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Keywords:

  • motor symptoms;
  • MPTP ;
  • noradrenaline;
  • Parkinson's disease;
  • thalamus

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We recently found severe noradrenaline deficits throughout the thalamus of patients with Parkinson's disease [C. Pifl, S. J. Kish and O. Hornykiewicz Mov Disord. 27, 2012, 1618.]. As this noradrenaline loss was especially severe in nuclei of the motor thalamus normally transmitting basal ganglia motor output to the cortex, we hypothesized that this noradrenaline loss aggravates the motor disorder of Parkinson's disease. Here, we analysed noradrenaline, dopamine and serotonin in motor (ventrolateral and ventroanterior) and non-motor (mediodorsal, centromedian, ventroposterior lateral and reticular) thalamic nuclei in MPTP-treated monkeys who were always asymptomatic; who recovered from mild parkinsonism; and monkeys with stable, either moderate or severe parkinsonism. We found that only the symptomatic parkinsonian animals had significant noradrenaline losses specifically in the motor thalamus, with the ventroanterior motor nucleus being affected only in the severe parkinsonian animals. In contrast, the striatal dopamine loss was identical in both the mild and severe symptom groups. MPTP-treatment had no significant effect on noradrenaline in non-motor thalamic nuclei or dopamine and serotonin in any thalamic subregion. We conclude that in the MPTP primate model, loss of noradrenaline in the motor thalamus may also contribute to the clinical expression of the parkinsonian motor disorder, corroborating experimentally our hypothesis on the role of thalamic noradrenaline deficit in Parkinson's disease.

Abbreviations used
DA

dopamine

NA

noradrenaline

PD

Parkinson's disease

In a recent neurochemical study on brains of patients dying with Parkinson's disease (PD), we reported marked deficits of noradrenaline (NA) in various thalamic nuclei, with the losses in thalamic motor areas being particularly profound (Pifl et al. 2012). Previous experimental in vitro and in vivo studies furnished evidence for the functionally significant influence of NA on the thalamic neuronal activity. It was shown that lack of NA in thalamic tissue preparations as well as in vivo resulted in abnormal neuronal activity and pathologic discharge patterns of thalamic neuronal populations (ref. see McCormick et al. 1991 and Berridge and Waterhouse 2003). As analogous neuronal discharge abnormalities have been observed in the motor thalamus of patients with PD (ref. see Galvan and Wichmann 2008), we proposed the hypothesis that NA loss in the motor thalamus of PD patients (Pifl et al. 2012) may interfere with motor information transfer from the basal ganglia to the cortex and represent a relevant pathophysiological feature of the parkinsonian state.

Here, we now describe the thalamic concentrations of NA, dopamine (DA) and serotonin associated with different degrees of motor impairment in the MPTP monkey model, providing further evidence in support of the role of thalamic NA in the parkinsonian state.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In this neurochemical study, brain tissue of 32 male macaque monkeys (Macaca fascicularis) was used, derived from our recent study in which four distinct stages of MPTP-induced neurodegeneration mimicking more closely the progression of PD proper, were characterized by in vivo imaging, as well as follow-up histology and neurochemistry of the nigrostriatal DA system (Blesa et al. 2012). MPTP (Sigma, Saint Louis, MO, USA) was systemically administered using a dose regimen of 0.5 mg/kg (i.v.) every 2 weeks. Animals, provided by RC Hartelust (Tilburg, The Netherlands), were housed in an animal room under standard conditions and treated in accordance with the European and Spanish guidelines (86/609/EEC and 2003/65/EC European Council Directives; and the Spanish Government). The Bioethics Committees of the Universidad de Navarra and the Universidad Autónoma de Madrid approved the experiments. Monkeys were classified into experimental subgroups according to the degree of motor impairment: Asymptomatic group – monkeys that did not develop any motor signs after two injections of MPTP; recovered group – monkeys that developed moderate parkinsonian features but showed complete motor recovery and maintained normal motor features 4 weeks later and thereafter; mildly parkinsonian group – monkeys with persistent, clearly recognizable but not very intense parkinsonian features; severely parkinsonian group – monkeys that exhibited very prominent motor features and remained severely affected thereafter (for details of criteria for subgroups and histological findings see Blesa et al. 2012). Each animal had to remain stable for at least 1 month in the corresponding motor state. Accordingly, all monkeys outlived for at least 4 weeks after the last MPTP injection (Blesa et al. 2012). None of these monkeys received treatment with any anti-parkinsonian drug or any other therapeutic intervention at any time during the study. Monkeys were killed in the morning and early afternoon by deep anaesthesia with pentobarbital, the brains removed and divided by a midsagittal section into two hemispheres, one of which was immediately frozen at −80°C and kept frozen until biochemical analyses. Tissue samples of an average wet weight of 25 mg each were taken from five consecutive, between 1 and 2 mm thick frozen coronal slices cut by hand from the frozen hemispheres, starting at the rostral pole of the thalamic mass. Because in the frozen material the boundaries between the various thalamic territories and their subdivisions are not clearly apparent, we examined only six topographically prominent whole nuclei (reticular; ventroanterior; ventrolateral; dorsomedial: centromedian; ventroposterior lateral), identified according to the figures 10–16 of the Macaca fascicularis stereotaxic brain atlas of Szabo and Cowan (1984). Individual thalamic nuclei were in most instances pooled from several slices. For measuring monoamine neurotransmitters, samples were prepared and analysed by two different HPLC and electrochemical detection systems, one for NA and DA after extraction with alumina oxide, the other for serotonin by direct injection of supernatants of centrifuged perchloric acid homogenates (Pifl et al. 1991, 2012; Blesa et al. 2012). Differences in NA, serotonin and DA, expressed in ng/g fresh tissue, were calculated using analyses of variance (anova) followed by the Bonferroni t-test.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Thalamic biogenic amine neurotransmitters in control monkeys

