Parkinson’s disease (PD) is a progressive neurodegenerative disorder producing a clinical syndrome of bradykinesia, rigidity and resting tremor. These motor symptoms appear due to the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) and loss of dopamine in the striatum, which subsequently leads to an imbalance of the basal ganglia motor circuit. The most effective pharmacological treatment for PD is L-3,4-dihydroxyphenylalanine (L-DOPA), the immediate metabolic precursor of dopamine, which effectively restores motor function. L-DOPA is catabolised into dopamine and replaces neurotransmitter loss in PD. However, long-term L-DOPA treatment leads to abnormal involuntary movements (AIMs), such as L-DOPA-induced dyskinesia (LID), which reduces the quality of life in PD patients. Currently, there are no reliable pharmacological treatments for these motor complications. Clinical and preclinical studies have shown that development and expression of LID is linked to unregulated dopamine release and plasticity-induced changes of striatal dopaminergic and non-dopaminergic signalling pathways. The activities of these pathways can be modulated by neurotransmitter receptors of a specific classification, the G-protein-coupled receptor (GPCR) family. In turn, GPCRs are regulated by certain endogenous proteins, the regulators of G-protein signalling (RGS) proteins. Numerous RGS protein subtypes are expressed in the striatum but their roles in PD and LID remain poorly understood. Given the modulatory function of RGS proteins in the striatum, these endogenous factors may have pathophysiological roles in the expression of motor symptoms in PD and LID. The studies presented in this thesis investigated the roles of RGS proteins in the unilateral 6-hydroxydopamine (6-OHDA)-lesioned rat model of PD and LID. Rats received unilateral 6-OHDA lesions of the right medial forebrain bundle to induce severe dopamine denervation. L-DOPA/benserazide (6/15 mg/kg) was then administered once daily for at least 21 days to induce stable abnormal involuntary movements (AIMs). In Chapter 2 of this thesis, increased levels of RGS2 and RGS4 mRNA were found in the rostral striatum of the unilateral 6-OHDA-lesioned rat model of LID. Moreover, elevated levels of RGS4 mRNA were specific to sensorimotor regions and positively correlated with AIMs severity. These molecular and behavioural data suggest that RGS4 proteins are involved in the expression of LID. In Chapters 3 and 4, behavioural studies conducted in the unilateral 6-OHDA-lesioned rat model of LID showed that acute inhibition of striatal RGS4 proteins reduced the expression of AIMs and improved overall motor function. Moreover, repeated de novo treatment with RGS4 protein inhibitors, in combination with L-DOPA, attenuated the development of AIMs and reduced the overexpression of preproenkephalin-B, a molecular marker of LID. These behavioural and molecular data suggest that blockade of RGS4 proteins can reduce the induction of LID. In Chapter 5, in vivo microdialysis conducted in the unilateral 6-OHDA-lesioned rat model of LID showed that systemic administration of RGS4 protein inhibitors, in combination with L-DOPA, attenuated unregulated striatal dopamine efflux. These data suggest that RGS4 proteins may regulate specific G-protein coupled receptors, such as 5-HT1A receptors, that modulate striatal dopamine release. In conclusion, the work presented in this thesis shows that RGS4 proteins play a pathophysiological role in the expression and development of LID. These proteins could mediate regulation of key neurotransmitter receptors involved in LID, making them a potential therapeutic target for the development of future treatments.

The ontogenic destruction of dopamine (DA) neurons in rat brain is associated with supersensitization of DA D1 receptors. This effect is attenuated when rats are cotreated in ontogeny with the serotonin (5-HT) neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT). In an attempt to determine whether 5-HT fibers might have a similar modulatory role on the sensitivity of the DA D2 receptor complex, we pretreated rats with desipramine HCl (20 mg/kg IP, base), 1 h before the DA neurotoxin, 6-hydroxydopamine (6-OHDA; 134 μg ICV, base) and/or 5,7-DHT (75 μg ICV) and/or vehicle. At about 3 months after birth dose-effect curves for quinpirole-induced oral activity were constructed for each group of rats. We found that quinpirole, an agonist for the DA D2 receptor complex, produced a dose-related increase in oral activity in all groups of rats. After a 200 μg/kg dose of quinpirole HCl, however, neonatal 5,7-DHT-lesioned rats had a peak oral response of 54.4 ± 5.1 (mean and SEM) vs. 22.6 ± 4.8 for control rats (p < 0.01). In neonatal 6-OHDA-lesioned rats this dose of quinpirole increased oral activity to 36.8 ± 5.8 oral movements (p < 0.05 vs. control). In rats lesioned with both 5,7-DHT and 6-OHDA, the oral response was not different from control. The enhanced oral response to quinpirole in 5,7-DHT-lesioned rats was attenuated by spiperone, an antagonist for the DA D2 receptor complex. These findings are believed to be the first to demonstrate that receptors of the DA D2 complex become sensitized after ontogenic injury to 5-HT fibers. This effect is opposite to the attenuated sensitivity of DA D1 receptors in rats with a similar 5-HT lesion.

