital developmental

disorders. Approximately 44% of individuals with Cornelia de Lange syndrome self-

injure by head banging, self-scratching and finger biting (Bemey et al., 1999). Among

girls with Rett syndrome, 50% compulsively wring or bite their hands until there are

lesions on the skin (Sansom et al., 1993). Estimates of SIB in autism are more variable, as

are all the characteristics of autism. One study found mild SIB in 21.5%, moderate SIB

in 17.1% and severe SIB in 14.6% of their autistic sample (Baghdadli et al., 2003).

Additionally, 80% of individuals with Prader-Willi syndrome will compulsively pick at

their skin, leading to sores and infection (Symons et al., 1999). SIB is almost always

observed in Lesch-Nyhan syndrome, but there have been rare cases where the expression

of SIB has been delayed or non-existent (Mitchell & Mclnnes 1984; Singh et al., 1986;

Hatanaka et al., 1990; Adler & Wrabetz, 1996). The severity of their SIB is usually

extreme and many individuals with Lesch-Nyhan syndrome exhibit self-injury that causes

tissue loss and deformity of the hands and face. (Nyhan, 1968; Anderson & Ernst, 1994).

The biological basis of Lesch-Nyhan syndrome is any single point mutation in the

HPRT enzyme, which renders the enzyme completely inactive. The biological

consequences of that deficiency that lead to SIB are not understood. There is

disregulation in a variety of neurotransmitter systems, and most studies have indicated a

prominent disregulation of dopamine. Studies of post-mortem brain tissue have shown a

significant loss of dopamine functioning in the nigrostriatal and mesolimbic dopamine

terminals, as measured by decreases in dopamine content and functional activity of

tyrosine hydroxylase and dopa decarboxylase (Lloyd et al., 1981). Significant reductions

in dopa decarboxylase activity and dopamine storage have also been found in the caudate,









putamen, frontal cortex and ventral tegmental complex in an in vivo investigation using

positron emission tomography (PET) imaging with a fluorodopa F 18 tracer (Ernst et al.,

1996). Using PET, Wong and colleagues (1996) found decreased binding of the

radiolabeled dopamine transporter ligand, WIN-35,428, (50-63% reduction in the caudate

and 64-75% in the putamen) in individuals with Lesch-Nyhan syndrome. This indicates a

significant loss of dopaminergic nerve terminals. There is also an upregulation of D1 and

D2 receptors in the striatum (Saito et al., 1999), suggesting post-synaptic supersensitivity

for dopamine in the striata of individuals with Lesch-Nyhan syndrome. Reduced

concentrations of the dopamine metabolite, homovanillic acid (HVA), have also been

seen in cerebrospinal fluid (Jankovic et al., 1988). Increased concentrations of serotonin

and the serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA), have also been found

in the putamen (Lloyd et al., 1981). There is evidence that decreased dopamine

functioning may cause increases in serotonin levels in the brain (Mrini et al., 1995).

Marked decreases in caudate, putamen and cerebral volume in individuals with Lesch-

Nyhan syndrome have also been seen using magnetic resonance imaging (Harris et al.,

1998). Overall, these data indicate that there is profound disregulation of monoamine

systems in the brains of Lesch-Nyhan syndrome, and that nigrostriatal and

mesocorticolimbic dopamine neurotransmission may play a particularly important role in

the etiology and expression of self-injury.

Many drugs have been prescribed to help reduce the incidence of SIB in clinical

populations. Unfortunately, no one medication, or class of medications, has proven

effective for all patients. This suggests that these medications may not be able to correct

the behavioral overlay (e.g., escape from a demanding task) that is associated with the









SIB. Clinical trials with typical neuroleptics such as haloperidol have produced

inconsistent results. Some studies report successful attenuation of SIB (Janowsky et al.,

2005) and some studies report failures to decrease SIB (Mace et al., 2001). Interestingly,

Durand (1982) found that neither haloperidol nor mild punishment reduced a case of

severe SIB, but a combination of haloperidol and behavioral intervention did significantly

decrease the occurrence of SIB. This suggests that the effectiveness of pharmacological

treatment in human self-injurers may be complicated by environmental conditions that

influence the expression of the behavior disorder. Clinical trials with fluoxetine, a

selective serotonin reuptake inhibitor (SSRI), have also produced inconsistent results. In

a blind, placebo-controlled experiment using fluoxetine in children with Obsessive

Compulsive Disorder, King and colleagues (1991) reported an emergence of both SIB

and obsessive self-injurious ideations in six children, four of whom had to be

hospitalized. Decreased SIB with fluoxetine treatment has also been reported (e.g.,

Ricketts et al., 1993), but many of those trials have not included placebo control or have

not used blind observers to measure the dependent outcomes. In addition, naltrexone, an

opioid antagonist, has produced conflicting results in its effectiveness to reduce SIB in

clinical trials. There are reports that naltrexone produced increases in SIB (Benjamin et

al., 1995), decreases in SIB (Symons et al., 2001) and no effects on SIB (Willemsen-

Swinkels et al., 1995). Valproate, an indirect GABA agonist (that also has actions in

other systems), has also reduced the incidence of SIB in small clinical trials with autistic

(Hollander et al., 2001) and intellectually handicapped individuals (Kastner et al., 1993).

Risperidone, an atypical neuroleptic that affects multiple monoaminergic systems, has

had the most consistent effects across different patient groups in reducing the amount of









aggression, directed at both the self and others, in children and adults with Lesch-Nyhan

syndrome (Allen & Rice, 1996), autism (McCracken et al., 2002; Caicedo & Williams,

2002) and mental retardation (Cohen et al., 1998). In summary, the results of these

clinical trials have provided evidence of the involvement of dopaminergic, serotonergic,

opioid and GABAergic systems in clinical SIB, and these data further suggest that there

are sub-groups of self-injurers, who may respond differently to different kinds of

pharmacotherapy.

Animal models of SIB have also provided important information regarding the

neurobiological basis of SIB. These animal models include neonatal lesions,

environmental deprivation and pharmacological manipulations. In one model, 6-

hydroxydopamine (6-OHDA) is used to lesion striatal dopamine neurons in neonatal rat

pups. When these lesioned rats become adults they begin to self-injure after

administration of either direct or indirect dopamine agonists (e.g., apomorphine, 1-dopa),

which affect multiple dopamine receptors (Breese et al., 1984b; Breese et al., 1984a).

Furthermore, agonists that are effective only at the D1 class of dopamine receptors (e.g.,

SKF 38393) will effectively induce SIB, whereas D2-selective agonists do not induce SIB

in the 6-OHDA model (Breese et al., 1985). Additionally, D1 antagonists block the SIB

(Breese et al., 1985). It has been hypothesized, therefore, that SIB in this model is due to

a supersensitivity at the D1 receptors. This is consistent with evidence of a dopamine

supersensitivity in individuals with Lesch-Nyhan syndrome (Saito et al., 1999).

Risperidone and nifedipine (an L-type calcium channel blocker, which decreases the

amount of dopamine released in the caudate (Okita et al., 2000) have lowered the

incidence of SIB in the 6-OHDA model (Blake et al., 2004). In summary, the 6-OHDA









model provides further evidence of dopamine's important role in SIB. It is unclear if

altered functioning of other neurotransmitter systems may also contribute to the induction

of SIB in the 6-OHDA model.

Early environmental and maternal deprivation of non-human primates has been

found to produce abnormal behaviors including stereotyped locomotion, abnormal

socialization and SIB (Harlow & Harlow, 1962). The occurrence of whole-body

stereotypes and the severity of SIB increase with an apomorphine challenge (Lewis et

al., 1990), which suggests dopamine supersensitivity. These changes in dopaminergic

functioning are similar to those found in individuals with Lesch-Nyhan syndrome and

provide further evidence that dopamine disregulation is an important contributor to SIB.

There is also a significant decrease in the density of immunoreactivity for tyrosine

hydoxylase, substance P and leucine-enkephalin in the striatum and related basal ganglia

regions in rhesus monkeys with a history of social isolation (Martin et al., 1991). This

suggests that early environmental deprivation directly affects the development of the

dopaminergic and peptidergic systems in the striatum. Once again, this resembles the

striatal disorganization seen in Lesch-Nyhan syndrome (Wong et al., 1996; Harris et al.,

1998).

A variety of different classes of pharmacological manipulations have been used to

induce self-injury in animals. Bay-K 8644, an L-type calcium channel agonist, causes

dose-orderly expression of self-biting in mice (Jinnah et al., 1999; Jinnah et al., 2003).

Moreover, Bay-K 8644- induced SIB is eliminated by administration of nifedipine

(Jinnah et al., 1999), which demonstrates that Bay-K 8644-induced SIB is specifically

due to actions on the L-type calcium channels. Injections of Bay-K 8644 directly into the









striatum produce significant increases in dopamine release in a dose dependent fashion

(Jinnah et al., 1999). Additionally, administration of fluoxetine (Kasim et al., 2002) and

amphetamine (Kasim & Jinnah, 2003) (serotonin and dopamine agonists, respectively)

each increase Bay-K 8644-induced SIB and administration of drugs that antagonize

serotonin (Kasim et al., 2002) or dopamine decrease Bay-K 8644-induced SIB (Kasim &

Jinnah, 2003). These results demonstrate that Bay-K 8644-induced SIB, like SIB seen in

clinical populations, is associated with changes in dopaminergic and serotonergic

neurotransmission.

Caffeine, a non-selective adenosine receptor antagonist, has also been reported to

induce SIB when administered repeatedly at very high doses (Mifiana et al., 1984).

However, it was recently reported that the caffeine-induced SIB is not dose orderly, that

the self-injury is extremely mild and only seen in a small percentage of the animals, and

the required doses are highly toxic (Kies & Devine, 2004).

GBR-12909, an indirect dopamine agonist that blocks the dopamine transporter

and the uptake of dopamine into synaptic vesicles, produces dose- and time-orderly

induction of SIB in rats. GBR-12909-induced SIB is blocked by 6-OHDA lesions of

nigrostriatal dopaminergic neurons, which suggests GBR-12909 produces SIB by altering

presynaptic dopamine (for review see Sivam, 1996). This is consistent with evidence of

altered presynaptic dopamine functioning in individuals with Lesch-Nyhan syndrome

(Lloyd et al., 1981).

