The development of catecholaminergic systems
Reports of the development of catecholaminergic cell populations have been published earlier. While some of these nicely report the timed appearance of catecholaminergic cell groups, they fail to deal with the diencephalic complex as separate populations (populations 5–6 and 8–13) (Guo et al. 1999; Holzschuh et al. 2001). Another report finely dissects these populations, but lacks detailed description of the time scale and hindbrain populations (Rink and Wullimann 2002). Comparison to these publications can be found in the Supporting information Table S2. Our analysis essentially fills these gaps and provides a method for counting the absolute number of cells. In addition, fibers can be readily detected and followed. Contrary to earlier reports, we found that all adult catecholaminergic cell populations are detectable already at 72 hpf, while they are more readily separable at 5 dpf. The difference in the appearance of the populations might be related to the fact that the method used is more sensitive and cells with lower concentration of TH can be detected.
The development of the catecholaminergic system in zebrafish followed a very similar spatiotemporal sequence of appearance to the other reported teleosts (Manso et al. 1993). In all these teleosts, the catecholaminergic cells are first detected in the diencephalon and the locus coeruleus, followed by cells in the suprachiasmatic/tuberal area and the rhombencephalon. Later, just before hatching catecholaminergic cells are found in the pretectum, rostral preoptic area and the periventricular hypothalamus. The telencephalic catecholaminergic cells are detected earlier in zebrafish than in the other teleosts (Manso et al. 1993). In mammals, appearance of the midbrain dopaminergic neurons preceed the appearance of diencephalic and telencephalic neurons (Di Porzio et al. 1990; Kalsbeek et al. 1992). The homologue of midbrain dopaminergic neurons in teleost is currently unknown. Generally, the zebrafish catecholaminergic systems share similarities with other vertebrates, but also differ from them significantly. These differences must be taken into account when using zebrafish to study the dopaminergic system.
MPTP induced behavioral deficit
The locomotor network of zebrafish develops through different steps starting with spontaneous tail coilings at 17 hpf and reaching a mature beat-and-glide swimming pattern at 5 dpf (Brustein et al. 2003). In our study, a large-scale behavioral motility analysis was applied to 5–7-day-old zebrafish. MPTP produced a delayed and transient locomotor activity deficit in larval zebrafish, which was attenuated by deprenyl treatment. MPTP decreased the levels of DA, NA and 5-HT, which all may contribute to the decrease in locomotion. Deprenyl restored the locomotion of MPTP treated fish at 5 dpf, and prevented the decrease in the amine levels significantly. We have noticed that deprenyl alone does not affect DA or NA levels but it increases the levels of 5-HT and the increased 5-HT levels decrease spontaneous locomotion (Sallinen et al., in preparation), indicating that deprenyl does not increase motility per se. Attenuation of the MPTP-induced motility deficit and restoration of amine levels by deprenyl could be due to inhibition of the MPTP conversion to MPP+. This is supported by the fact that MPP+ produced similar behavioral deficits as MPTP and also decreased the levels of catecholamines. Similar transient decline in the locomotor activity is seen in adult zebrafish after MPTP or 6-hydroxydopamine treatment (Anichtchik et al. 2004). Also, in adult zebrafish NA levels were decreased along with DA levels (Anichtchik et al. 2004), a finding similar to our findings in larval fish. An earlier report showed non-quantified slowness in tail touch response in 3-day-old zebrafish after exposure to MPTP (Lam et al. 2005). However, at 3 dpf the locomotor system is not mature yet, and 3-day-old zebrafish do not swim spontaneously. Thus, comparison of these results to our study is difficult. McKinley et al. (2005) reported that 4-day-old larvae were immobile and unresponsive to touch at 4 dpf. We have observed similar behavior, which could be caused by acute non-specific toxicity of MPTP. Hence, we analyzed the behavior earliest at 1 day after the treatment, i.e., at 5 dpf. Another report analyzed ten 7-day-old zebrafish larvae swimming in the same tank, and reported quantified decrease in the locomotion after 1–5 dpf treatment with 9 mg/L (42.9 μM) or 45 mg/L (214.6 μM) MPTP (Bretaud et al. 2004). We did not notice any decrease in the locomotion after treatment with 100 μM MPTP. It is also noteworthy that this dose did not affect catecholamine levels either. These differences in the dose–response to MPTP might be due to the differences in the MPTP lot and/or in the zebrafish strain used as it is the case with mice. None of the earlier studies report effects of deprenyl on the behavior of zebrafish larvae. The behavioral response may arise from the diencephalic dopaminergic population 5,6,11, since it is affected by both MPTP and MPP+.
Mechanism and targets of MPTP action
The zebrafish cell population homologous to mammalian substantia nigra is unclear at the moment. Immunohistochemical and tracer studies in adult zebrafish suggest that the strongest candidade for zebrafish substantia nigra is the dopaminergic cell group of the posterior tuberculum (larval population 5,6,11) (Kaslin and Panula 2001; Rink and Wullimann 2001).
