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Studies showed that the dopamine (DA) transporter (DAT) modulates changes in levodopa-derived synaptic dopamine levels (Δ(DA)) in Parkinson’s disease (PD). Here we evaluate the relationship between DAT and Δ(DA) in the 6-hydroxydopamine model of Parkinson’s disease to investigate these mechanisms as a function of dopaminergic denervation and in relation to other denervation-induced regulatory changes. 27 rats with a unilateral 6-hydroxydopamine lesion (denervation ∼20–97%) were imaged with 11C-dihydrotetrabenazine (VMAT2 marker), 11C-methylphenidate (DAT marker) and 11C-raclopride (D2-type receptor marker). For denervation <75%Δ(DA) was significantly correlated with a combination of relatively preserved terminal density and lower DAT. For denervation <90%, Δ(DA) was significantly negatively correlated with DAT with a weaker dependence on VMAT2. For the entire data set, no dependence on pre-synaptic markers was observed; Δ(DA) was significantly positively correlated with 11C-raclopride binding-derived estimates of DA loss. These findings parallel observations in humans, and show that (i) regulatory changes attempt to normalize synaptic DA levels (ii) a lesion-induced functional dependence of Δ(DA) on DAT occurs up to ∼ 90% denervation (iii) for denervation < 75% relative lower DAT levels may relate to effective compensation; for higher denervation, lower DAT levels likely contribute to oscillations in synaptic DA associated with dyskinesias.
Parkinson’s disease (PD), which affects approximately 300/100 000 of the general population (de Lau and Breteler 2006), is characterized by a progressive degeneration of the dopaminergic system leading to increasingly severe dopamine (DA) deficiency resulting in debilitating motor and often cognitive impairment. It is known that clinical symptoms generally occur when at least 50% of dopaminergic neurons have died; this implies the existence of a pre-clinical period during which disease-induced neurochemical changes take place (Adams et al. 2005). Increase in DA turnover in humans and animal models of PD (Zigmond et al. 1990; Sossi et al. 2002) and DA transporter (DAT) down-regulation (Lee et al. 2000; Adams et al. 2005) have been identified as possible early compensatory changes. It is also known that the majority of PD patients develop treatment-related motor complications within 5–10 years of treatment. While the neurochemical changes underlying motor complications are not completely understood, current knowledge favors a synergistic combination of altered DA release patterns and post-synaptic receptor sensitization in response to pulsatile DA stimulation (de la Fuente-Fernández et al. 2004a, Bibbiani et al. 2005; Cenci and Lundblad 2006) Experimental evidence suggests that an increase in both DA turnover and levodopa-induced changes in synaptic DA levels (Δ(DA)) is associated with increased risk of motor complications (de la Fuente-Fernández et al. 2001b, 2004a,b;Pavese et al. 2006). A link between an increase in DA turnover and/or Δ(DA) and lower DAT levels has also been recently suggested in human studies including symptomatic patients (Sossi et al. 2007a) as well as in 6-hydroxydopamine (6-OHDA) lesioned rats with dopaminergic denervation > 60% (Lee et al. 2008). These results suggest that once disease is present, the functional role of DAT is to preserve DA in the terminals and to reduce oscillations in synaptic DA levels that contribute to a pulsatile stimulation of the post-synaptic receptors. Indeed, in a recent clinical human study, down-regulated DAT levels have been directly linked to the occurrence of dyskinesia (Troiano et al. 2009).
The same mechanisms (i.e., an increase in Δ(DA) and/or DA turnover and regulatory DAT changes) seem to be involved in both delaying the onset of clinical symptoms in early disease as well as increasing the risk of treatment-induced motor complications as disease progresses. It is thus of interest to explore the relationship between DAT expression and Δ(DA) as a function of severity of dopaminergic denervation avoiding possible confounds of treatment, such as a reported differential responses of DAT to levodopa or DA agonists (Parkinson’s Study Group 2002, 2004), and to relate such changes to other denervation-induced effects, such as decrease of synaptic DA levels. In this study, we used a unilateral 6-OHDA-lesion rat model of PD and positron emission tomography (PET) imaging to investigate the relation between these mechanisms over a range of denervation corresponding to the pre-symptomatic to moderate/advanced symptomatic PD stage.