Pemoline, an indirect monoamine agonist, has also been used as a

pharmacological model of SIB (Genovese et al., 1969; Mueller & Hsiao, 1980). Chronic

administration of moderately high doses of pemoline produces dose-orderly self-injury in









a large majority of rats in a few days time (Kies & Devine, 2004). SIB in this model is

usually directed towards the forepaws and abdomen, but is occasionally directed at the

hindpaws and tail (Mueller & Hsiao, 1980; Kies & Devine, 2004). Pemoline-induced

SIB is potentiated by impoverished environmental conditions during development (Kies

et al., 2002) and by stress exposure (Kies et al., 2004). This is consistent with

characteristics of clinical SIB, which is more prevalent in environmentally impoverished

conditions, such as institutions than it is in community-based populations (Eyman et al.,

1972; Eyman & Call, 1977), and is commonly expressed in stressful situations (Anderson

& Ernst, 1994). An examination of brain structures affected by pemoline administration,

using an assay for cytochrome oxidase (the end product of the mitochondrial electron

transport chain and a marker of on-going neuronal activity) indicated that there is a

significant pemoline-induced down-regulation of neuronal activity in the caudate nucleus,

septum, bed nucleus of the stria terminalis, hippocampus, periaqueductal grey and some

hypothalamic nuclei (Kies & Devine, 2003). These results suggest that the pemoline acts

upon the dopaminergic nigrostriatal and mesolimbic pathways, and that there is

significant indirect impact on a variety oflimbic structures that are known to participate

in processing of emotionally-relevant stimuli (Herman et al., 1996, Herman & Cullinan

1997). Disregulation of the nigrostriatal dopamine pathway is strongly implicated in

clinical populations in which SIB is manifested and negative affect and SIB are highly

correlated in some individuals with intellectual handicaps (as previously discussed)

(Lindauer et al., 1999). In addition to dopaminergic actions, there is evidence that other

neurotransmitter systems are involved in pemoline-induced SIB. Specifically, King and

colleagues (1995) have found that pemoline-induced SIB is attenuated by MK-801









administration, which suggests glutamatergic involvement. Additionally, paroxetine (an

SSRI) significantly potentiated pemoline-induced SIB, suggesting a role of serotonin in

the pemoline model (Turner et al., 1999).

Based on the results from these investigations of SIB using the pemoline model,

we have begun to further characterize the model in rats by pharmacologically challenging

the induction of SIB with five specific drugs. Those drugs are risperidone (Risperdal),

valproate (Depakote), nifedipine, tramadol (Ultram) and memantine (Namenda). We

have examined the predictive validity of the pemoline model by investigating the

effectiveness of two drugs that have been clinically useful in reducing SIB, risperidone

and valproate.

Additionally, we have examined the effectiveness of nifedipine, an L-type

calcium channel antagonist, to lessen pemoline-induced SIB. Nifedipine has been used to

lower the incidence of self-injury in the 6-OHDA model and to decrease Bay-K 8644-

induced SIB (as previously discussed). The purpose of this investigation is to evaluate

the generalizability between animal models as this may help to reveal whether common

neurobiological mechanisms contribute to the induction and expression of SIB in the

various animal models. Commonality in these models may be useful in further

investigating the neurobiological basis of SIB. In all of these drug challenges, we are

also considering the specific biological mechanisms that are acted upon by the drug

challenges in order to further evaluate the neurobiological mechanisms that underlie

pemoline-induced SIB.

We have also evaluated the pharmacotherapeutic potential of two drugs that have

not previously been assessed in clinical populations, using the pemoline model. Since









SIB is a behavior disorder that appears highly stereotypic and compulsive in clinical

populations (Bodfish et al., 1995; King, 1993) and in the pemoline model, we

investigated the effectiveness of tramadol to reduce the incidence of pemoline-induced

SIB. Tramadol is a low affinity mu-opioid receptor agonist (Dhasmana et al., 1989),

which also blocks reuptake of serotonin (Driessen & Reimann, 1992) and norepinephrine

(Driessen et al., 1993). It has been shown to reduce the amount of compulsive behaviors

in individuals with Obsessive Compulsive Disorder (OCD) (Goldsmith et al., 1999) and

to reduce the amount of motor tics in individuals with Tourette's syndrome (Shapira et

al., 1997).

Additionally, we evaluated the effectiveness of memantine, a non-competitive

NMDA receptor antagonist, to lessen SIB in the pemoline model. MK-801, another non-

competitive NMDA receptor antagonist with high affinity, blocks SIB in the pemoline

model (King et al., 1995). MK-801, however, cannot be used as a clinical

pharmacotherapy because of its psychotomimetic side effects (Koek et al., 1988).

Memantine lacks these side effects because it has a lower affinity for the NMDA

receptor, and was recently approved by the FDA for use in Alzheimer's patients

(Molineuvo et al., 2005). In light of the effects of MK-801, we hypothesized that

memantine could be clinically effective for treatment of clinical SIB.














CHAPTER 2
METHODS

Animals

Male Long Evans (LE) rats weighing 225-275 grams were housed in an

AAALAC-approved, climate controlled vivarium. The rats were maintained on a 12-

hour light/dark schedule with lights on at 7 am. Standard laboratory rat chow (Lab Diet

5001) and tap water were available adlibitum. The rats were pair-housed in standard

polycarbonate cages (43 x 21.5 x 25.5 cm) during 5-7 days of acclimation to the housing

facility. After the acclimation period the rats were singly-housed in similar

polycarbonate cages. All procedures were conducted in accordance with the Guide for

the Care and Use of Laboratory Animals published by the National Institutes of Health

and all procedures were pre-approved by the Institutional Animal Care and Use

Committee at the University of Florida.

Drugs

Pemoline (Spectrum Chemicals, New Brunswick, New Jersey) was suspended at a

concentration of 50 mg/ml in warm peanut oil (held at approximately 360 Celsius), with

constant stirring. Risperidone was purchased from Sigma-Aldrich Co. (St. Louis,

Missouri) and was suspended in a solution of 0.45% (w/v) hydroxypropyl-beta-

cyclodextrin. The risperidone was suspended at concentrations of 0, 0.1, 0.5 and 1.0

mg/ml. Sodium valproate was purchased from Sigma-Aldrich Co. and was suspended in

a solution of 0.04% (w/v) Na2EDTA. The valproate was suspended at concentrations of

0, 50, 100 and 200 mg/ml and was adjusted to a neutral pH of 7.4. Nifedipine was









purchased from Sigma-Aldrich Co. and was suspended in a solution consisting of 40%

propylene glycol (v/v), 10% ethanol (v/v), 15% benzyl alcohol (v/v), 5% sodium

benzoate (w/v) and approximately 35% distilled water (v/v). Nifedipine was suspended

at concentrations of 0, 1.5, 5 and 15 mg/ml. Tramadol hydrochloride was purchased from

Sigma-Aldrich Co. and was dissolved in sterile saline at concentrations of 0, 1.0, 10 and

100 mg/ml. Memantine was purchased from Sigma-Aldrich Co. and was dissolved in

sterile saline at concentrations of 0, 3, 10 and 30 mg/ml.

Experimental Procedures

Drug Treatments-Experiment 1: Risperidone

Twenty-three male LE rats (Charles River Laboratories, Raleigh, NC) were

weighed and injected with pemoline (200 mg/kg s.c.) at approximately 8:00 a.m. on each

of five consecutive days. These injections were administered at the nape of the neck and

either flank on a rotating basis, using 21 gauge needles. The rats were also injected twice

daily with risperidone (0, 0.1, 0.5 or 1.0 mg/kg, i.p.) on each of the five days (n = 5-6 per

group), using 26-gauge needles. The risperidone injections were administered at

approximately 8:00 am (immediately after the pemoline injection) and approximately

6:00 pm.

Drug Treatments-Experiment 2: Valproate

Thirty-six male LE rats (Charles River Labs) received daily pemoline injections at

200 mg/kg and twice-daily injections ofvalproate (0, 50, 100 or 200 mg/kg, i.p.) for five

days (n = 9 rats per group), following the same procedures as in Experiment 1.









Drug Treatments-Experiment 3: Nifedipine

Twenty-three male LE rats (Charles River Labs) received daily pemoline

injections at 200 mg/kg and twice-daily injections of nifedipine (0, 3, 10 or 30 mg/kg,

s.c.) for five days (n = 6 rats per group), following the same procedures as in Experiment

1.

Drug Treatments-Experiment 4: Tramadol

Seventy-two male LE rats (Harlan Inc., Indianapolis, Indiana) received daily

pemoline injections at 200 mg/kg and twice-daily injections oftramadol (0, 0.1, 1.0 or 10

mg/kg, s.c.) for five days (n = 18 rats per group), following the same procedures as in

Experiment 1.

Drug Treatments-Experiment 5: Memantine

Twenty-two male LE rats (Charles River Labs) received daily pemoline injections

at 200 mg/kg and twice-daily injections of memantine (0, 3, 10 or 30 mg/kg, i.p.) for five

days (n = 5-6 rats per group). The injection procedures were similar to the procedures in

Experiment 1 except that memantine was administered 30 minutes before pemoline each

day, and then again at approximately 6:00 p.m.

Behavioral and Histological Assays All Experiments

The rats were visually inspected each time they were injected (i.e. twice per day

for five days), and the inspections were videotaped. These inspections were also

performed on the morning of the sixth day, but no injections were administered on the

sixth day. The rats were held in front of a video camera and the head, forepaws,

hindpaws, ventrum and tail were displayed. An injury score (see Table 1) was assigned

to describe the presence (or absence) and severity of all injuries. An observer blind to the









drug treatment independently re-scored the injuries from the videotapes, and inter-

observer reliability was assessed.

After the morning injections of pemoline and challenge drug (i.e. risperidone,

valproate, nifedipine, tramadol or memantine) each rat was placed back into its home

cage. A locomotor monitor (San Diego Instruments, San Diego, CA) was then raised

around each cage in order to measure the locomotor activating (or inhibiting) effects of

the pemoline and challenge drug. Each locomotor monitor had four LED sensors spaced

along the length of the cage. A locomotor count was recorded each time the rat

interrupted a photocell sensor, and then no further counts were recorded from that sensor

until the rat interrupted another photocell sensor. Accordingly, each locomotor count

represented actual movement across the cage (approximately 3.5 inches) rather than

repetitive interruption of any single photocell sensor. The rats' locomotion was

monitored for two hours after the injections each morning.

A variety of behaviors were recorded using night-vision cameras, where a camera

was focused on the cage of each rat each night. Five-minute time samples were recorded

once per hour for eight hours each night. These recordings were scored for duration of

self-injurious oral contact, duration of grooming, duration of inactivity and the amount of

locomotion. Self-injurious oral contact was defined as all oral contact that stayed fixed

on any one body part for longer than two seconds. Grooming was defined as oral contact

with any part of the body that continued to move from site to site on the body (e.g., oral

contact with the paws, then moving up each arm and continuing to the ventrum, in which

the contact was not sustained in any spot on the body for longer than two seconds).

Inactivity was defined as complete lack of movement except respiratory movements.









Locomotion was counted by sectioning the cage into three equal parts (along the length

of the cage) and tallying the number of times the rat entered into a different section

without returning to the section that he occupied immediately prior to that movement.

On the morning of the sixth day each rat was inspected and assigned an injury

score. Immediately after this inspection the rat was rapidly decapitated. Each rat's brain

was removed and rapidly frozen in 2-methylbutane at -400C and later stored at -800C.

