1 Pemoline-Induced Self-Injurious Behavior in the BALB/C Mouse

Pemoline-Induced Self-Injurious Behavior in the BALB/c Mouse Strain

Kanita Beba

Interdisciplinary Studies in Neuroscience, University of Florida

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).

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 have complex etiologies and various symptoms and triggers.

Thus it is often difficult to model all of the symptoms and characteristics of a disorder in a single animal model (Devine & Muehlmann, 2009). Instead, recent research has focused on identifying and targeting specific endophenotypes that are common across various disorders. This strategy improves translational power and validity of the results.

SIB is one of the studied endophenotypes. However, most of the animal models of the behavior, including the pemoline model, are currently studied in rats. Conversely, most genetic research has been done in mice. Having a mouse model of SIB would allow for novel genetic manipulations and might give new insight into the etiology of the disorder.

The purpose of this study is to investigate whether a pemoline-induced mouse model of SIB can be created in an inbred BALB/c mouse strain. Since there may be metabolic differences between this mouse strain and the Long-Evans rat strain used in the established pemoline rat model of SIB, it was important to characterize the mouse strain at different doses in order to find the optimal model of SIB.

METHODS

Animals

Six male BALB/c mice weighing between 18 and 21 grams were purchased from Charles

River Laboratories and were housed in an AAALAC-approved vivarium. The humidity,

8 temperature and light schedule were automatically controlled, and the mice were kept on a 12-hour light cycle with the lights on at 7:00 am. All animals were given free access to food and tap water, and were housed in standard polycarbonate cages (25.4 x 15.2 x 12.7 cm). The mice were housed three per cage for about 6-7 days prior to the first injection, and were subsequently singly-housed to ensure that any SIB was self-inflicted. All experimental procedures were conducted in accordance with the Guide for the Care and

Use of Laboratory Animals and were pre-approved by the Institutional Animal Care and

Use Committee at the University of Florida.

Drugs

Pemoline (2-amino-5-phenyl-1,2-oxazol-4-one) purchased from Spectrum Chemical Co.

(New Brunswick, NJ) was suspended in peanut oil at 25 mg/ml (for the lower two doses) and at 50 mg/ml (for the highest dose) and administered at three different doses: 150 mg/kg/day , 200 mg/kg/day and 250 mg/kg/day. The mice were weighed and injected at approximately 9:30 am. The injections were administered subcutaneously using 23 gauge needles at the nape of the neck.

Experimental Procedures

After the mice were acclimated to the housing facility, they were subjected to three consecutive experimental conditions: The mice were injected daily with pemoline at 150 mg/kg for five days, then with pemoline at 200 mg/kg for six days, and then with pemoline at 250 mg/kg for six days. There were 9 days without pemoline treatment between the 150 mg/kg and 200 mg/kg doses, and 22 days without pemoline treatment

9 between the 200 mg/kg and 250 mg/kg doses. Each mouse was weighed at approximately

9:30 am daily, and immediately injected with pemoline. On the second day following the last day of pemoline administration (day 8 of the 250 mg/kg condition), the mice were inspected for injury for the final time. They were then immediately placed in a sealed plastic chamber and terminated by CO2 gas. The chest cavity of each animal was then opened with scissors to create a pneumothorax.

Assays of Self-Injury and Other Behaviors

The mice were inspected twice a day during the injection regimen: in the morning immediately before injections and again at approximately 5:30 pm. Injury scores (see

Table 1.) were assigned to each mouse at both daily inspection times. The mice in the 250 mg/kg dose group were also inspected on two additional days after the 6-day regimen of pemoline injections. In addition to being closely inspected by physical manipulation, the mice were also video recorded twice a day during the inspections. Each mouse was held approximately 30 cm away from the camera, while the head, forepaws, hindpaws, ventrum and tail were displayed. Images of each injured tissue site were digitally captured from the video footage and calibrated with a 5 mm marker on the gloved index finger in order to measure the size of the tissue injury area. The calibration and tissue injury area measurements were determined using MCID Image Analysis Software.