Neonatal destruction of rat nigrostriatal dopaminergic fibers results in an enhanced oral activity response to both dopamine (DA) D1 and serotonin (5-HT) agonists. Because cholinergic systems represent another one of the neural circuits involved in oral behavior, it was of interest to determine whether muscarinic receptors might also be sensitized in the lesioned rats. At 3 days after birth, rats were pretreated with desipramine HCl (20 mg/kg, IP) 1 h before 6-hydroxydopamine (6-OHDA) HBr (100 μg in each lateral ventricle) or saline-ascorbic acid (0.1%) vehicle. Between 2 and 4 months, behavioral supersensitivity to a D1 agonist (SKandF 38393) and 5-HT agonist (m-chlorophenylpiperazine; m-CPP) was established before rats were challenged with the muscarinic receptor agonist, pilocarpine HCl (0.125 to 10.0 mg/kg, IP). The pilocarpine dose-effect curve was shifted to the left, with a maximal effect of 63.7 ± 8.6 oral movements being produced by a 1.0 mg/kg pilocarpine HCl dose in the 6-OHDA lesioned rats, versus 15.0 ± 2.4 oral movements in the control group (p < 0.001). The enhanced response to pilocarpine was attenuated by the muscarinic receptor antagonist, scopolamine HCl (0.1 mg/kg IP). These findings indicate that neonatal 6-OHDA treatment produces supersensitization of muscarinic receptors in rats.

Neonatal 6-hydroxydopamine (6-OHDA) treatment of rats is associated with supersensitization of the dopamine D1 agonist induction of oral activity. The present study was conducted to determine whether induced oral responses to serotonin (5-HT) agonists would be similarly altered in this rat model. At 3 days after birth, rats received desipramine HCl (20 mg/kg, IP) 1 h before 6-OHDA HBr (100 μg in each lateral ventricle) or saline-ascorbic acid (0.1%) vehicle. At approximately 9 mo, rats were challenged with the mixed 5-HT1C and 5-HT2 receptor agonist, m-chlorophenylpiperazine diHCl (m-CPP 2HCl; 0.30-6.0 mg/kg, IP) and were then observed for 1 min every 10 min over a 60-min period. m-CPP induced oral activity in both the vehicle and 6-OHDA groups, with the responses of the 6-OHDA group being much greater. An m-CPP dose of 3.0 mg/kg produced a maximal response of 63.6 ± 3.2 oral movements in the 6-OHDA group. A bell-shaped response curve was obtained, with lower and higher doses of m-CPP producing less of an effect. Attenuation of the m-CPP-induced response by the 5-HT receptor antagonist, mianserin HCl (1.0 mg/kg, IP, 30 min before m-CPP), indicates that the m-CPP effect is receptor mediated. These findings demonstrate that neonatal 6-OHDA treatment produces ontogenic long-lived supersensitization of a 5-HT receptor system in rats.