These brains are being retained for histochemical analyses to compare a variety of

potential neurochemical differences between self-injurious and non-injurious rats that

were treated with the pemoline and challenge drugs. These analyses are not included in

this thesis. The thymus and adrenal glands were also removed, frozen on dry ice and

stored at -800C. These glands were later weighed in order to determine the health status

of the rats.

Statistical Analyses

The induction of self-injury was determined by graphing the injury scores of each

rat across the six days of the experiment. The onset of SIB was defined for each

experiment at the time the first rat in that experiment exhibited self-injury (i.e. received

an injury score of 1 or more). A linear regression line was then plotted from the time of

onset to the time when injury scores began to asymptote, and the slope of that line was

determined for each rat. Between groups differences in the slopes of the regression lines

were then compared using a one-way analysis of variance (ANOVA). All significant

effects were further analyzed with Fisher's Least Significant Difference (LSD) post-tests

for each experiment.









The maintenance of the self-injury was compared between the groups by taking

the mean of the injury scores that were recorded for each rat during the final days of the

experiment (i.e. all days after the asymptote was reached). On those days, the injury

scores leveled off or began to decline in most groups. A one-way ANOVA was used to

compare the mean injury scores for the rats in each treatment group in each experiment.

All significant effects were further analyzed with Fisher's LSD post-tests.

The percentage of time that the rats exhibited self-injurious oral contact (data

scored from the 40 minutes of videotaped recordings for each night) was used to quantify

the behavioral expression of self-injury. The induction of self-injurious oral contact was

assessed from the first night until the night that the most self-injurious group reached the

peak mean duration of self-injurious oral contact. The duration of self-injurious oral

contact data were then transformed by calculating the square root of the percentage of

time spent with self-injurious oral contact to equalize the variability between the

treatment groups (see below). A linear regression line was then plotted from the first

night to the night when the duration of self-injurious oral contact peaked, using the

square root transformed scores, and the slope of that line was determined for each rat.

Between groups differences in the slopes of the regression lines were then compared

using an ANOVA for each experiment. All significant effects were further analyzed with

LSD post-tests.

The maintenance of the self-injurious oral contact was compared by taking the

mean of the transformed percentage of oral contact data for the final nights of the

experiment, when the percent of self-injurious oral contact duration started to decline in

the groups that were treated with pemoline and vehicle. The mean of the transformed









duration of self-injurious oral contact was determined for each rat. These data were

compared for each treatment group using a one-way ANOVA. All significant effects

were further analyzed with LSD post-tests.

The dose of pemoline (200 mg/kg/day) that was used in these experiments

induces self-injury in approximately 75% of the rats. Consequently, there was substantial

variability of the duration of self-injurious oral contact within each treatment group. In

fact, in each experiment we observed some rats with self-injurious oral contact for 100%

of the time sampled and other rats, in the same treatment group, with no self-injurious

oral contact whatsoever (see Results). Accordingly, square root transformations of the

duration of self-injurious oral contact were used to control for this variability (Ott &

Longnecker, 2001). The power of the statistical analyses of injury scores and oral contact

duration was also diminished by the fact that the range of doses for the challenge drugs

included doses that were ineffective at reducing the different measures of pemoline-

induced SIB. In these cases, the statistical outcome was confounded by the ineffective

dose(s). We therefore set the acceptable alpha error level at 0.1 for all analyses of injury

scores and oral contact durations.

The duration of grooming behavior, inactivity, and the amounts of overnight

locomotion and post-injection locomotion (measured by the locomotor monitors) were

compared for each group using 4x5 (group x day) repeated measures ANOVAs. All

significant effects were further analyzed with LSD post-tests.

Body weights were analyzed by two-way (4 groups x 5 days) repeated measures

ANOVA. Glandular weights were compared using a one-way ANOVA for each

experiment. All significant effects were further analyzed with LSD post-tests.









Some rats were euthanized before the end of the experiment because they had an

injury score of 4 (open lesion or amputated digit). In these cases, the missing data were

replaced by repeating the final score that was attained for each dependent measure

through the end of the experiment. This strategy was used to avoid the potential that the

group means would underestimate the injury scores and self-injurious oral contact scores,

and to avoid the potential that the group means would over- or under-estimate the

locomotor, inactivity, and grooming scores when the most severe self-injurers were

removed from any group.

The prevalence of pemoline-induced SIB is dose-dependent (Kies and Devine,

2004), and we chose to work with a dose (200 mg/kg/day) that would induce SIB in some

but not all of the rats. Accordingly, the variability in expression of SIB is high in all the

groups of rats, including the control (pemoline + vehicle) groups. Furthermore, the doses

of challenge drugs included ineffective doses in most of the experiments. These factors

decreased the power of our ANOVAs to identify effective treatments. In light of these

statistical issues, treatment-associated decreases in injury scores and self-injurious oral

contact scores were treated as statistically reliable when the p-values were less than 0.10.

Between-groups differences in all other dependent measures were treated as statistically

reliable when the p-values were less than 0.05.











Table 2.1. Injury score rating scale (adapted from Turner et al., 1999).


Score Classification Description

0 no self-injury

1 very mild self-injury denuded skin, edema or erythema; involves small area

S. denuded skin, edema or erythema; involves medium area or
2 mild self-injurymultiple small sites
multiple small sites
S. denuded skin, edema or erythema; involves large area or
3 moderate self-injury multiple medium sites
Smultipele medium sites

4 severe self-injury open lesion, amputated digit. Requires euthanasia.














CHAPTER 3
RESULTS

Experiment 1: Risperidone

In the experiment with risperidone, there were no signs of self-injury until the

second night during the pemoline treatment. On the morning of day 3, mild injury was

observed in one rat in the group that was treated with pemoline plus vehicle, and in one

rat that was treated with pemoline plus the medium dose (0.5 mg/kg) of risperidone. The

injury scores of the vehicle-treated rats reached an asymptote around the morning of day

4 (see Fig. 3.1). During the experiment, two rats were assigned an injury score of 4, and

both were in the vehicle-treated group. One was put down on day 4; the other reached an

injury score of 4 on day 6 and was terminated with the other rats.

By the final morning of the experiment, 5 of the 6 vehicle-treated rats exhibited

injury scores of 1 or higher (i.e. had self-induced tissue damage). Fewer rats in each of

the risperidone-treated groups exhibited injury scores of 1 or higher, and this effect was

dose-orderly. The prevalence of positive injury scores throughout the experiment is

depicted in Fig. 3.2.

The rats that were treated with risperidone exhibited lower injury scores than the

vehicle-treated rats did, and this effect was dose-orderly (Fig. 3.1). The induction of self-

injury (i.e. the slope of the injury scores starting with the onset on day 3 to the asymptote

on day 4) occurred at a significantly lower rate in all the risperidone-treated groups, than

in the vehicle-treated group (F(3, 22) = 7.340, p< 0.01; Fig. 3.1 and Fig. 3.3a). The mean

injury scores during the maintenance phase (i.e. the asymptotic expression from day 4 to









day 6) were significantly lower in the risperidone-treated rats than they were in the

vehicle-treated rats, and this effect was roughly dose-orderly (F(3,22) = 5.276, p< 0.01;

Fig. 3.1 and Fig. 3.3b).

The duration of self-injurious oral contact was also significantly less in the

risperidone-treated rats than it was in the vehicle-treated rats (Fig. 3.4). The induction of

self-injurious oral contact (taken from night 1 until the peak on night 3) was significantly

lower in the risperidone-treated rats than in the vehicle-treated rats (F(3, 22) = 4.380, p<

0.10; Fig. 3.4 and Fig. 3.5a). The maintenance of the self-injurious oral contact (taken

from night 3 until night 5) appeared to be lower in the risperidone-treated rats, but this

effect did not reach statistical significance (F(3,22) = 2.336, p> 0.10; Fig. 3.4 and Fig.

3.5b).

Risperidone did not significantly affect the duration of time spent grooming

(F(12,76) = 0.7929, p> 0.05; Fig. 3.6a). Although risperidone did significantly increase the

duration of inactivity overnight (F(3,76) =3.393, p< 0.05; Fig. 3.6b), there were no

significant time or group by time interaction effects. The amount of locomotion recorded

on videotapes overnight decreased significantly across the days of the experiment (F(4,76)

=10.67, p< 0.05; Fig. 3.6c), but there were no significant between-groups differences, and

there were no group by time interaction effects. The amount of post-injection locomotion

(counts taken from the photocell monitors each morning) was not significantly affected

by risperidone treatment (F(12,76) = 0.9274, p> 0.05; Fig. 3.6d).

All the groups exhibited weight loss for the first four days, followed by a slight

weight gain. There was a significant interaction between group and time for body weight

(F(15,95) =1.947, p< 0.05; Fig 3.7a), wherein the risperidone-treated rats exhibited less









weight loss than the vehicle-treated rats did. Risperidone treatment, however, did not

appear to impact the health of the rats. There were no significant between-groups

differences in thymus (F(3,22) =2.630, p> 0.05; Fig. 3.7b), or adrenal weights (left adrenal:

F(3,22) =.4862, p> 0.05; right adrenal: F(3,22) =.5737, p> 0.05; Fig. 3.7 c,d).



4 -E- 0 mg/kg
0.1 mg/kg
--0.5 mg/kg
3- 1.0 mg/kg
0


2- 1-



0-
0 1 2 3 4 5 6
day


Fig. 3.1. Effects of risperidone on pemoline-induced self-injury: Risperidone
dose-dependently delayed the onset of self-injury, with the highest dose of
risperidone blocking the pemoline-induced self-injury until day 4. Risperidone
also dose-dependently attenuated the severity of pemoline-induced self-injury, as
measured by the injury scores. All values expressed are group means + S.E.M.















-B- 0 mg/kg
S. -F-O0.1 mg/kg
S75 ---0.5 mg/kg

-- 1.0 mg/kg
S 50-


25-



0 1 2 3 4 5 6
day



Fig. 3.2. Effects of risperidone on the incidence of self-injury: Risperidone dose-
dependently lowered the incidence of self-injury, as measured by the percentage of
rats that exhibited injury on day 6 of the experiment.


I


*
*


0 0.1 0.5


a)
C' 3-
E

3 8 2-



o 1-
4-*

C
1.0 E


dose of risperidone (mg/kg)


**

r4-1' *


0 0.1 0.5 1.0
dose of risperidone (mg/kg)


Fig. 3.3. Effects of risperidone on the induction and maintenance of pemoline-
induced self-injury: (a) Risperidone dose-dependently reduced the induction of
pemoline-induced self-injury. (b) Risperidone also dose-dependently reduced the
maintenance of pemoline-induced self-injury. All values expressed are group
means + S.E.M. Significant differences between the pemoline plus vehicle and
pemoline plus risperidone treated groups (LSD) are depicted with asterisks, where
* indicates p < 0.10.


o
g 2.5-

'E 2.0-
00
1.5-
2 -c
o" 1.0-

o
a)
0 0 0.0
Co


'


'






























0 1 2 3 4 5
night


Fig. 3.4. Effects of risperidone on the duration of pemoline-induced self-injurious
oral contact: Risperidone reduced the overall percent duration of self-injurious oral
contact. Self-injurious oral contact peaked on day 4 for most groups. All values
expressed are group means S.E.M.