Although the images from the video were also used to confirm the injury scores previously assigned during the daily inspections, all of the original injury scores from the inspections were reported in the results.

Table 1. Injury Score Assessment Score Classification Description 0 no SIB 1 very mild SIB denuded skin, edema or erythema; involves small area denuded skin, edema or erythema; involves medium area or 2 mild SIB multiple small sites denuded skin, edema or erythema; involves large area or multiple 3 moderate SIB medium sites 4 severe SIB open lesion. Requires euthanasia.

No animals had to be euthanized prior to the conclusion of the experiment.

Statistical Analyses

Linear regression was used to determine whether there was a relationship between

pemoline administration and the injury scores, areas of injury, and weights of the mice

over time. A significant effect was inferred if the slope of the regression line for any

measure was significantly different from zero.

RESULTS

A pemoline-induced model of SIB was created in the BALB/c mouse strain, since

the mice that were treated with daily injections of pemoline at 250 mg/kg exhibited self-

injury. However, it appears that the lower doses of pemoline (150 mg/kg and 200 mg/kg),

were not high enough concentrations to induce SIB in the particular mouse strain. The

percentage of mice injuring (Fig 1A), the tissue injury scores (Fig. 1B), and the tissue

11 injury areas (Fig. 1C) all remained zero in the 150 mg/kg and 200 mg/kg conditions.

A B 100 4 pemoline 250 mg/kg pemoline 200 mg/kg 3 75 pemoline 150 mg/kg

50 pemoline 250 mg/kg 2 pemoline 200 mg/kg pemoline 150 mg/kg

exhibited injuryexhibited 25 1

percentage of mice that percentage

0 injurytissue score average 0 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 day day

C D 30 pemoline 250 mg/kg 150 pemoline 200 mg/kg 25 pemoline 150 mg/kg

) 20 2 100 15

(mm pemoline 250 mg/kg 10 50 pemoline 200 mg/kg

average weight (g) average 5 pemoline 150 mg/kg

average tissue injurytissue area average 0 0 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 day day

Fig 1. Effects of pemoline administration over time (150 mg/kg/day, 200 mg/kg/day and 250 mg/kg/day), on the percentage of mice exhibiting injury, tissue injury scores, tissue injury areas, and body weights. The percentage of mice exhibiting self-injury increased over time in the 250 mg/kg/day dose group, while it remained zero in the 150 mg/kg/day and 200 mg/kg/day dose groups (A). There is a positive relationship between time and tissue injury score in the 250 mg/kg/day dose group, while there is no induction of tissue injury in the 150 mg/kg/day and 200 mg/kg/day dose groups (B). There is a positive relationship between time and tissue injury area in the 250 mg/kg/day dose group, while there is no increase in tissue injury area over time in the150 mg/kg/day and 200 mg/kg/day dose groups (C). There was no significant relationship between time and weight of the mice in the 250 mg/kg/day dose group, while there was a significant relationship between time and weight of the mice in the 150 mg/kg/day and

200 mg/kg/day dose groups (D). Values expressed in B, C and D are group means ± S.E.M.

However, the percentage of mice exhibiting injury increased significantly over days, and reached 100% by the second day after discontinuation of the 250 mg/kg/day regimen (Fig. 1A). There was a significant increase in the average tissue injury score over time in the 250 mg/kg/day condition [F(1,82)= 58.23, p<0.0001](Fig. 1B). These mice also exhibited a significant increase in the average area of injury over time [F(1,82)=

19.34, p<0.0001](Fig. 1C). However, the average area of injury decreased during the final two days of the experiment, during which the mice received no pemoline (days 7 and 8).

The average weight of the mice increased significantly over time during the days of pemoline administration in the 150 mg/kg dose group [F(1,58)= 10.65, p=0.0018] and the 200 mg/kg dose group [F(1,70)= 52.54, p< 0.0001], but did not change over days in the 250 mg/kg dose group [F(1,82)= 0.02370, p=0.8780] (Fig. 1D). None of the mice showed any signs of sickness or toxicity at any of the dosages of pemoline.