Enhanced oral responses have been observed in rats that are treated shortly after birth with 6-hydroxydopamine (6-OHDA). A series of studies was conducted to characterize this effect. A dose-response curve demonstrated that the dopamine D1 receptor agonist, SKF 38393, produced a maximal response in 6-OHDA-treated rats at a dose of 0.10 mg/kg (IP). With the D2 receptor antagonist, spiperone, a bell-shaped dose-response curve was seen, with a maximal effect in the 6-OHDA group occurring at 80 μg/kg. There were only slight increases in oral activity with different SKF 38393 or spiperone doses in the saline group, indicating that there was an overt supersensitization of D1 receptors in the 6-OHDA-treated rats. The D1 antagonist SCH 23390 (0.30 mg/kg, IP) attenuated the response to both SKF 38393 and spiperone. The oral response to the D2 agonist, quinpirole (0.10 mg/kg, IP) was not preferentially increased in the 6-OHDA group of rats. These findings indicate that the enhanced oral response in neonatal 6-OHDA-treated rats is mediated by supersensitive dopamine D1 receptors. The persistence of the enhanced oral ersponse in 6-OHDA-treated rats at 8 months demonstrates that this sensitization of D1 receptors is a long-lived phenomenon.

The influence of central dopaminergic fibers on drug-induced oral activity in rats has not been well studied. Rats were treated 3 days after birth with bilateral intracerebroventricular 6-hydroxydopamine (6-OHDA; 134 ωg total, base form) to destroy dopaminergic fibers in the brain. Control rats received vehicle by the same route. At about 10 weeks of age, a challenge dose of the dopamine D2 receptor antagonist, spiperone (40 ωg/kg, IP), produced an 8-fold increase in the number of oral movements during a 60-minute observation period, vs. the control group. SKF 38393 (3.0 mg/kg, IP), a D1 agonist, produced the same number of oral movements as spiperone in the 6-OHDA group, representing a 2.4-fold increase over the controls. The Bmax and Kd for both D1 and D2 receptors was not changed in rat striatum by neonatal 6-OHDA treatment, even though dopamine content was reduced by 96%. These findings demonstrate that oral activity in rats can be greatly altered, even when there is no change in absolute numbers of D1 and D2 receptors and no change in the ratio of D1:D2 receptors.

Neonatal 6-hydroxydopamine (6-OHDA) treatment is associated with destruction of dopamine (DA) fibers and subsequent sprouting of serotonin (5-HT) fibers in the striatum of rats. Enhanced oral activity responses to SKF 38393 and m-chlorophenylpiperazine (ifm-CPP), respective agonists for the DA D1 receptor complex and 5-HT2C receptor complex, ensue. To study the ontogenetic nature of this effect, rats were treated at birth, 3 days, 7 days, 10 days or 14 days with 6-OHDA-HBr (200 μg i.c.v.; salt form), following desipramine-HCl pretreatment (20 mg/kg i.p., 1 h; base form). Another group of rats was treated at 35 days and again at 42 days with 6-OHDA-HBr (300 γg i.c.v.), following desipramine-HCl (20 mg/kg i.p., 1 h) and pargyline-HCl (50 mg/kg i.p., 30 min). In rats treated from birth to 10 days, 6-OHDA reduced striatal DA content at 5 months by ≥ 94%. Striatal 5-HT content was elevated by 28% to 51%, but only in rats treated with 6-OHDA at 7 days from birth or earlier. An enhanced oral activity response to SKF 38393-HCl (0.03 to 1.0 mg/kg i.p.) was absent in rats treated 7 days or later, and the change in SKF 38393 effect was correlated with a change in striatal DA content. An enhanced response to m-CPP-2HCl (0.3 to 6.0 mg/kg i.p.) was absent after treatment at 14 or 35 days, when striatal DA content was reduced only 44% to 63% and 5-HT content was not changed. Loss of the enhanced m-CPP response was not directly correlated with the magnitude of change in striatal content of either DA or 5-HT. The findings indicate that SKF 38393 and m-CPP-enhanced oral activity responses are dependent on the age at which 6-OHDA is administered to rats, and that the enhanced response to m-CPP can persist when there is no enhanced response to SKF 38393.