0
0o
o
O
o


*
.U)




0

0 0.1 0.5 1.0
dose of risperidone (mg/kg)
E


0 0.1 0.5 1.0
dose of risperidone (mg/kg)


Fig. 3.5. Effects of risperidone on the induction and maintenance of pemoline-
induced self-injurious oral contact: (a) Risperidone dose-dependently decreased
the induction of pemoline-induced self-injurious oral contact. (b) Although it
appeared that risperidone also reduced the maintenance of pemoline-induced self-
injurious oral contact in a dose-orderly manner, this effect was not statistically
significant. All values expressed are group means S.E.M. Significant differences
between pemoline plus vehicle and pemoline plus risperidone treated groups
(LSD) are depicted with asterisks, where indicates p < 0.10.













--0 mg/kg 12500 mg/kg
--0 mg/kg
200- T--0.1 mg/kg 1000 -0 1 mg/kg
Si -4-0.5 mg/kg -0 5 mg/kg
t; 5 1 0 mg/kg
oE 100- 500
150 +1.0 mg/kg 750- mg/kg
50-I 250-

0 2345 012345
0 1 2 3 4 5 0 1 2 3 4 5
night night

C D
.2 100- --0 mg/kg c 1000-
0 -80mg/kg
S---0.1 mg/kg mg/kg
S75 0.5mg/kg 750- -- 0. mg/kg
E: --1.0 mg/kg 750 --0.5 mg/kg
50 50 1.0 mg/kg
50- 0u 500-
E


0 MI I
'E0 25 250-

S0 0 0
0 1 2 3 4 5 0 1 2 3 4 5
night day



Fig. 3.6. Effects of risperidone on grooming, inactivity and locomotion: (a)
Risperidone did not significantly affect time spent grooming. (b) Risperidone-treated
rats did exhibit more inactivity as compared to vehicle-treated rats. Significant
differences between vehicle- and risperidone-treated rats (LSD) are depicted as
follows: p< 0.05 for comparisons between risperidone at 1.0 mg/kg and vehicle.
Risperidone had no significant effect on locomotion, either (c) overnight or (d) after
injections. All values expressed are group means + S.E.M.











A --0 mg/kg 200 B
275- --V0.1 mg/kg
5 *# -*-0.5 mg/kg
250 .0 mg/kg 1C Z


S200 100

50 E
lroo-
0 0. 1 1 1 0 5 1.
0 1 2 35 0 0.1 0.5 1.0
day dose of risperidone (mg/kg)

C D
15- 12.5-







0 o.I I 1 0.0o
10 10.
Co
s 5.0-


01 0.0
0 0.1 0.5 1.0 0 0.1 0.5 1.0
dose of risperidone (mg/kg) dose of risperidone (mg/kg)



Fig. 3.7. Effects of risperidone on the health status of the rats: (a) there was a significant
interaction between time and group for body weight. Pemoline plus vehicle treated rats
lost more weight than pemoline plus risperidone treated rats did. Significant differences
between vehicle- and risperidone-treated rats (LSD) are depicted as follows: p< 0.05
for comparisons between risperidone at 1.0 mg/kg and vehicle; 7 p< 0.05 for
comparisons between risperidone at 0.5 mg/kg and vehicle; # p< 0.05 for comparisons
between risperidone at 0.1 mg/kg and vehicle. Risperidone did not significantly affect
the weights of the (b) thymus glands or (c,d) adrenal glands. All values expressed are
group means + S.E.M.









Experiment 2: Valproate

In the experiment with valproate, there were no signs of self-injury during the first

day or night of pemoline treatment. The onset of self-injury occurred on day 2, with a rat

in the pemoline plus vehicle group exhibiting mild injury. The onset of self-injury was

further delayed in all the valproate treated groups. The injury scores of the vehicle-

treated rats reached an asymptote around the morning of day 5 (see Fig. 3.8). Five rats

were assigned an injury score of 4, two in the vehicle-treated group (one euthanized on

day 4, the other on day 5), two rats in the lowest dose (50 mg/kg) of valproate group (one

euthanized on day 4, the other on day 5) and one in the medium dose (100 mg/kg) of

valproate group (euthanized on day 4).

By the final morning of the experiment, 8 of the 9 vehicle-treated rats exhibited

injury scores of 1 or higher (i.e. had self-induced tissue damage). Fewer rats in the

groups treated with the middle and high doses of valproate exhibited injury scores of 1 or

higher, and this effect was dose-orderly. The prevalence of positive injury scores

throughout the experiment is depicted in Fig. 3.9.

The rats that were treated with valproate exhibited lower injury scores than the

vehicle-treated rats did, and this effect was primarily observed at the highest dose of

valproate (Fig. 3.8). The induction of self-injury (i.e. the slope of the injury scores

starting with the onset on day 2 to the asymptote on day 5) occurred at a significantly

lower rate in the valproate-treated groups, than in the vehicle-treated groups (F(3,35)

=2.593, p< 0.10; Fig. 3.8 and 3.10a), and this effect was primarily observed at the highest

dose of valproate. The mean injury scores during the maintenance phase (i.e. the

asymptotic expression from day 5 to day 6) were significantly lower in the valproate-









treated rats than they were in the vehicle-treated rats (F(3,35) =2.420, p< 0.10; Fig. 3.10b),

and this effect was seen primarily in the highest dose of valproate.

In contrast to the effects of valproate on actual tissue damage (i.e. injury scores),

valproate administration did not significantly affect the duration of self-injurious oral

contact (Fig. 3.11). The induction of self-injurious oral contact (taken from night 1 until

the peak on night 4) was not significantly affected by valproate treatment (F(3,35) =.8013,

p> 0.10; Fig. 3.12a). The maintenance of self-injurious oral contact (taken from night 4

until night 5) was also not significantly reduced by valproate (F(3,35) =.3864, p> 0.10; Fig.

3.12b).

Valproate administration did not significantly affect any of the other behaviors

that were measured during the experiment. Although the time spent grooming (F4,128)

=5.378, p> 0.05; Fig. 3.13a), or inactive (F(4,128) =3.784, p> 0.05; Fig. 3.13b), and the

amount of locomotion recorded on videotapes overnight (F(4,128) =20.19, p> 0.05; Fig.

3.13c) decreased significantly across the days of the experiment, there were no significant

between-groups differences and there were no group by time interaction effects. Post-

injection locomotion, recorded with the photocell monitors immediately after pemoline

and valproate injections each morning, was not significantly affected by valproate

treatment (F(3,35) =1.991, p> 0.05; Fig. 3.13d).

All groups exhibited weight loss for the first four days, followed by a slight

weight gain. This weight loss was significant across days of the experiment (F(5,160)

=10.79, p< 0.05; Fig. 3.14a). There were no significant between-groups differences or

group by time interaction effects. Thymus weights were found to be significantly

different between groups (F(3,35)=5.841, p< 0.05; Fig. 3.14b), but no significant









differences were found between the valproate-treated groups and the vehicle-treated

group. There were also no significant between-groups differences on adrenal weights

(left: F(3,35) =.6223, p> 0.05; right: F(3,35) =.8762, p> 0.05; Fig. 3.14c,d).





4- -- 0 mg/kg
50 mg/kg
3 100 mg/kg
3-
S -- 200 mg/kg




"- 1-

0-
0 1 2 3 4 5 6
day


Fig. 3.8. Effects of valproate on pemoline-induced self-injury: Valproate delayed
the onset of self-injury. The two highest doses of valproate (100 and 200 mg/kg)
attenuated the severity of pemoline-induced self-injury, as measured by the injury
scores. All values expressed are group means + S.E.M.











-B- 0 mg/kg
-1- 50 mg/kg
S-*-- 100 mg/kg
S75 --200 mg/kg
O -0
50-







0 1 2 3 4 5 6
day


Fig. 3.9. Effects of valproate on the incidence of self-injury: The middle and high
doses of valproate lowered the incidence of self-injury, as measured by the
percentage of rats that exhibited injury on day 6 of the experiment.


5*-
50 100 200
dose of valproate (mg/kg)


B
E



4-
co
cnuW
0


E
E


50 100 200
dose of valproate (mg/kg)


Fig. 3.10. Effects of valproate on the induction and maintenance of pemoline-
induced self-injury: (a) Valproate reduced the induction of pemoline-induced self-
injury. (b) Valproate also reduced the maintenance of pemoline-induced self-
injury. All values expressed are group means + S.E.M. Significant differences
between the pemoline plus vehicle and pemoline plus valproate treated groups
(LSD) are depicted with asterisks, where indicates p< 0.10.


-ff
T
a)

.) 1.
1o.
U) 0.
._ o.

o

0.
.2
T!











100. -B- 0 mg/kg
-1 50 mg/kg
80- -4- 100 mg/kg
S-- 200 mg/kg


1 2 3 4 5
night


Fig. 3.11. Effects of valproate on the duration of pemoline-induced self-injurious
oral contact: Valproate did not significantly reduce the overall percent duration of
self-injurious oral contact. Self-injurious oral contact peaked on night 4 for all
groups. All values expressed are group means + S.E.M.


0 50 100 200
dose of valproate (mg/kg)


B










0 50 100 2(
dose of valproate (mg/kg)


Fig. 3.12. Effects of valproate on the induction and maintenance of pemoline-induced
self-injurious oral contact: (a) Valproate did not significantly affect induction of
pemoline-induced self-injurious oral contact. (b) Valproate also did not significantly
affect the maintenance of pemoline-induced self-injurious oral contact. All values
expressed are group means + S.E.M.


o
C
0
o 3-

0





U)
S-02-





o
- -

4c












-- 0 mg/kg
-- 50 mg/kg
--100 mg/kg
-- 200 mg/kg


nigh0 1 2 3 4
night


-B- 0 mg/kg
-- 50 mg/kg
-- 100 mg/kg
-A-200 mg/kg


1 2 3
night


C 2000-
0
.O2
E 1500-
0
o
c 1000-
0=

500-

0
0. n-


4 5


day 1 2 3
day


Fig. 3.13. Effects ofvalproate on grooming, inactivity and locomotion: Valproate

did not significantly affect (a) time spent grooming or (b) time spent inactive.

Valproate also had no significant effect on locomotion, either (c) overnight or (d)

after injections. All values expressed are group means + S.E.M.


