1 Pemoline-Induced Self-Injurious Behavior in the BALB/c Mouse Strain Kanita Beba Interdisciplinary Studies in Neuroscience, University of Florida 2 ABSTRACT Self-injurious behavior (SIB) is a devastating behavior disorder that is highly prevalent in the intellectually handicapped, those with specific genetic syndromes, and in certain psychiatric disorders. Although various theories of its basis have been proposed, the biochemical mechanisms leading to SIB remain largely unknown. Various animal models are used to investigate the etiology of SIB, among them the pemoline model of SIB in rats. In this model, repeated administration of pemoline induces stereotyped behavior and dose-orderly SIB. The purpose of this study was to investigate whether a pemoline- induced model of SIB can be created in an inbred BALB/c mouse strain. We found that mice did not exhibit SIB when administered pemoline at 150 mg/kg/day and 200 mg/kg/day, but that they do injure at 250 mg/kg/day, and the expression of the behavior appeared quite similar to that which we have previously observed in rats. All the mice injured after the administration of the 250 mg/kg/day concentration. Furthermore, the average injury score, average injury area and the percentage of animals injuring all increased over time, suggesting that there is a progression in injury over time in the pemoline mouse model. In addition, none of the animals became sick or showed signs of toxicity. These results could allow for novel genetic manipulations in future studies of SIB, but further characterization of the model with additional mouse strains is necessary. INTRODUCTION Self-injurious behavior (SIB) is commonly exhibited by the intellectually handicapped, those with specific genetic syndromes, and by individuals with certain psychiatric disorders (Rojahn & Esbensen, 2002). Examples of SIB include head-banging, 3 self-biting, self-punching and a variety of other forms (Symons & Thompson, 1997). The biochemical mechanisms leading to SIB remain unknown, although various theories have been proposed to explain its origins. Furthermore, only a sub-population of individuals with the aforementioned genetic disorders exhibit SIB, suggesting that there are individual differences contributing to the vulnerability to self injure that still need to be studied and which may possibly give insight into the etiology of the behavior (Thompson & Caruso, 2002). The genetic disorders in which SIB is common include Prader-Willi Syndrome, Cornelia de Lange Syndrome, Autism and Lesch-Nyhan Syndrome (LNS). Although these disorders have differing phenotypes and even differing topographies of injury (Symons & Thompson, 1997), there is much overlap between them in the regulation of certain neurochemical substrates and in the responsiveness to pharmacological methods. This has helped to confirm the involvement of specific neurotransmitter systems and to demonstrate the effectiveness of specific drug therapies. Dopaminergic neurotransmission may be disrupted in disorders in which SIB is common. There is evidence of altered dopamine function in LNS, and perhaps in some individuals with autism (Wong et al., 1996;Ernst, Zametkin, Matochik, Pascualvaca & Cohen, 1997; Garcia, Puig & Torres, 2009) In LNS, where all or nearly all individuals exhibit self- biting (Nyhan, 1968), PET scans show decreased dopamine transporters (Wong et al., 1996). In addition, mRNA expression in peripheral blood lymphocytes shows altered adenosine (ODORA2A) and dopamine (DRD5) receptor expression (Garcia et al., 2009), and post-mortem studies show abnormal dopamine receptor expression (Saito et al., 1999). 4 Dopaminergic neurotransmission also appears to be disrupted in all of the animal models of SIB. There is evidence of altered dopamine function in the neonatal 6- hydroxydopamine (6-OHDA) animal model, pemoline model, clonidine model, Bay K 8644 model, and in isolation reared animals (Breese et al., 1984; Kasim & Jinnah., 2003; Tiefenbacher, Novak, Lutz & Meyer, 2005; Muehlmann, Brown & Devine., 2008a; Devine & Muehlmann, 2009). In the 6-OHDA animal model, SIB in the form of self- biting is induced by deliberately destroying dopaminergic neurons in neonatal rats and subsequently administering dopamine agonists such as apomorphine or L-dopa in adulthood (Breese et al., 1984). Studies of animal models of SIB also show that serotonergic neurotransmission may be involved in the regulation of SIB, as seen in the 6-OHDA model, pemoline model, clonidine model and Bay K 8644 model (Breese et al., 1984; Bhattacharya, Jaiswal, Mukhopadhyay & Datla, 1988; Towle et al., 1989; Turner, Panksepp, Bekkedal, Borkowski & Burgdorf, 1999; Kasim, Egami & Jinnah, 2002; Muehlmann et al., 2008a). Overlap in efficacies of drugs such as SSRIs and