DISCUSSION

In this study, we investigated whether a pemoline-induced mouse model of SIB can be created in the BALB/c strain. Average tissue injury score, average tissue injury area and the percentage of animals exhibiting injury all increased over time during the pemoline administration at 250 mg/kg/day. The timing of the onset of SIB in the mouse model was similar to that which has been reported for the rat model. SIB in rats usually occurs within 48 hours with a 250-300 mg/kg pemoline administration (Muehlmann &

Devine, 2008b; Muehlmann et al., 2008a; Kies & Devine, 2004; Mueller, Hollingsworth

& Pettit, 1986), and this onset timing was also observed at the 250 mg/kg/day dosage in

13 the mouse model. Although self-injury was not seen at lower doses, this does not mean that the mouse model doesn’t show dose-orderly differences, and it is possible that dose- orderly effects might have been observed if higher doses were used. Although injury was apparent at 250 mg/kg/day, the mice were not administered 300 mg/kg/day to see if the increase in dose induced quicker and more severe injury. Other limitations of the study include the small sample size, and that only one group of mice was used over the three drug dosing conditions. Since the average weight of the mice increased significantly over time during administration of the 150 mg/kg/day and 200 mg/kg/day doses, but did not increase further during administration of the 250 mg/kg/day dose regimen, it could be interpreted that the high dose was suppressing ongoing weight gain. However, examination of the growth characteristics of BALB/c mice (Harlan Laboratories) indicates that they reach a maximum weight at about 13 weeks or 27 grams. Thus, the mice were likely at their full adult weights at the time of the high dose administration, and the lack of weight gain should not be interpreted as an adverse health effect of the drug.

Not only do animal models provide great translational power and strength of validity when convergence is found among the different models of SIB (Blake et al.,

2007), but the findings are further strengthened once they confirm the known abnormalities in the genetic syndromes (Muehlmann & Devine, 2008a). This study shows that a mouse model of SIB can be created using the pemoline drug in the BALB/c strain, and thus that novel genetic manipulation may be feasible in future studies of SIB.

Furthermore, it shows convergence. Even in a small sample of mice, the pemoline- induced SIB shares trends and characteristics with the established rat model of the drug.

Future studies should focus on further characterization of the pemoline-induced mouse model of SIB. In addition to adding a larger sample size in order to investigate whether nearly 100% of mice will exhibit SIB within four days of pemoline treatment

(Kies & Devine, 2004), using varying strains of mice, and expanding the range of doses administered in order to investigate whether there is a dose-orderly difference in injury, future research should also focus on finding out whether the mouse model confirms what has been observed in the rat model of pemoline induced injury. For example, it should be investigated whether injury in the mouse model is enhanced by early developmental deprivation and exacerbated by stress. It should also be investigated whether there are metabolic differences of the drug between mice vulnerable and invulnerable to SIB, as has been found in the pemoline rat model (Muehlmann & Devine, 2007). The mouse model should also be tested to see whether it shows predictive validity for pharmacotherapeutic effects when tested with drugs that have been effective in human patients exhibiting SIB. In the long term, these studies could lead to the use of novel transgenic research methods to identify and better understand the genetic factors that may confer increased vulnerability or resistance to SIB.

References

Beckett, C., Bredenkamp, D., Castle, J., Groothues, C., O'Conner, T. G., & Rutter, M. (2002). Behavior patterns associated with institutional deprivation: A study of children adopted from romania. Journal of Developmental & Behavioral Pediatrics, 23(5), 297.

Bhattacharya, S.K., Jaiswal, A.K., Mukhopadhyay, M. & Datla, K.P. (1988). Clonidine-induced automutilation in mice as a laboratory model for clinical self-injurious behaviour. Journal of Psychiatric Research, 22, 43-50.