700-

600-

U 500-

400-

. 300-

S200-

100-


night


70-

S60
S50-

o

S20-

> 10
0 -
-- 0 -"


o 0
o


-- 0 mg/kg
--50 mg/kg
-.-100 mg/kg
--200 mg/kg


4 5










-B- 0 mg/kg
A -50 mg/kg B
300- -4- 100 mg/kg 200-
-- 200 mg/kg
S250- '
100-
E-6


'2____00_L __-
20-i E I I

0 1 2 3 4 5 6 0 50 100 200
Day dose of valproate (mg/kg)




C D
12.5- 12.5-

10.0- 10.0-


S 2.5- > 2.5
O.o '1 o 1
-5-

S 0.0 0.0 I I
0 50 100 200 0 50 100 200
dose of valproate (mg/kg) dose of valproate (mg/kg)


Fig. 3.14. Effects of valproate on the health status of the rats: (a) There was a significant
effect of time on body weight, wherein all groups lost weight during the first four days of
the experiment and then exhibited a slight weight gain. (b) Valproate did significantly
affect the weights of the thymus glands, however, no significant differences were found
between the valproate-treated groups and the vehicle-treated group. (c,d) Valproate did
not significantly affect the weights of the adrenal glands. All values expressed are group
means + S.E.M.









Experiment 3: Nifedipine

In the experiment with nifedipine, there were no signs of self-injury until the

second day during the pemoline treatment. On the afternoon of day 2, mild injury was

observed in one rat that was treated with pemoline plus the medium dose (10 mg/kg) of

nifedipine. The injury scores of the vehicle-treated rats reached an asymptote around the

morning of day 5 (Fig. 3.15). During the experiment, thirteen rats were assigned an

injury score of 4 and were euthanized before the end of the experiment. The rats in the

vehicle-treated group exhibited extremely severe self-injury. In fact, all the rats in the

vehicle-treated group received an injury score of 4 and were terminated early (one on day

3, four on day 4 and one on day 5). Three rats in the group treated with pemoline plus the

low dose (3 mg/kg) of nifedipine reached an injury score of 4 (one on day 4 and two on

day 6). One rat from the group treated with pemoline plus the middle dose (10 mg/kg) of

nifedipine reached an injury score of 4 on day 4 and three rats in the group treated with

pemoline plus the high dose (30 mg/kg) of nifedipine group were euthanized early (one

on day 3 and two on day 4).

By the final morning of the experiment, all rats exhibited injury scores of 1 or

higher (i.e. had self-induced tissue damage) in the group treated with pemoline plus

vehicle and the group treated with pemoline plus the low dose (3 mg/kg) of nifedipine.

All but one rat in each of the groups that were treated with pemoline plus the middle (10

mg/kg) and high (30 mg/kg) doses of nifedipine also exhibited injury scores of 1 or

higher. The prevalence of positive injury scores throughout the experiment is depicted in

Fig. 3.16.

The rats that were treated with nifedipine exhibited lower injury scores than the

vehicle-treated rats did (Fig. 3.15). The induction of self-injury (i.e. the slope of the









injury scores starting with the onset on day 2 to the asymptote on day 4) occurred at a

significantly lower rate with all the nifedipine-treated groups, than in the vehicle-treated

group (F(3,22) =2.626, p< 0.10; Fig. 3.15 and Fig. 3.17a). The mean injury scores during

the maintenance phase (i.e. the asymptotic expression from day 4 to day 6) were

significantly lower in the nifedipine-treated rats than they were in the vehicle-treated rats

(F(3,22) =3.041, p< 0.10; Fig. 3.15 and Fig. 3.17b).

In contrast to the effects of nifedipine on actual tissue damage (i.e. injury scores),

nifedipine administration did not significantly affect the duration of self-injurious oral

contact (Fig. 3.18). The induction of self-injurious oral contact (taken from night 1 until

the peak on night 3) was not significantly affected by nifedipine treatment (F(3,21)

=0.4573, p> 0.10; Fig. 3.18 and Fig. 3.19a). The maintenance of self-injurious oral

contact (taken from night 3 until night 5) was also not significantly reduced by nifedipine

(F (3,22) =0.7444, p> 0.10; Fig. 3.18 and Fig. 19b).

Nifedipine did not significantly affect any of the other behaviors that were

measured during the experiment. Time spent grooming (F(4,72) =4.676, p< 0.05; Fig.

3.20a), inactive (F(4,72) =6.579, p< 0.05; Fig. 3. 20b) and the amount of locomotion

recorded on videotapes overnight (F(4,72) =23.94, p< 0.05; Fig. 3.20c) or recorded with the

photocell monitors immediately after pemoline and nifedipine injections each morning

(F(4,76) =10.79, p< 0.05; Fig. 3. 20d) all changed significantly across the days of the

experiment, but no significant group by time interaction effects were found.

All groups exhibited weight loss during the experiment. This weight loss was

significant across days of the experiment (F(5,95) =86.06, p< 0.05; Fig. 3.21a). There were

no significant between-groups differences or group by time interaction effects.









Nifedipine treatment significantly affected thymus weights (F(3,21) =6.917, p< 0.05; Fig.

3.21b), with the thymus glands of nifedipine-treated animals weighing significantly less

than the glands of the vehicle-treated animals did. Adrenal weights, however, were not

different between groups (left: F(3,21) =.4816, p> 0.05; right: F(3,21) =1.095, p> 0.05; Fig.

3.21c,d).



-E- 0 mg/kg
4 3 mg/kg
--- 10 mg/kg
S-A-30 mg/kg
0

2-




0-
0 1 2 3 4 5 6
day


Fig. 3.15. Effects of nifedipine on pemoline-induced self-injury: Nifedipine dose
dependently attenuated the severity of pemoline-induced self-injury, as measured
by the injury scores. Nifedipine did not delay the onset of pemoline-induced self-
injury. All values expressed are group means + S.E.M.












S100- -E- 0 mg/kg
S- 3 mg/kg
75. -4- 10 mg/kg
0o-
S-A- 30 mg/kg
.| 50
X
W 25-


0-Y
0 1 2 3 4 5 6
day


Fig. 3.16. Effects of nifedipine on the incidence of self-injury: The highest doses
of nifedipine lowered the incidence of self-injury, as measured by the percentage
of rats that exhibited injury on day 6 of the experiment.


S 3 10 30
dose of nifedipine (mg/kg)


S 3 10 30
dose of nifedipine (mg/kg)


Fig. 3.17. Effects of nifedipine on the induction and maintenance of pemoline-
induced self-injury: (a) Nifedipine reduced the induction of pemoline-induced self-
injury. (b) Nifedipine also decreased the maintenance of pemoline-induced self-
injury. All values expressed are group means + S.E.M. (Significant differences
between pemoline plus vehicle and pemoline plus nifedipine treated groups (LSD)
are depicted with asterisks, where indicates p< 0.10).

























0 1 2 3 4 5
night


Fig. 3.18. Effects of nifedipine on the duration of pemoline-induced self-injurious
oral contact: Nifedipine did not significantly reduce the overall percent duration of
self-injurious oral contact. Vehicle-treated rats exhibited self-injurious oral
contact for 100% of the night, beginning on night 3. All values expressed are
group means S.E.M.


0 3 10 30
dose of nifedipine (mg/kg)


I 3 10 30
dose of nifedipine (mg/kg)


Fig. 3.19. Effects of nifedipine on the induction and maintenance of pemoline-
induced self-injurious oral contact: (a) Nifedipine did not significantly affect the
induction of pemoline-induced self-injurious oral contact. (b) Nifedipine also did
not significantly affect the maintenance of pemoline-induced self-injurious oral
contact. All values expressed are group means S.E.M.














700-
600-
S500.
400-
> 300-
S200-
100-
0-






1250-


--0 mg/kg
-- 3 mg/kg
---10 mg/kg
--30 mg/kg







1
0 1 2 3 4 5
night

--0 mg/kg
3 mg/kg
-4- 10 mg/kg
--30 mg/kg T


day


Fig. 3.20. Effects of nifedipine on grooming, inactivity and locomotion: Nifedipine did
not significantly affect (a) time spent grooming or (b) time spent inactive. Nifedipine
also had no significant effect on locomotion, either (c) overnight or (d) after injections.
All values expressed are group means + S.E.M.


- 0 mg/kg
-- 3 mg/kg
-*-10 mg/kg
-- 30 mg/kg


2 3
night


4 5


- 0 mg/kg
-F-3 mg/kg
--10 mg/kg
--30 mg/kg


0-

. 750-

- w 500-
Ua

- 250-
0


night


a











-- 0 mg/kg
--3 mg/kg
300 -*-10 mg/kg
--30 mg/kg

p250-



530-

0 1 2 3 4 5 6
day




20-



0 1
.Co


0 3 10 30
dose of nifedipine (mg/kg)


30
(mg/kg)


Fig. 3. 21. Effects of nifedipine on the health status of the rats: (a) There was a
significant effect of time on body weight, wherein all groups lost weight during the six
days of the experiment. Nifedipine significantly affected the (b) thymus gland
weights. Significant thymus involution was seen in all rats in the nifedipine-treatment
groups. Nifedipine, however, had no significant effect on (c,d) adrenal gland weights.
All values expressed are group means + S.E.M. (Significant differences between
pemoline plus vehicle and pemoline plus nifedipine treated groups (LSD) are depicted
with asterisks, where indicates p< 0.05).









Experiment 4: Tramadol

In the experiment with tramadol, there were no signs of self-injury until the

second day of pemoline treatment. On day 2, mild injury was observed in one rat in the

group treated with pemoline plus vehicle, and one rat that was treated with pemoline plus

the medium dose (1.0 mg/kg) of tramadol. The injury scores of the vehicle-treated rats

reached an asymptote around the morning of day 4 (Fig. 3.22). During the experiment,

five rats were assigned an injury score of 4. Two rats in the vehicle-treated group (one

euthanized on day 2, the other on day 3), one rat in the group treated with the lowest dose

(0.1 mg/kg) of tramadol (euthanized on day 6), one rat in the group treated with the

medium dose (1.0 mg/kg) of tramadol (euthanized on day 4) and one rat in the group

treated with the highest dose (10 mg/kg) of tramadol (euthanized on day 3) were assigned

an injury score of 4.

By the final morning of the experiment, 12 of 18 vehicle-treated rats exhibited

injury scores of 1 or higher (i.e. had self-induced tissue damage). More rats in each of

the tramadol-treated groups exhibited positive injury scores than in the vehicle-treated

group. The prevalence of positive injury scores throughout the experiment is depicted in

Fig. 3.23.

The rats that were treated with tramadol did not exhibit lower injury scores than

did the vehicle-treated rats (Fig. 3.22). The induction of self-injury (i.e. the slope of the

injury scores starting with the onset on day 2 to the asymptote on day 4) was not

significantly affected by tramadol treatment (F(3,71) =1.532, p> 0.10; Fig. 3.22 and Fig.

3.24a). The mean injury scores during the maintenance phase (i.e. the asymptotic

expression from day 4 to day 6) was also not significantly affected by tramadol treatment

(F(3,71) =.7915, p> 0.10; Fig. 3.22 and Fig. 3.24b).