In the six thalamic nuclei analysed in normal monkeys (n = 8), the concentrations of NA and serotonin were of comparable magnitude, with the mean values ranging between 385 and 628 ng/g (Table 1) and 277 and 480 μg/g (Table 2) respectively. Thalamic DA concentrations were considerably lower, with means ranging between 31 and 109 μg/g (Table 3). Centromedian nucleus had the highest concentration of NA (628 ± 81 ng/g) and serotonin (480 ± 38 ng/g), closely followed by ventroanterior nucleus (NA, 529 ± 89; serotonin, 449 ± 46 ng/g); the ventrolateral and ventroposterior lateral nuclei had the lowest NA (about 400 ng/g) and serotonin (about 300 ng/g) levels. Significant differences were found for NA and serotonin in the centromedian versus ventroposterior lateral nucleus, and for serotonin between centromedian and ventrolateral nucleus (< 0.05, by paired Student's t-test). Serotonin levels in ventroanterior nucleus were significantly higher than that in the ventrolateral and ventroposterior lateral nuclei (< 0.05, by paired Student's t-test). There were no significant differences for DA among the various nuclei analysed here; individual DA values had a much higher scatter than NA and serotonin values.

Table 1. Thalamic NA (ng/gww) in various states of MPTP parkinsonism
Thalamic nucleiControlAsymptomaticRecoveredMildly parkinsonianSeverely parkinsonian
  1. *< 0.05 versus control, **< 0.05 versus asymptomatic (anova followed by Bonferroni t-tests).

  2. Values are given as Mean ± SEM (n).

Ventrolateral412 ± 36 (6)365 ± 48 (5)299 ± 38 (5)181 ± 41 (5)*,**189 ± 25 (5)*,**
Ventroanterior529 ± 89 (8)413 ± 74 (6)383 ± 51 (6)484 ± 104 (6)161 ± 11 (5)*
Mediodorsal504 ± 70 (8)569 ± 124 (6)550 ± 146 (6)664 ± 159 (6)325 ± 92 (6)
Centromedian628 ± 81 (8)987 ± 199 (6)692 ± 102 (6)808 ± 99 (6)393 ± 108 (2)
Ventroposterior lateral385 ± 45 (8)443 ± 94 (6)301 ± 53 (5)332 ± 88 (5)379 (1)
Reticularis573 ± 142 (8)581 ± 225 (6)427 ± 123 (6)563 ± 155 (5)518 ± 65 (6)
Table 2. Thalamic serotonin (ng/gww) in various states of MPTP parkinsonism
Thalamic nucleiControlAsymptomaticRecoveredMildly parkinsonianSeverely parkinsonian
  1. Values are given as Mean ± SEM (n).

Ventrolateral277 ± 27 (6)286 ± 37 (5)353 ± 49 (5)234 ± 36 (5)318 ± 33 (5)
Ventroanterior449 ± 46 (8)446 ± 42 (6)531 ± 55 (6)468 ± 68 (6)423 ± 56 (6)
Mediodorsal398 ± 48 (8)375 ± 51 (6)481 ± 45 (6)441 ± 46 (6)312 ± 46 (6)
Centromedian480 ± 38 (8)451 ± 41 (6)499 ± 49 (6)409 ± 48 (6)328 ± 74 (2)
Ventroposterior lateral309 ± 39 (8)284 ± 29 (6)309 ± 16 (5)238 ± 38 (5)150 (1)
Reticularis356 ± 48 (8)344 ± 47 (6)316 ± 39 (6)410 ± 81 (5)357 ± 50 (6)
Table 3. Thalamic DA (ng/gww) in various states of MPTP parkinsonism
Thalamic nucleiControlAsymptomaticRecoveredMildly parkinsonianSeverely parkinsonian
  1. Values are given as Mean ± SEM (n).