Blake, B. L., Muehlmann, A. M., Egami, K., Breese, G. R., Devine, D. P., & Jinnah, H. A. (2007). Nifedipine suppresses self-injurious behaviors in animals. Developmental Neuroscencei, 29(3), 241-250.

Breese, G. R., Baumeister, A. A., McCown, T. J., Emerick, S. G., Frye, G. D., Crotty, K., & Mueller, R. A. (1984). Behavioral differences between neonatal and adult 6- hydroxydopamine-treated rats to dopamine agonists: Relevance to neurological symptoms in clinical syndromes with reduced brain dopamine. Journal of Pharmacology and Experimental Therapeutics, 231(2), 343.

Cassano, P., Lattanzi, L., Pini, S., Dell'Osso, L., Battistini, G. & Cassano, G.B. (2001). Topiramate for self-mutilation in a patient with borderline personality disorder. Bipolar Disorders, 3(3),161.

Cromwell, H.C., King, B.H. & Levine, M.S. (1997). Pemoline alters dopamine modulation of synaptic responses of neostriatal neurons in vitro, Developmental Neuroscience, 19:,497- 504.

Devine, D. P. & Muehlmann, A. M. (2009). Tiermodelle für selbstverletzendes Verhalten (Animal models of self-injurious behavior), In: Selbstverletzendes Verhalten bei stressassoziierten Erkrankungen (Self-Injurious Behaviour in Stress-Associated Disorders), In C. Schmahl and C. Stiglmayr, (Eds.). pp 39-60, Verlag W. Kohlhammer, Stuttgart.

Devine, D.P., Wilkinson, J.A., & Muehlmann, A.M. (2007). Sensory thresholds in an animal model of self-injurious behavior, Society for Neuroscience Abstracts. 33: 799.21

Ernst, M., Zametkin, A.J., Matochik, J.A., Pascualvaca, D. & Cohen, R.M. (1997). Low medial prefrontal dopaminergic activity in autistic children. Lancet, 350, 638.

García, M. G., Puig, J. G., & Torres, R. J. (2009). Abnormal adenosine and dopamine receptor expression in lymphocytes of Lesch–Nyhan patients. Brain Behavior and Immunity, 23(8), 1125-31.

Kabbaj, M., Devine, D. P., Savage, V. R., & Akil, H. (2000). Neurobiological correlates of individual differences in novelty-seeking behavior in the rat: Differential expression of stress-related molecules. Journal of Neuroscience, 20(18), 6983.

Kasim, S., & Jinnah, H. A. (2003). Self-biting induced by activation of L-type calcium channels in mice: Dopaminergic influences. Developmental Neuroscience, 25(1), 20-25.

Kasim, S., Egami, K., & Jinnah, H. A. (2002). Self-biting induced by activation of L-type calcium channels in mice: Serotonergic influences. Developmental Neuroscience, 24(4), 322-327.

Kies, S. D., & Devine, D. P. (2004). Self-injurious behaviour: A comparison of caffeine and pemoline models in rats. Pharmacology, Biochemistry and Behavior, 79(4), 587-598.

Molina, V.A. & Orsingher, O.A. (1981). Effects of Mg-pemoline on the central catecholaminergic system. Archives Internationales de Pharmacodynamie et de Therapie, 251(1):66-79.

Muehlmann, A. M., Brown, B. D., & Devine, D. P. (2008a). Pemoline (2-amino-5-phenyl-1, 3- oxazol-4-one)-induced self-injurious behavior: A rodent model of pharmacotherapeutic efficacy. Journal of Pharmacology and Experimental Therapeutics, 324(1), 214.

Muehlmann, A.M. & Devine, D.P. (2008a). Self-injurious behavior: Individual differences in neurotransmitter concentrations using an animal model, Keystone Symposium: Towards Identifying the Pathophysiology of Autistic Syndromes. C2: 206

Muehlmann, A. M., & Devine, D. P. (2008b). Glutamate-mediated neuroplasticity in an animal model of self-injurious behaviour. Behavioural Brain Research, 189(1), 32-40.