The same three PET tracers used in similar human studies (Sossi et al. 2007a) were used here: (+) 11C-dihydrotetrabenazine (DTBZ, a vesicular monoamine transporter type 2 (VMAT2) marker) to estimate the degree of dopaminergic denervation, 11C-methylphenidate (MP) to evaluate DAT expression and 11C-raclopride (RAC, a D2-type receptor marker) to evaluate Δ(DA) as well as denervation-induced changes in RAC binding, taken to be an estimate of synaptic DA loss. For a subset of rats data obtained from the pre-synaptic PET markers were also compared to post-mortem autoradiography with DTBZ (Strome et al. 2006) and [3H]WIN 35,428 (a DAT marker) to confirm the accuracy of the in vivo denervation assessment (n = 25 for VMAT2 and for DAT). [3H]WIN 35 428 was used for DAT autoradiography because of its accepted use as DAT marker and ready availability.
Lesion-induced changes in baseline RAC binding were used for two purposes, the first of which was a relative estimation of baseline synaptic DA levels. RAC binding has been indeed shown to be sensitive to synaptic DA levels (de la Fuente-Fernández et al. 2001a,Piccini et al. 2003; Pavese et al. 2006). An increase in baseline RAC binding has been shown to occur in early symptomatic disease in untreated human subjects (Rinne et al. 1995) and in symptomatic MPTP lesioned non-human primates (Doudet et al. 2002), while no change has been observed in lesioned, but asymptomatic non-human primates. It is thus reasonable to assume that as long as synaptic DA levels are sufficient to maintain adequate motor function, RAC binding remains at control levels, while an increase in baseline RAC binding can be directly (by competition) or indirectly (by receptor regulation) related to lower endogenous synaptic DA levels (Doudet et al. 2002; Schiffer et al. 2005). The second purpose was to compare denervation-induced baseline RAC binding changes to those observed with the pre-synaptic markers to gain some insight into their respective dependence on the level of denervation.
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This study represents, to our knowledge, the first in vivo multi-tracer study evaluating levodopa-derived changes in synaptic DA levels and comparing them to changes in pre-synaptic and synaptic markers for a wide range of denervation, ranging from 20% to almost complete denervation. A most useful approach to the interpretation of these results is to consider the difference between BPND_RAC0_L and BPND_RAC0_C as a reflection of the lesion-induced loss of endogenous synaptic DA levels, while Δ(DA) as a measure of the capacity of the lesioned system to release DA in response to exogenous levodopa. Likewise, it is useful to examine how the nature of the relationships between the variables may be affected by the degree of denervation, with a threshold effect on Δ(DA) seen at denervation levels of 75%, based on an examination of the raw data. The examination of the entire data set provides an opportunity to examine the consistent overall effects of the dopaminergic denervation; on the other hand, the analysis of isolated subsets provides an opportunity to identify mechanisms that are most relevant at specific ranges of denervation, and thus, by extrapolation, at particular stages of human PD.
Keeping this approach in mind, the analysis of the entire data set indicates that DA loss was significantly correlated with log(BPND_DTBZ) and thus degree of denervation. This finding indicates that most of the endogenously derived synaptic DA originates from the dopaminergic terminals even in the case of severe denervation. On the other hand, the relationship between Δ(DA) and dopaminergic denervation was not significant, despite a significant positive correlation of Δ(DA) with the degree of synaptic DA loss. Interpretation of this finding suggests that (i) throughout the entire range of denervation, the primary response of the system to exogenously administered levodopa is an attempt to normalize synaptic DA levels (de la Fuente-Fernández et al. 2004a) and (ii) at high levels of denervation, the engagement of other DA synthesis and release mechanisms might become relevant, as suggested by others (Carta et al. 2007; Lee et al. 2008).