Neonatal 6-hydroxydopamine (6-OHDA) treatment of rats is associated with supersensitization of the dopamine (DA) D1 agonist induction of stereotyped and locomotor behaviors. The present study was conducted to determine whether ontogenetic treatments of these rats with the DA D1 receptor agonist, SKF 38393, would produce a maximal DA D1 receptor supersensitivity, as measured by locomotor behavior in adulthood. Rat pups were treated daily with SKF 38393-HCl (3.0 mg/kg per day, i.p.) or saline vehicle for 28 consecutive days from birth. These animals were additionally treated at 3 days after birth with 6-OHDA-HBr (100 μg, in each lateral ventricle, salt form) or its vehicle. Between 6 and 9 weeks locomotor activity or stereotyped behaviors were observed after weekly challenge doses of SKF 38393-HCl (3.0 mg/kg, i.p.). In the neonatal 6-OHDA group, successive SKF 38393 treatments produced progressively greater locomotor activity. In the group of rats treated during postnatal ontogeny with both 6-OHDA and SKF 38393 daily treatments, the first adult challenge dose of SKF 38393 produced an enhanced locomotor response, greater than that seen in other groups (P < 0.01). Subsequent SKF 38393 treatments of this group produced increasingly greater locomotor responses. SKF 38393-induced stereotyped behavioral effects were greater in the 6-OHDA-lesioned groups, whether or not SKF 38393 was administered ontogenetically. Profound reductions (> 99%) of DA and its metabolites were found in the striatum of neonatal 6-OHDA treated rats, regardless of whether SKF 38393 was co-administered ontogenetically. A marked elevation in striatal 5-HT (> 50%) accompanied the DA depletion in the striatum. These findings indicate that neonatal 6-OHDA treatment produces the expected destruction of striatal DA fibers with associated sprouting of 5-HT fibers, while repeated ontogenetic treatments of these rats with a D1 agonist produces partial sensitization of the DA D1 receptors in adulthood.

Dopamine (DA) neurons are implicated in the hyperlocomotion of neonatal 6-hydroxydopamine (6-OHDA)-lesioned rats, an animal model of attention deficit hyperactivity disorder (ADHD). Because serotonin (5-HT) neurons mediate some DA agonist effects, we investigated the possible role of 5-HT neurons on locomotor activity. Rats were treated at 3 days after birth with vehicle or 6-OHDA (134 μg ICV; desipramine pretreatment, 20 mg/kg IP, 1 h), and at 10 weeks with vehicle or 5,7-dihydroxytryptamine (5,7-DHT; 75 μg ICV; pretreatment with desipramine and pargyline, 75 mg/kg IP, 30 min), to destroy DA and/or 5-HT fibers. Intense spontaneous hyperlocomotor activity was produced in rats lesioned with both 6-OHDA and 5,7-DHT. Locomotor time in this group was 550 ± 17 s in a 600 s session, vs. 127 ± 13 s in the 6-OHDA group and <75 s in 5,7-DHT and intact control groups (p < 0.001). Oral activity dose-effect curves established that 5,7-DHT attenuated DA D1 receptor supersensitivity and further sensitized 5-HT2c receptors. Acute treatment with dextroamphetamine (0.25 mg/kg SC) reduced locomotor time in 6-OHDA+5,7-DHT-lesioned rats to 76 ± 37 s (p < 0.001). Striatal DA was reduced by 99% and 5-HT was reduced by 30% (vs. 6-OHDA group). Because combined 6-OHDA (to neonates) and 5,7-DHT (to adults) lesions produce intense hyperlocomotion that is attenuated by amphetamine, we propose this as a new animal model of ADHD. The findings suggest that hyperactivity in ADHD may be due to injury or impairment of both DA and 5-HT neurons.

The amino acid, 6-hydroxydopa (6-OHDOPA), found at the active site of amine oxidases, exists as a keto-enol. Exogenously administered 6-OHDOPA is an excitotoxin like β-N-oxalylamino-L-alanine (BOAA) and β-N-methylamino-L-alanine (BMAA), acting at the non-N-methyl-D-aspartate (non-NMDA) α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor. BMAA and BOAA are causal factors of neurolathyrism in humans. Much exogenously administered 6-OHDOPA is biotransformed by aminoacid decarboxylase (AADC) to the highly potent and catecholamine-(CA) selective neurotoxin, 6-hydroxydopamine (6-OHDA). 6-OHDOPA destroys locus coeruleus noradrenergic perikarya and produces associated denervation of brain by norepinephrine-(NE) containing fibers. Opiopeptides and opioids enhance neurotoxic effects of 6-OHDOPA on noradrenergic nerves, by a naloxone-reversible process. An understanding of mechanisms underlying neurotoxic effects of 6-OHDOPA can be helpful in defining actions of known and newfound amino acids and for investigating their potential neurotoxic properties.