Muehlmann, A.M., Wolfman, S., & Devine, D.P. (2008b). Examining the effects of chronic stress on self-injurious behavior in an animal model, Society for Neuroscience Abstracts. 34: 446.29.

Muehlmann, A.M.& Devine, D.P. (2007). Characterization of drug titers and neurotransmitter concentrations during induction and maintenance of pharmacologically-induced self- injurious behavior, Society for Neuroscience Abstracts. 33: 799.20

Mueller, K., Hollingsworth, E., & Pettit, H. (1986). Repeated pemoline produces self-injurious behavior in adult and weanling rats. Pharmacology, Biochemistry and Behavior, 25, 933- 938.

Mueller, K. & Hsiao, S. (1980). Pemoline-induced self-biting in rats and self-mutilation in the de Lange syndrome. Pharmacology, Biochemistry and Behavior, 13(5), 627-31.

Nyhan, W.L. (1968). Clinical features of the Lesch-Nyhan syndrome. Introduction--clinical and genetic features. Federation Proceedings, 27(4), 1027-33

Ramirez, A., Vial, H., Barailler, J. & Pacheco, H. (1978). [Effects of levophacetoperane, pemoline, fenozolone, and centrophenoxine on catecholamines and serotonin uptake in various parts of the rat brain]. Comptes Rendus Hebdomadaires des Séances de l'Académie des Sciences. Série D: Sciences naturelles, 187(1), 53-6.

Rojahn, J. & Esbensen, A.J. (2002). Epidemiology of self-injurious behavior in mental retardation; a review, In S.R. Schroeder, M.L. Oster-Granite, & T. Thompson (Ed.), Self- Injurious Behavior: Gene-Brain-Behavior Relationships. (pp. 41-77).Washington, D.C.: APA Books

Saito, Y., Ito, M., Hanaoka, S., Ohama, E., Akaboshi, S. and Takashima, S. (1999). Dopamine receptor upregulation in Lesch-Nyhan syndrome: a postmortem study. Neuropediatrics, 30(2):66-71.

Shapira, N. A., Lessig, M. C., Murphy, T. K., Driscoll, D. J., & Goodman, W. K. (2002). Topiramate attenuates self-injurious behaviour in Prader–Willi syndrome. The International Journal of Neuropsychopharmacology, 5(02), 141-145.

Symons, F.J. & Thompson, T. (1997). Self-injurious behavior and body site preference. Journal of Intellectual Disability Research, 41:456-68.

Thompson, T., & Caruso, M. (2002). Self-injury: knowing what we're looking for. In S.R. Schroeder, M.L. Oster-Granite, & T. Thompson (Ed.), Self-Injurious Behavior: Gene-Brain- Behavior Relationships. (pp. 41-77).Washington, D.C.: APA Books.

Tiefenbacher, S., Novak, M. A., Lutz, C. K., & Meyer, J. S. (2005). The physiology and neurochemistry of self-injurious behavior: A nonhuman primate model. Frontiers in Bioscience, 10, 1-11.

Towle, A.C., Criswell, H.E., Maynard, E.H., Lauder, J.M., Joh ,T.H., Mueller, R.A. & Breese GR. (1989). Serotonergic innervation of the rat caudate following a neonatal 6- hydroxydopamine lesion: an anatomical, biochemical and pharmacological study, Pharmacology, Biochemistry and Behavior; 34, 367-374.

Turner, C.A., Panksepp, J., Bekkedal, M., Borkowski, C. & Burgdorf J. (1999). Paradoxical effects of serotonin and opioids in pemoline-induced self-injurious behavior, Pharmacology, Biochemistry and Behavior, 63: 361-366.

Wong, D. F., Harris, J. C., Naidu, S., Yokoi, F., Marenco, S., Dannals, R. F., Ravert, H. T., Yaster, M., Evans, A., & Rousset, O. (1996). Dopamine transporters are markedly reduced in lesch-nyhan disease in vivo. Proceedings of the National Academy of Sciences, 93(11), 5539.