Ventrolateral31 ± 6 (6)29 ± 7 (5)52 ± 14 (5)25 ± 4 (5)23 ± 5 (5)
Ventroanterior109 ± 42 (8)115 ± 59 (6)127 ± 48 (6)115 ± 78 (6)73 ± 23 (6)
Mediodorsal34 ± 8 (8)19 ± 4 (6)34 ± 10 (6)28 ± 7 (6)31 ± 10 (6)
Centromedian49 ± 8 (8)51 ± 8 (6)50 ± 18 (6) 53 ± 14 (6)28 ± 14 (2)
Ventroposterior lateral47 ± 12 (7)28 ± 7 (6)33 ± 17 (5)24 ± 7 (5)  22 (1)
Reticularis49 ± 12 (6)67 ± 10 (5)43 ± 19 (6)37 ± 9 (5)42 ± 9 (6)

Effect of MPTP

The ‘asymptomatic’ group (= 6) and the ‘recovered’ group (= 6) showed normal NA, serotonin and DA levels in all six thalamic nuclei analysed (Fig. 1a–c, Tables 1-3). The mildly parkinsonian group (n = 6) had significantly reduced NA levels by 56% in the ventrolateral nucleus (Fig. 1a) but unchanged in the remaining five nuclei (Fig. 1a–c, Table 1), as well as unchanged serotonin and DA levels in all thalamic nuclei investigated (Table 2, 3). In contrast, the severely parkinsonian group (n = 6) had significantly reduced NA levels in both the ventrolateral and ventroanterior motor nuclei (Fig. 1a) (−54 % and −70 % respectively), and not significantly reduced NA in the mediodorsal and centromedian (Fig. 1b) nucleus (−36 % and −37% respectively); neither serotonin nor DA was significantly affected in any of the thalamic nuclei of the severe parkinsonian group (Table 2, 3). All four MPTP-treated groups had significantly reduced DA levels in caudate and putamen; the asymptomatic group: −33 % and −28 % respectively; the recovered group: −90 and −86 %; the mildly parkinsonian group: −96 and −98 % and the severely parkinsonian group: −97 and −95 %; the levels of the latter groups being also significantly lower versus the asymptomatic group (Fig. 1d).

image

Figure 1. Tissue levels of noradrenaline in thalamic nuclei (a–c) and of dopamine in caudate/putamen (d) of Macaca fascicularis monkeys staying after MPTP treatment either asymptomatic (asymp), recovered from parkinsonian symptoms (recovered), mildly or severely parkinsonian. Shown are mean values ± SEM in percent of controls of 5–6 animals, if not indicated differently. *p < 0.05 versus control, +< 0.05 versus asymp by Bonferroni t-test after anova.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The main finding of our study is the significant loss of NA in the motor thalamus of both the mildly and the severely parkinsonian MPTP-treated Macaca fascicularis monkeys but normal values in monkeys without detectable parkinsonian features at the end of the MPTP experiment (i.e., the ‘asymptomatic’ and the ‘recovered’ groups). However, whereas in the ventrolateral motor thalamus, NA was already maximally reduced in the mildly parkinsonian group with no further reduction in the severely parkinsonian monkeys, the NA loss in the ventroanterior motor thalamus was only apparent in the severely parkinsonian monkeys, suggesting a possible causal connection between the ventroanterior motor thalamus NA loss and the severity of the MPTP-induced parkinsonian condition. In sharp contrast to this differential behaviour of the NA loss in the ventrolateral versus ventroanterior thalamus, discriminating between the mildly and severely parkinsonian monkeys, the striatal DA reduction pathognomonic for PD, was of the same magnitude (only 2–5% DA remaining) in both parkinsonian groups.

MPP+, the toxic agent proper of MPTP, is a substrate of monoamine transporters with higher affinity to the NA than to the DA transporter (Buck and Amara 1994; Pifl et al. 1996). The substantially higher in vivo susceptibility to MPTP of the DA system can be explained by the about ten-fold higher turnover numbers of the DA transporter than the NA transporter for the substrate MPP+ (Pifl et al. 1996) and is reflected by DA losses of up to 98 % (in the putamen of parkinsonian MPTP-treated monkeys in this study) as compared with NA losses of up to 70 % (in the ventroanterior thalamus of the severely parkinsonian group). Whether differences in the density of NA uptake sites might explain differences in the susceptibility of ventroanterior/ventrolateral nuclei as compared with the resistant reticular or ventroposterior lateral nuclei, can only be speculated. The unchanged serotonin levels are in agreement with a very low affinity of MPP+ to the human serotonin transporter which is seven fold lower than its affinity to the DA transporter (Bryan-Lluka et al. 1999).