No substantial loss of synaptic DA was observed for denervation levels lower than 75% indicating that pre-synaptic compensatory changes are effective at maintaining adequate synaptic DA levels up to this level of denervation. Increase in Δ(DA), presumably reflecting elevated release of exogenously derived DA (Zigmond et al. 1990), was found to be positively correlated with log(BPND_DTBZ) and negatively with log(BPND_MP). This indicates a more effective compensation for relatively milder degrees of denervation [higher log(BPND_DTBZ)] and shows that, once denervation is present, DAT assumes a functional role in the regulation of synaptic DA levels, which was found to be maintained until at least 90% denervation, such that lower DAT was consistently associated with higher Δ(DA).
The analysis of the subset of animals with denervation < 90% indicated that as more severe denervation is included, a similar dependence on the pre-synaptic markers is still observed, but now the only significant correlate is DAT; there is a only trend towards significance for the Δ(DA) dependence on VMAT2. This finding likely indicates a decreasing ability of the pre-synaptic mechanism to provide adequate compensation for the denervation-induced DA loss and a potential gradual engagement of other sources of DA synthesis and release (Carta et al. 2007; Lee et al. 2008). In this situation of DA deficiency, larger changes in Δ(DA), lead to oscillatory behavior of synaptic DA levels. Thus, levodopa treatment in this scenario leads to a pulsatile stimulation of post-synaptic receptors and a heightened risk of dyskinesia (de la Fuente-Fernández et al. 2004a; Cenci and Lundblad 2006). The consistent negative correlation between Δ(DA) and DAT levels explains how, at this stage lower DAT levels become a risk factor.
Interestingly, our current results obtained in rodents agree very well with human studies showing a significant negative correlation between Δ(DA) and DAT levels (Sossi et al. 2007a) and a relative DAT down-regulation has been observed to be linked to a higher occurrence of dyskinesia in both humans (Troiano et al. 2009) and rodents (Lee et al. 2008).
Furthermore, since no significant relation was observed for the unlesioned side and no treatment had been administered to the animals, the correlation between DAT expression and DA release must clearly be determined by the dopaminergic deficit and not by treatment.
A potentially interesting finding is the increase of the BPND_MP to BPND_DTBZ ratio for very high levels of denervation (Fig. 1). Obviously more complex studies involving animals with very high degrees of denervation are needed to investigate this behavior and no conclusions can be drawn from this data set, especially, since at such high level of denervation the measured parameters are numerically very small. However, it can be stated that no obvious imaging-related methodological problems have been observed: as mentioned, partial volume effects are unlikely to explain this trend and neither do these animals appear as obvious outliers when PET data are compared with post-mortem binding (Fig. 4). Abnormal behavior of DAT in the presence of severe VMAT2 deficiency have been however suggested previously (Patel et al. 2003).
There are clearly limitations to this study. First, it was performed on a rat model of PD that differs from human disease. Nonetheless, the virtually identical relationships among the PET data measured in the current study and in a similar human study argue towards strong similarities between this model and human disease. Second, PD-like symptoms have been induced by a well-defined acute mechanism in rats, while the etiology of PD remains unknown and the disease itself progresses; in this study, we are not following progressive adaptive changes, but rather taking a snapshot of the relationships between regulatory changes at different, fixed levels of denervation. There are also data showing that a unilateral lesion might affect the contralateral side as well (Nikolaus et al. 2002), while in this study the contralateral side is used for control values. While the results presented here do not eliminate the possibility of some contralateral effect, such effect must be limited, since no significant relations between the observed variables were found for the unlesioned side.
Despite these caveats, the present study provides a plausible scenario that might be applicable to at least a subset of PD patients. Similar studies performed on different animal models of PD are required to ascertain whether this is a common mechanism or limited to specific disease origins.
In summary, this study shows, using multi-tracer in vivo imaging and the 6-OHDA rodent model of PD, that regulatory changes strive to normalize synaptic DA levels throughout the range of denervation examined and that DAT is consistently functionally related to levodopa-derived changes in synaptic DA levels for levels of dopaminergic denervation ranging from 20% to 90% with lower DAT levels being associated with higher changes. This may explain why relatively low DAT levels may provide an effective compensatory mechanism in a situation of mild dopaminergic deficit, when the system is still able to maintain sufficient endogenous DA levels in the synapse, while increasing the propensity to treatment-induced motor complication at more advanced levels of dopaminergic denervation.