The duration of self-injurious oral contact was also not significantly altered in the

tramadol-treated rats (Fig. 3.25). The induction of self-injurious oral contact (taken from

night 1 until the peak on night 3) was not significantly affected by tramadol treatment

(F(3,42) = 1.942, p> 0.10; Fig. 3.25 and Fig. 3.26a). The maintenance of self-injurious

oral contact (taken from night 3 until night 5) was also not significantly reduced by

tramadol (F(3,42) =0.8608, p> 0.10; Fig. 3.25 and Fig. 3.26b).

Tramadol did not significantly affect the other behaviors that were measured

during the experiment. The time spent grooming (F(4,164) =9.596, p< 0.05; Fig. 3.27a),

inactive (F(4,164) =5.113, p< 0.05; Fig. 3.27b) and the amount of locomotion recorded on

the videotapes overnight (F(4,164) = 7.454, p< 0.05; Fig. 3.27c) or recorded with the

photocell monitors immediately after pemoline and tramadol injections each morning

(F(4,272) = 3.050, p< 0.05; Fig. 3.27d) were significantly changed across days of the

experiment. However, there were no significant between-groups differences or group by

time interaction effects for any of these behaviors.

All groups exhibited weight loss during the first four days of the experiment,

followed by a slight weight gain. This weight loss was significant across days of the

experiment (F(5,340) =22.90, p< 0.05; Fig. 3.28a). No between-groups differences or

interaction effects were significant. Tramadol treatment, however, did not appear to

impact the health of the rats. There were no significant between-groups differences in

thymus (F(3,71) = 0.7867, p> 0.05; Fig. 3.28b), or adrenal weights (left: F(3,71) = 0.5434,

p> 0.05; right: F(3,71) = 1.016, p> 0.05; Fig. 3.28c,d).










-- 0 mg/kg
--0.1 mg/kg
-- 1.0 mg/kg
-A- 10 mg/kg


1 2 3
day


4 5 6


Fig. 3.22. Effects oftramadol on pemoline-induced self-injury: Tramadol did not
affect pemoline-induced self-injury, as measured by the injury scores. All values
expressed are group means + S.E.M.









0 mg/kg
100 0. 1 mg/kg
-- 1.0 mg/kg
S*- -A-10 mg/kg
"U. 75-

o-
50-

S25-



0 1 2 3 4 5 6
day


Fig. 3.23. Effects oftramadol on the incidence of self-injury: Tramadol did not
significantly affect the incidence of self-injury, as measured by the percentage of
rats that exhibited self-injury on day 6 of the experiment.











SA B
o 0.8- 2.5-
U 0.8 E-
0.7- 6 2.0-
0 .6
S0.5- 1.5-
= o 0.4- -r 0,
C o.
'5 O- 0 2) 0.-
0.2 0.
S o 0.1 1.0 10 0 0.1 1.0 10
S dose of tramadol (mg/kg) dose of tramadol (mg/kg)



Fig. 3.24. Effects of tramadol on induction and maintenance of pemoline-
induced self-injury: Tramadol did not significantly affect the induction of
pemoline-induced self-injury. (b) Tramadol also did not significantly affect the
maintenance of pemoline-induced self-injury. All values expressed are group
means S.E.M.











100 -- 0 mg/kg
S- -0.1 mg/kg
S-1.0 mg/kg
8 75- +10 mg/kg


-)c
0


25-

'I-

0 1 2 3 4 5
night


Fig. 3.25. Effects of tramadol on the duration of pemoline-induced self-injurious
oral contact: Tramadol did not significantly affect the overall percent duration of
pemoline-induced self-injurious oral contact. Self-injurious oral contact peaked
on day 3 for most groups. All values expressed are group means S.E.M.














A B
o U
o 40 70-
2 60-
30 50-
2.2 T0

4 300
E 20
20- i1

CU






Fig. 3.26. Effects of tramadol on the induction and maintenance of pemoline-
induced self-injurious oral contact: (a) Tramadol did not significantly affect the
induction of pemoline-induced self-injurious oral contact. (b) Tramadol also did
not significantly affect the maintenance of pemoline-induced self-injurious oral
contact. All values expressed are group means + S.E.M.







47




A -B-0 mg/kg B -- 0 mg/kg
700- -7-0.1 mg/kg 2000- ---0.1 mg/kg
1.0 mg/kg
600- -10 mg/kg --1.0 mg/kg
1500- -- T 10 mg/kg
500-
400-
30 1000-


100-
0 0
0 1 2 3 4 5 0 1 2 3 4 5
night night


S C --0mg/kg D -E-0 mg/kg
S100- 0.1 rng/kg 2000- ----0.1 rng/kg
-4-- 1.0 mg/kg -1.0 mg/kg
.2 -*- 10 mg/kg -A--10 mg/kg


o 7




0 2 3 4 5 0 2 3 4 5
night day



Fig. 3.27. Effects oftramadol on grooming, inactivity and locomotion: (a) Tramadol
2 0










did not significantly affect time spent grooming. (b) Tramadol did significantly alter
the amount of time spent inactive. However, no tramadol-treated group differed
0 0 a. 0

night day



Fig. 3.27. Effects of tramadol on grooming, inactivity and locomotion: (a) Tramadol
did not significantly affect time spent grooming. (b) Tramadol did significantly alter
the amount of time spent inactive. However, no tramadol-treated group differed
significantly from the vehicle-treated group. Tramadol had no significant effect on
locomotion, either (c) overnight or (d) after injections. All values expressed are group
means + S.E.M.











-8- 0 mg/kg
A -- 0.1 mg/kg B
27- -- 1.0 mg/kg 200
-- 10 mg/kg
o 250- B *' -


E 0-
oa200-L 0 00
HE 0

0 1 2 3 4 5 6 0 0.1 1.0 10
day dose of tramadol (mg/kg)


C D
12.5 12.5




S5.0 5.0



0 0.1 1.0 10 0 0.1 1.0 10
dose of tramadol (mg/kg) dose of tramadol (mg/kg)


Fig. 3.28. Effects of tramadol on the health status of the rats: (a) There was a
significant effect of time on body weight, wherein all groups lost weight during the
first four days of the experiment. Tramadol did not significantly affect the weights
of the (b) thymus glands or (c,d) adrenal glands. All values expressed are group
means + S.E.M.









Experiment 5: Memantine

In the experiment with memantine, there were no signs of self-injury until the

second night during the pemoline treatment. On the morning of day 3, moderate injury

was observed in one rat in the group treated with pemoline plus vehicle. The injury

scores of the vehicle-treated rats reached asymptote around the afternoon of day 4 (Fig.

3.29). During the experiment, five rats were assigned an injury score of 4, one in the

vehicle-treated group (euthanized on day 3), two rats in the group treated with the lowest

dose (3 mg/kg) of memantine (one euthanized on day 3, the other on day 4) and two rats

in the group treated with the medium dose (10 mg/kg) of memantine (one euthanized on

day 4, the other on day 6).

By the final morning of the experiment, 5 of the 6 vehicle-treated rats exhibited

injury scores of 1 or higher (i.e. had self-induced tissue damage). All rats in the groups

treated with the lowest and medium doses (3 and 10 mg/kg) of memantine exhibited

injury scores of 1 or higher, and 4 out of the 6 rats in the group treated with the highest

dose (30 mg/kg) of memantine exhibited injury scores of 1 or higher. The prevalence of

positive injury scores throughout the experiment is depicted in Fig. 3.30.

The rats that were treated with memantine did not exhibit lower injury scores than

the vehicle-treated rats did. The induction of self-injury (i.e. the slope of the injury

scores starting with the onset on day 3 to the asymptote on day 4) was not significantly

affected by memantine treatment (F(3,21) = 1.042, p> 0.10; Fig. 3.29 and Fig. 3.31a). The

mean injury scores during the maintenance phase (i.e. the asymptotic expression from

day 4 to day 6) was also not significantly affected by memantine treatment (F(3,21)

0.4803, p> 0.10; Fig. 3.29 and Fig. 3.31b).









The duration of self-injurious oral contact was also not significantly affected in

the memantine-treated rats, as compared to the oral contact durations in the vehicle-

treated rats (Fig. 3.32). The induction of self-injurious oral contact (taken from night 1

until the asymptote on night 4) was not significantly affected by memantine treatment

(F(3,21) = 0.5208, p> 0.10; Fig. 3.32 and Fig. 3.33a). The maintenance of the self-

injurious oral contact (taken from night 4 until night 5) was also not significantly affected

by memantine (F(3,21) = 0.1132, p> 0.10; Fig. 3.32 and Fig. 3.33b).

Although there were significant changes in the duration of grooming (F(4,72)

5.442, p< 0.05; Fig. 3.34a) and inactivity (F(4,72) = 4.869, p< 0.05; Fig. 3.34b) across

the days of the experiment, there were no significant between-groups differences or

interaction effects with memantine treatment. There were significant group by day

interactions for both overnight locomotion (taken from the overnight videotapes) (F(12,72)

= 2.948, p< 0.05; Fig. 3.34c)and post-injection locomotion (counts taken from the

locomotor monitors) (F(12,72)= 7.753, p< 0.05; Fig. 3.34d), wherein memantine-treated

rats exhibited greater locomotion than did vehicle-treated rats.

All groups exhibited weight loss for the first four days, followed by a slight

weight gain. There was a significant interaction between group and time for body weight

(F(30,180)=2.243, p< 0.05; Fig. 3.35a), wherein the memantine-treated rats exhibited more

weight loss than the vehicle-treated rats did. Thymus weights were found to be

significantly different between groups (F(3,21) =3.657, p< 0.05; Fig. 3.35b), but no

significant differences were found between the weights in the memantine-treated and

vehicle-treated groups. Adrenal weights were not significantly affected by memantine






51


administration (left adrenal: F(3,21) =0.5132, p> 0.05; right adrenal: p> 0.05, F(3,21)

2.857, p> 0.05; Fig. 3.35c,d).



4- -B- 0 mg/kg
-- 3 mg/kg
-*- 10 mg/kg
a 3- 30 mg/kg
0



CT T
u,





0' !
0 1 2 3 4 5 6
day

Fig. 3.29. Effects of memantine on pemoline-induced self-injury: Memantine
did not affect pemoline-induced self-injury, as measured by the injury scores.
All values expressed are group means + S.E.M.



-B- 0 mg/kg
1,00- --3 mg/kg
-- 10 mg/kg
SE- 75- -A- 30 mg/kg

.iE 50-

L 25 B2


0 1 2 3 4 5 6
day


Fig. 3.30. Effects of memantine on the incidence of self-injury: Memantine did not
significantly affect the incidence of self-injury, as measured by the percentage of
rats that exhibited self-injury on day 6 of the experiment.













2 A
0) U)
E -
c 2.0.
S1.5

1.0-



a. 0 3 10 30
0
'r doses of memantine (mg/kg)


doses of memantine (mg/kg)


Fig. 3.31. Effects of memantine on induction and maintenance of pemoline-induced
self-injury: Memantine did not significantly alter either (a) induction or (b)
maintenance of pemoline-induced self-injury. All values expressed are group means +
S.E.M.