NA in thalamic tissue stems from noradrenergic nerve terminals of noradrenergic neurons with the majority of cell bodies situated in the locus coeruleus (Lindvall et al. 1974). We are not aware of any reports in the literature on the presence in the thalamus of monoaminergic fibres of passage. As in a study on Macaca fascicularis intoxicated with MPTP less than 25 % of the locus coeruleus tyrosine hydroxylase positive neurons were lost (Herrero et al. 1993), a retrograde degenerative mechanism of MPTP action on the locus coeruleus NAergic system appears to be at work, analogous to that demonstrated for the nigrostriatal dopamine system (Herkenham et al. 1991).

There are similarities and differences in our findings on thalamic NA in monkeys as compared with humans. In controls, the highest level of NA was found in centromedian nucleus, both in monkeys and in humans; however, the difference between the centromedian and the ventrolateral nuclei was only 1.2 fold in monkeys but five fold in humans. In the parkinsonian condition, NA was significantly decreased only in motor thalamus of MPTP-treated Macaca fascicularis by 60 % at most, whereas in human PD the thalamic NA loss was much greater, with reductions between 80 and 90 % in the centromedian and mediodorsal nucleus as well (Pifl et al. 2012). Furthermore, in the present MPTP study the NA in the analysed non-motor thalamic nuclei was not statistically significantly reduced unlike the substantial NA losses in many thalamic regions in patients with PD (Pifl et al. 2012). These qualitative as well as quantitative differences may be due to species differences and/or the longer disease process in the PD patients, or reflect principal differences between the MPTP-induced and the PD-induced neurodegeneration.

In our MPTP study, neither DA nor serotonin levels were consistently affected in thalamic nuclei by treatment with MPTP. In contrast, one study using intracarotid administration of MPTP to Macaca mulatta (rhesus) monkeys reported loss of thalamic DA innervation as determined by loss of DA transporter immunoreactive axon elements (Freeman et al. 2001). Considering the density of DA transporter immunoreactivity in primate thalamus (Freeman et al. 2001; Melchitzky and Lewis 2001; García-Cabezas et al. 2007), DA in DA transporter expressing fibres might be only a fraction of the total DA measured in tissue samples, where a good part of the DA might be in DA axons devoid of the DA transporter (García-Cabezas et al. 2007) and in NAergic axons as precursor of NA. This alternative is supported by data in Oke et al. (1997) for the human thalamus and Palkovits et al. (1979) for the rat cortex, and evidenced by co-release of both catecholamines in rat cortex in vivo (Devoto et al. 2001). In addition, partial noradrenergic denervation resulting in a (compensatory) up-regulation of the metabolic DA in the remaining NA terminals, may further increase the discrepancies between DA transporter versus total DA measurements in brain regions such as the primate thalamus or the cortex.

To our knowledge, this is the first study to provide experimental support for our earlier suggested hypothesis (Pifl et al. 2012) about the role of NA, specifically in motor thalamus, in the parkinsonian motor disorder. Thus, in our MPTP monkey study only the symptomatic animals had statistically significant NA loss in the motor ventroanterior and ventrolateral nuclear regions. Although, in contrast to our findings in PD (Pifl et al. 2012), the degree of this NA loss was not extreme, ranging between 54 % and 70 % loss, the observation that the NA loss in the ventroanterior nucleus distinguished between the mildly (no NA loss) and the severely (70 % NA loss) parkinsonian animals, suggests that this degree of NA loss was already sufficient to substantially add to the pathophysiologic dysfunction caused by the severe striatal DA deficit which, however, failed to distinguish between the mildly and severely parkinsonian MPTP groups. We interpret this as indicating that, given a severe enough striatal DA loss, already moderate thalamic NA losses are sufficient to significantly exaggerate the parkinsonian disability. Moreover, advanced PD is associated with profound nigro-striatal lesion and motor severity but also with a number of other manifestations that are not sensitive to dopaminergic therapies, such as gait and equilibrium problems, reduced attention, day time sleepiness and cognitive impairment. It is possible that involvement of thalamic NA may play a role in the origin of some of such unresolved problems in the management of PD.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Ing. Harald Reither for technical assistance with biogenic amine analysis. The authors have no conflict of interest to declare. This study was supported in part by the Plan Nacional de Investigación (SAF2005-08416; SAF2008-04276), and by the UTE-CIMA agreement with the University of Navarra.

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  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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