Risperidone in both the disorders and animal models further confirms that serotonergic and dopaminergic neurotransmission is disrupted and that these neurochemical substrates may be involved in the etiology of SIB (Muehlmann et al., 2008a). Topiramate has not only successfully attenuated SIB in Prader-Willi Syndrome (Shapira, Lessig, Murphy,Driscoll & Goodman, 2002), but it has also decreased SIB in psychiatric disorders such as borderline personality disorder and bipolar disorder (Cassano et al., 2001). Pharmacological convergence has also been observed in nifedipine, an L-type Calcium channel antagonist which reduces SIB in four unrelated animal models: the neonatal 6-OHDA, Bay K 8644, 5 methamphetamine and pemoline models (Blake et al., 2007). Early social development and deprivation have repeatedly shown to contribute to the etiology of SIB in many species of animals, including humans (Devine & Muehlmann., 2009; Beckett et al., 2002). This has been evident in many studies of early experience in primates, although the primate model doesn’t necessarily require early isolation or pharmacological manipulation to induce SIB (Tiefenbacher et al., 2005). The success of inducing SIB in the 6-OHDA model is dependent on the age at which the neurotoxin is administered (Breese et al., 1984). Stress has also been shown to play a role in the expression of SIB in the 6-OHDA model, and chronic stress has shown to exacerbate SIB in the pemoline model (Muehlmann, Wolfmann & Devine, 2008b). Furthermore, studies of stress in the pemoline model have suggested that the difference in responsiveness to stress in rats may be a factor in the individual vulnerability to self injure (Devine, Wilkinson & Muehlmann, 2007). Rats that are highly responsive (HRs) have higher levels of corticosterone as well as a unique gene expression in brain regions that control stress responsiveness (Kabbaj, Devine, Savage & Akil 2000), and were found to have higher nociceptive thresholds than the low responsive rats (LRs) (Devine et al., 2007). The pemoline rat model is an effective pharmacological model of SIB (Muehlmann et al., 2008a). An indirect dopamine agonist, pemoline is thought to block neuronal uptake of dopamine and norepinephrine (Molina & Orsingher, 1981), while its effects on serotonin levels are less clear (Ramirez, Vial, Barailler, & Pacheco, 1978). SIB in rats usually occurs within 48 hours with a 250-300 mg/kg injection, or after 3-12 daily injections at smaller dosages (Mueller, Hollingsworth & Pettit, 1986; Kies & Devine, 2004; Muehlmann & Devine, 2008b; Muehlmann et al., 2008a). It is accompanied by an 6 increase in stereotyped behaviors, and locomotor and exploratory activity (Mueller & Hsiao, 1980). The injury is enhanced by neonatal isolation, and chronic stress also appears to play a role (Muehlmann et al., 2008b). A comparison study between the caffeine and pemoline models of SIB in rats demonstrates the advantages of the latter. All of the caffeine treated rats showed caffeine toxicity at high doses. Whereas those high doses of caffeine produced only mild SIB and in only a small percentage of the rats, nearly 100% of the rats treated with pemoline exhibited SIB within four days of treatment. Pemoline was effective at a broader range of doses (100-300 mg/kg/day), showed dose-orderly differences, and the treated rats demonstrated individual variability in injury that is reminiscent of what has been documented in human patients. Pemoline treated rats showed mild signs of toxicity only at the highest dose (Kies & Devine, 2004). One of the advantages of the pemoline model when compared to other animal models of SIB, is that it has shown predictive validity for pharmacotherapeutic effects when tested with drugs (valproate, risperidone and topiramate) that have been effective in human patients exhibiting SIB (Muehlmann et al., 2008a). Additional investigation revealed that an antagonist of glutamatergic neurotransmission (MK-801) blocked the induction of SIB in the pemoline model, and lead to the hypothesis that pemoline-induced SIB may be a result of altered interactions between glutamatergic and monoaminergic systems (Muehlmann & Devine, 2008b). Similar findings were found when the NMDA antagonist was used in the 6-OHDA model. There appear to be no individual differences in metabolism of pemoline between rats that are vulnerable or resistant to pemoline-induced SIB (Muehlmann & Devine, 2007), although the glutamatergic and dopaminergic inputs 7 are altered in the striatal medium spiny neurons of the vulnerable rats (Cromwell, King & Levine, 1997). Traditional research has focused on creating animal models that would exhibit the entire range of human symptoms that are characteristics of those disorders like autism. However, these disorders