0 1 2 3
night


4 5


Fig. 3.32. Effects of memantine on the duration of pemoline-induced self-injurious
oral contact: Memantine did not significantly affect pemoline-induced self-
injurious oral contact. Self-injurious oral contact peaked on day 4 for most groups.
All values expressed are group means S.E.M.







53












S 0 3 10 30 0 3 10 30
S25- T

dose of memantine (mg/kg) dose of memantine (mg/kg)







E
15-
o 150

7015 w






Fig. 3.33. Effects of memantine on the induction and maintenance of pemoline-



induced self-injurious oral contact: Memantine did not significantly affect either
(a) induction or (b) maintenance of pemoline-induced self-injurious oral contact.
All values expressed are group means S.E.M.











A B
250- -- 0 mg/kg 700 -0 mg/kg
-- 3 mg/kg 600
*-- 10 mg/kg 0- -3 mg/kg
S-+ 30 mg/kg U 500- --10 mg/kg
150- 400- -30 mg/kg

S1 300
ooo
0
0 200-
50- -
100

0 1 2 3 4 5 0 1 2 3 4 5
night night


C D --0 mg/kg
200 --0 mg/kg5000
r i -M-3 mg/kg #
o 4-- 10 mg/kg
--10 mg/kg 4000 --30 mg/kg
El 30 mg/E


W 2000-

S. 0 o
SI I I M 0 I
0 1 2 3 4 5 0 1 2 3 4 5
night day



Fig. 3.34. Effects of memantine on grooming, inactivity and locomotion:
Memantine did not significantly affect (a) time spent grooming or (b) time spent
inactive. Memantine-treated rats exhibited significantly greater counts of
locomotion, both (c) overnight and (d) post-injection. Significant differences
between vehicle- and memantine-treated rats (LSD) are depicted as follows: p<
0.05 for comparisons between memantine at 30 mg/kg and vehicle; 7 p< 0.05 for
comparisons between memantine at 10 mg/kg and vehicle; # p< 0.05 for
comparisons between memantine at 3 mg/kg and vehicle. All values expressed are
group means + S.E.M.











-- Pem + veh
--Pem + 3 Mem
A --Pem + 10 Mem
-- Pem + 30 Mem
300- *




225-

200-
o 100


0 1 2 3 4 5 6


200-




0 100.



E


H

C

0)
a0)

EE


U 3 10 30
dose of memantine (mg/kg)


I I I I
0 3 10 30
dose of memantine (mg/kg)


U 3 10 30
dose of memantine (mg/kg)


Fig. 3.35. Effects of memantine on the health status of the rats: (a) There was a
significant interaction between time and group for body weight. The rats in the
pemoline plus memantine groups lost more weight than the rats in the pemoline
plus vehicle group did. Significant differences between vehicle- and memantine-
treated rats (LSD) are depicted as follows: p< 0.05 for comparisons between
memantine at 30 mg/kg and vehicle; p< 0.05 for comparisons between
memantine at 10 mg/kg and vehicle; # p< 0.05 for comparisons between
memantine at 3 mg/kg and vehicle. (b) There were significant between-groups
differences in the weights of the thymus glands, however, no significant
differences were found between the memantine-treated groups and the vehicle-
treated group. Memantine had no affect adrenal gland weight (c,d). All values
expressed are group means + S.E.M.


C>
Bo.

v E
0-






56


Inter-observer reliability

Inter-observer reliability for injury scores across the five experiments was as

follows. In 93% of cases, the two observers' scores matched exactly. In 6% of cases, the

scores were mismatched by one point on the 5-point scale (e.g., one observer assigned a

score of 2, and the other observer assigned a score of 3). In less than 1% of cases, the

scores were mismatched by 2 points, and the scores were never mismatched by 3 points

or more.














CHAPTER 4
DISCUSSION

The results of the current study replicate previous findings that approximately

75% of the rats exhibit SIB when treated with pemoline at 200 mg/kg/day, and most of

the self-injury was targeted at the forepaws and ventrum (Kies and Devine, 2004). In

four of the five current experiments, approximately 75-80% of the rats exhibited tissue

injury when injected daily with 200 mg/kg pemoline (i.e. those that were injected with

pemoline plus vehicle). The only exception was the nifedipine experiment in which

100% of the rats self-injured when treated with pemoline and the vehicle (see discussion

below). The fact that some of the rats in the groups that were treated with pemoline and

vehicle did not self-injure in four of the five experiments, suggests that there are

individual differences in vulnerability to develop pemoline-induced SIB. Individual

differences in vulnerability to develop pemoline-induced SIB resemble the expression of

SIB in clinical populations. Even in disorders with a high prevalence of SIB, there are

individuals who do not self-injure. Individual differences in the vulnerability to develop

pemoline-induced SIB may provide a useful tool for investigating the neurobiological

differences between rats that exhibit pemoline-induced SIB and those that do not.

In these experiments we used multiple measures to characterize pemoline-induced

SIB. These included injury scores that detail the severity and the prevalence of injury in

each treatment group. We also evaluated a measure of the behavioral expression of SIB,

using the duration of self-injurious oral contact on the body, and we used the injury

scores and the oral contact scores to evaluate the rate of onset and the ongoing









maintenance of the pemoline-induced self-injury. An analysis of the behavioral

expression, or duration, of SIB has generally not been used (for an exception, see King et

al., 1995). It has even been proposed that the behavioral expression of pemoline-induced

SIB could not be quantified because of its resemblance to normal grooming (Mueller &

Hsiao, 1980). In fact, we found that there was general concordance between the

measures of self-injury, and that the measure of prolonged oral contact reliably

discriminates between grooming and SIB. Additionally, we found that the quantification

of the behavioral expression of SIB highlighted important information about the

pemoline-induced SIB; information that the injury scores alone were not sensitive enough

to decipher. The oral contact data from the overnight videos indicated that there was

pemoline-induced self-biting behavior before there were any signs of injury. The

quantification of SIB also indicated that the behavioral expression of SIB actually peaks

and then declines during the five nights of the experiment in most of the groups of rats

that were treated with pemoline plus vehicle. The injury scores remained at asymptotic

levels around this time and so they did not accurately reflect this eventual decrease in

self-biting behavior. The reason for the decline in behavioral expression of pemoline-

induced SIB is not known. Perhaps the decline in self-injurious oral contact results from

tolerance to the injury-inducing effect of pemoline. This decline could also be a response

to the pain of injuring tissue that has been traumatized. This interesting finding will

require further investigation. On the other hand, the analysis of the self-injurious oral

contact was not very sensitive to the severity of biting. In two of the experiments

(valproate and nifedipine), the rats that were treated with the drug challenges exhibited

lowered injury scores, but no significant effect on oral contact scores. Apparently, these









rats engaged in prolonged oral contact, with less severe self-biting so that they exhibited

lower amounts of tissue damage than the vehicle-treated rats did. Overall, these multiple

dependent variables each measure different aspects of pemoline-induced SIB and allow

for a more thorough characterization of the self-injury.

The drugs that were evaluated in these experiments were designed to provide

specific information about the pemoline model of SIB, and about the potential effects of

drug challenges in this model. In particular, the drug challenges were designed to assess

the predictive validity of the model (i.e. risperidone and valproate), the generalizability of

the pemoline model in relation to other animal models of SIB (i.e. risperidone and

nifedipine), and pharmacotherapies that may have clinical potential (i.e. tramadol and

memantine). The results of these pharmacological studies generated support for the

predictive validity and generalizability of the pemoline model. They did not yield any

promising leads for previously untested pharmacotherapy, but these studies revealed

interesting information about the potential use of the model to uncover the

neurobiological basis of SIB. The fact that the rats expressed lower amounts of SIB if

they were treated with some of the challenge drugs (risperidone, valproate, and

nifedipine) suggests that the neurochemical mechanisms that are directly or indirectly

addressed by these drugs may be important mediators of the induction and expression of

SIB, and these mechanisms may be important targets for future development of

pharmacotherapies.

The fact that risperidone attenuates pemoline-induced SIB suggests that the model

has predictive validity. Risperidone decreased SIB in both clinical samples (Allen &

Rice, 1996; Cohen et al., 1998; McCracken et al., 2002; Caicedo &Williams, 2002) and









in this animal model. Additionally, risperidone has lessened the occurrence of SIB in

another animal model (Allen et al., 1998). These results provide evidence for the

generalizability between the different animal models of SIB. This generalizability will

allow for neurobiological analyses that can highlight the common substrates that lead to

SIB in these models. The results from the risperidone experiment also provide further

evidence that disregulated monoaminergic neurotransmission is important for the

etiology and maintenance of SIB. Pemoline is a monoamine agonist (Molina &

Orsinghen, 1981) and risperidone is a monoamine antagonist (Leysen et al., 1988). Thus,

the opposing actions of these drugs, as they induce SIB and block SIB, suggests that

disregulated monoaminergic systems cause SIB in the pemoline model. This is

consistent with the evidence of disregulated monoaminergic neurotransmission in clinical

populations that exhibit SIB (Lloyd et al., 1981; Ernst et al., 1996; Wong et al., 1996).

The inconsistency between the effects of valproate on injury scores and its effects

on duration of self-injurious oral contact suggests that the effects of valproate were more

subtle than were the effects of risperidone. These results show that valproate lessened the

severity of the pemoline-induced self-injury, as measured by the injury scores, but did not

reduce the expression of stereotyped oral behaviors, as quantified from the overnight

videotaped samples. The significant valproate-induced decrease in the severity of the

tissue injury further indicates that the pemoline model has predictive validity since this

finding is consistent with decreases in SIB in autistic and intellectually handicapped

patients treated with valproate (Kastner et al., 1993; Hollander et al., 2001). Valproate

increases extracellular GABA concentrations by blocking the degradation of GABA by

GABA transaminase (Loscher, 1993). It also blocks voltage-gated sodium channels









(McLean & McDonald, 1986), calcium channels (Kito et al., 1994) and protein kinase C

(Chen et al., 1994). Accordingly, it is not clear whether valproate reduced pemoline-

induced self-injury through GABAergic mechanisms, ion channels actions or through

alterations in cell signaling pathways. But, it did not appear that the injury-reducing

effects of valproate were due to general sedation because the duration of self-injurious

oral contact, grooming behaviors, inactivity and amount of locomotion were not different

between the groups.

The fact that nifedipine attenuated pemoline-induced self-injury, and appeared to

produce an overall inhibition of the self-injurious oral contact (although this did not reach

statistical significance), concurs with the findings that nifedipine lowers the SIB in the 6-

OHDA (Blake et al., 2004) and Bay-K 8644 (Jinnah et al., 1999) models. This indicates

that there is generalizability between these different animal models of SIB. This could

indicate that there are commonalities in the neurobiological actions that initiate SIB in

each of these animal models. Identification of these common mechanisms could help to

characterize the neurobiological conditions that are necessary and sufficient to induce

SIB. One obvious factor is dopamine disregulation, and this investigation using

nifedipine contributes further evidence that dopamine is important in the expression of

SIB. L-type calcium channels are found predominantly on the presynaptic neurons

(Okita et al., 2000) in the striatum and cortex (Hirota and Lambert, 1997) and are

associated with increased release of dopamine from the caudate when activated (Okita et

al., 2000). Additionally, blocking these channels decreases the rate of action potentials of

midbrain dopaminergic neurons (Mercuri et al., 1994).









One potential confounding variable in this study is that the vehicle for the

nifedipine contained ethanol. Ethanol was not used in any of the other experiments in

this or previous studies, and the effect of ethanol on pemoline-induced SIB is not known.

The rats that just received pemoline plus vehicle exhibited more severe self-injury than

the rats did in any other experiment, even though all the groups of rats were treated with

pemoline at 200 mg/kg/day. All the rats in the pemoline plus vehicle group were

euthanized before the end of the experiment because they reached an injury score of 4.

Accordingly, it is possible that this anomalous outcome may have actually resulted from

an interaction between the ethanol and the pemoline in this experiment. This possibility

will require further study. Nevertheless, nifedipine significantly reduced the overall SIB,

demonstrating the effectiveness of this drug challenge.

Significant thymus involution was observed in all the nifedipine-treated groups.

This is consistent with reports that nifedipine administration causes thymic apoptosis

(Balakumaran et al., 1996). Accordingly, it does not appear that nifedipine could have

any clinical potential for treatment of SIB, but further studies with this drug may help to

reveal neurobiological mechanisms that underlie the induction and maintenance of SIB in

the various animal models in which it is effective.

Although tramadol has been reported to decrease compulsive behaviors in

individuals with OCD and Tourette's syndrome (Goldsmith et al., 1999; Shapira et al.,

1997) it did not significantly affect any of our dependent measures of SIB in the

pemoline model. One potential interpretation of these results is that pemoline-induced

SIB is not a compulsive behavior. This is contradictory to our casual observations, where

we have noticed that the rats are extremely difficult to distract after they have initiated









self-biting behavior and that even if the injury site is out of reach for the rat (i.e. when the

paw is being shown to the camera during the visual inspections) the rat will begin to

nibble on other items, such as the examiners glove or lab coat. Another possibility is that

tramadol does not disrupt compulsive behavior patterns with enough strength to combat

the compulsive nature of pemoline-induced SB. The two studies that describe the

effectiveness of tramadol to combat compulsive behaviors in OCD and Tourette's

syndrome are open-labeled and they included small sample sizes. Thus, the clinical

efficacy of tramadol is not yet well established. Moreover, it is possible that the dosage

(0, 0.1, 1.0, 10 mg/kg) or dosing regimen (b.i.d.) of tramadol that we used in this study

may not have been aggressive enough. We chose doses below 25 mg/kg/day because

higher doses have been shown to induce abnormal chewing movements, vigorous

grooming and spasms (Matthiesen et al., 1998). Accordingly, it seems unlikely that

higher doses of tramadol would be effective in the pemoline model of SIB. The potential

that compulsions play an important role in pemoline-induced SIB merits further

evaluation.

The fact that memantine did not affect any of our dependent measures of

pemoline-induced SIB is disappointing from a clinical perspective. Although the

pemoline model is not a definitive screening tool for the efficacy of therapeutic drugs, the

results from the memantine study suggest that it may not be an effective drug for

reducing SIB in clinical populations. This negative outcome is somewhat surprising

since memantine and MK-801 are both non-competitive NMDA receptor antagonists

(Wong et al., 1986; Bormann, 1989) and MK-801 blocked pemoline-induced SIB (King et

al., 1995). This difference in findings could be explained by the difference in









experimental procedures. King and colleagues investigated interactions between MK-

801 and pemoline only acutely, as MK-801 and pemoline were administered only once in

their study. In the present study the effects of blocking the NMDA receptor were studied

with repeated administration of memantine (b.i.d.) and pemoline (q.d.) for five days.

Although there is no specific reason to suspect that these different dosing regimens would

produce differing outcomes, it is possible that higher doses or more frequent

administration of memantine may have had some effect. However, memantine has been

found to produce some learning deficits and ataxia when administered at 20 mg/kg

(Hesselink et al., 1999) and in our study we used doses up to 30 mg/kg b.i.d.

Accordingly, higher doses of memantine may not be appropriate in this model.

Another potential reason for the effectiveness of MK-801 and the ineffectiveness

of memantine is that the actions of these drugs at the NMDA receptor differ substantially

from each other. MK-801 exhibits high affinity binding to activated NMDA receptors,

and becomes trapped in the ionophore where it cannot be displaced. Accordingly, MK-

801 blocks the physiological actions that result from transient release of glutamate (e.g.,

learning and memory) and it also blocks pathological actions that result from prolonged

glutamate stimulation (e.g., apoptotic cascades in neurodegenerative diseases).

Memantine, on the other hand, exhibits low affinity binding to the ionophore of the

activated NMDA receptors. As such, the binding is transient. Accordingly, memantine

is neuroprotective in conditions where there is prolonged glutamate stimulation (e.g.,

neurodegenerative disorders such as Alzheimer's disease) because it decreases the

calcium influx during the prolonged stimulation. However, under basal conditions, when

the postsynaptic NMDA receptor is generally quiescent, memantine is not bound to the









receptors, which allows transient binding of glutamate and the opening of the ion channel

to occur. If memantine does bind, the binding is rapidly reversible, and so the next time

normal transient glutamate stimulation occurs, the process is repeated. Accordingly,

memantine is only a very low potency antagonist against normal physiological actions of

glutamate at NMDA receptors (for review see Sonkusare et al., 2005). In fact, a

comparative analysis reveals that MK-801 exhibits very high potency blockade of

NMDA receptor-dependent hippocampal long term potentiation (LTP), whereas

memantine has only very low potency and does not affect LTP (Frankiewicz et al., 1996).

The discrepancy between the effects of MK-801 (King et al., 1995) and the lack

of effects of memantine (present results) coupled with the observation that there is a lag

between the onset of treatment and the onset of SIB when the rats are treated with a

moderately high dose of pemoline (Kies and Devine, 2004; present results), raises the

interesting possibility that glutamate-mediated neuroadaptations may play an important

role in pemoline-induced SIB. In this case, the MK-801 was effective because it blocked

the ability of pemoline to initiate these adaptations, and memantine was ineffective

because it did not block the neuroadaptations. An analysis of the systems in which

pemoline induces neuroadaptation through glutamate-mediated actions may reveal

neuropathological disregulation in the pemoline-treated rats that exhibit SIB.

Identification and characterization of disregulated systems that differentiate self-injurious

from non-injurious rats may provide interesting leads to examine neuropathological

substrates that underlie SIB in clinical populations.

In the valproate, nifedipine and tramadol experiments, the drug challenges had no

significant impact on any of the measures of grooming, inactivity or locomotion









(overnight videotapes and photocell counts). In the risperidone experiment, the duration

of inactivity on the last night of the experiment was significantly greater in rats treated

with the highest dose of risperidone, as compared to vehicle-treated rats. It does not

appear that risperidone was exerting its effect through sedative actions, however, because

duration of grooming and amount of locomotion were not different between risperidone-

and vehicle-treated groups. During the first two days of the memantine experiment, the

memantine-treated rats exhibited greater locomotor counts, both overnight and after the

morning injections, compared to that of the vehicle-treated rats. Beginning on day 3, the

locomotor counts of the memantine-treated rats no longer differed from that of the

vehicle-treated rats. The differences in locomotor counts between memantine- and

vehicle-treated rats are mostly inconsequential because memantine had no significant

effect on pemoline-induced self-injury.

The rats remained reasonably healthy throughout the experiments. Pemoline

caused some weight loss, which began to level off or improve on day 3 or 4 in most

experiments. This may be due to the psychomotor stimulating effects of pemoline or

altered feeding behaviors during pemoline treatment. Chromodaccryorrhea (porphyrin

containing secretions around the eyes; Payne, 1994) was noticed in some of the rats.

These secretions have been associated with stress (Harkness & Ridgway, 1980; Ross,

1994; Chen et al., 1997) and with toxic actions of a variety of drugs (Moser, 1991; Sauer

et al., 1995; Pegg et al., 1996; Graziano et al., 1996; Sauer et al., 1997). The presence of

chromodaccryorrhea replicates a previous finding when rats were treated daily with 200

mg/kg pemoline (Kies & Devine, 2004), but as in the previous study, this was a minor









effect that occurred in a very small number of the rats, and did not appear to be associated

with the induction or maintenance of SIB.

In summary, these projects provide evidence of the predictive validity of the

pemoline model to mimic the SIB seen in clinical populations as risperidone and

valproate lessened pemoline-induced SIB. They also provide more evidence of

monoamine involvement in SIB because risperidone and nifedipine each reduced the self-

injury in the pemoline model. Results from these experiments also indicate that common

neurobiological substrates may underlie the induction of SIB in multiple animal models,

as evidenced by the fact that nifedipine consistently blocks SIB in these various animal

models. Also, the current evidence suggests that glutamate-mediated neuroadaptations

may be involved in producing pemoline-induced SIB since memantine did not affect

pemoline-induced SIB. This interesting possibility merits further attention.

Unfortunately, the tramadol experiment suggests (within the limits that the model may

predict clinical efficacy) that this treatment does not hold therapeutic promise.

In accordance with the observations of these experiments, the predictive validity

and pre-clinical screening potential of the pemoline model should be investigated further

using drugs that are effective in decreasing SIB in different clinical populations. In light

of the fact that the monoamine agonist pemoline only produced SIB after days of

treatment, and that a monoamine antagonist blocked the effect, the dynamic regulation of

neurotransmission in dopaminergic, serotonergic, and noradrenergic systems should be

examined in relation to the onset and expression of the SIB. Additionally, the role of

specific glutamate-mediated neuroadaptations should also be investigated. Challenging

the pemoline model of SIB with drugs that inhibit the protein kinases that mediate









neuroadaptations, such as a protein kinase B and protein kinase C, will begin to elucidate

the changing mechanisms that produce pemoline-induced SIB. Additionally, challenging

the pemoline model with a protein kinase C inhibitor and with another GABA agonist,

topiramate, will also help decipher whether valproate reduced the pemoline-induced self-

injury through its actions on GABA or its actions on the kinase. These future

experiments will improve our knowledge about the neurobiological mechanisms that are

producing and maintaining this devastating behavior disorder and will lead to the

elucidation of potential targets for new pharmacotherapies.















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BIOGRAPHICAL SKETCH

Amber Marie Muehlmann graduated cum laude from San Diego State University

with a Bachelor of Arts in May 2002. She began her graduate education in August 2003

working towards her Master of Science degree in the behavioral neuroscience program in

the Psychology Department at the University of Florida.