In larval zebrafish, MPTP has been reported to cause cell death of the pretectal population at 5 dpf (McKinley et al. 2005), putative posterior tuberal nucleus at 2 dpf (Lam et al. 2005) or diencephalic dopaminergic neurons at 5 dpf (Bretaud et al. 2004). All these studies lack the description of the exact populations affected and diencephalic populations are considered as a whole, which renders it difficult to compare them with our results. Loss of cells is quantified by counting only in Bretaud et al. 2004;. Lam et al. (2005) describe cell loss at 2–3 dpf. Analysis at this early age presents issues that need consideration. Firstly, several dopaminergic cell populations have not developed yet, among them the putative group corresponding to mammalian mesencephalic neurons, population 5,6,11. Secondly, MAO activity is very weak before 2 dpf (Sallinen et al., in preparation), suggesting that conversion of MPTP to toxic MPP+ is modest. A recent study, which was published after the completion of this study, reported moderate, yet non-quantified, loss of cells in the VMAT2-positive neural clusters (Wen et al. 2008). A detailed comparison of the findings of previous reports is shown in Supporting information Table S3.
In line with previous reports, we detected TH-ir cell loss in the pretectal population (7) and hypothalamic population (13) following MPTP. Additionally, we also detected TH-ir cell loss in the diencephalic (5,6,11) and preoptic (3–4) populations, which were not described in earlier reports. Pretectal (7) and diencephalic (5,6,11) populations were the most severely affected populations. Concerning 5-HT populations we found [contrary to the findings of Lam et al. (2005) and Wen et al. (2008)] that MPTP affected only PVOi population, while MPP+ did not affect the 5-HT populations significantly. We also showed decrease in the DA, NA and 5-HT levels in the larval zebrafish after MPTP exposure. In line with the immunohistochemical results, MPP+ did not affect 5-HT levels significantly, while it decreased DA and NA levels. Despite several approaches we failed to show actual cell death in the brain. Furthermore, the loss of TH-ir recovered rapidly and completely. The quick recovery might be the reason why no cell loss has been detected in adult zebrafish after MPTP exposure, although a decline in the DA and NA content has been reported (Anichtchik et al. 2004; Bretaud et al. 2004). These results suggest that MPTP is incapable of producing dopaminergic cell death in zebrafish, but causes a transient loss-of-function of the cells.
As MPP+ in mammals gains intracellular access via DAT, the expression of DAT might affect the susceptibility of different TH-ir populations to MPTP or MPP+. TH-ir cells in the preoptic do not express putative DAT, whereas pretectal (7) and hypothalamic (13) TH-ir populations express it (Holzschuh et al. 2001). The data regarding DAT expression in the region of population 5,6,11 is vague. DAT positive, but TH negative cells are reported at this site along with TH positive but DAT negative cells (the region is termed as PT in Holzschuh et al. 2001). It could be that the TH signal is below detection level in DAT-positive TH-negative cells and these cells corresponds to TH-ir cells in population 5,6,11. It is noteworthy that even though putative zebrafish DAT protein has high similarity to human DAT, the functional properties, such as substrate specificity, are unknown. Further studies are needed to confirm functional homology.
In our study, deprenyl prevented the MPTP toxicity partly, raising a question whether MPTP-toxicity is MAO-dependent in larval zebrafish. MPTP is a relatively weak substrate for zebrafish MAO in contrast to mammalian MAO B (Anichtchik et al. 2006), suggesting that MPTP oxidation to MPP+ may not be very efficient. On the other hand, deprenyl inhibits developing zebrafish MAO only partially (Sallinen et al., in preparation), which might result in incomplete rescue. So far, one form of MAO has been found in zebrafish genome (Setini et al. 2005; Anichtchik et al. 2006), but there may be other enzymes present in zebrafish capable of converting MPTP into MPP+. Interestingly, MAO activity and mRNA expression were detected in close proximity to population 5,6,11, but not pretectal population 7. Also MAO activity was detected in the rostral cells of the diencephalic population 13, but not in the preoptic population 3–4 (this study and Sallinen et al., in preparation). This suggests that loss of TH-ir cells in some of the dopaminergic populations might be MAO-independent. The close proximity of MAO activity to the serotonergic PVOi might be the reason for PVOi being the only serotonergic population affected by MPTP. One of the sites for conversion of MPTP to MPP+ in mammals is the histaminergic neurons in the hypothalamus (Nakamura and Vincent 1986). MAO was detected in the histaminergic neurons in the PVOp, and thus these cells might contribute to the conversion of MPTP to MPP+ also in larval zebrafish. To address the question whether the MPTP-toxicity is due to direct toxicity of MPTP itself or a MAO-dependent toxicity in which MPTP is converted to MPP+, we exposed the larvae to MPP+. In mammals MPP+ cannot cross the blood–brain barrier (BBB). Thus it is not neurotoxic if administered systemically (Perry et al. 1985). Available data regarding the properties and development of zebrafish BBB is scarce. Intraperitoneal injections of 75 mg/kg MPP+ do not induce loss of TH-ir or behavioral deficit in adult zebrafish (Bretaud et al. 2004), suggesting that BBB develops later to prevent the entry of MPP+ into CNS. In larval zebrafish, MPP+ caused similar locomotor deficit as MPTP, but loss of TH-ir was detected only in the population 5, 6, 11. These results suggest that the MPTP toxicity towards population 5,6,11 is MAO-dependent and due to the conversion to MPP+, whereas the MPTP-neurotoxicity affecting the pretectal population 7 may be more MAO-independent. On the other hand, permeability of the zebrafish BBB might have regional differences and thus MPP+ might only penetrate the BBB in certain areas. These findings also support the concept that the population 5,6,11 is most sensitive to MPTP/MPP+ and also in this respect corresponds to mammalian substantia nigra.
The development of catecholaminergic system in zebrafish is similar to that of other vertebrates and thus zebrafish might be a good model to study the disorders of the dopaminergic and noradrenergic systems. Identification and detailed cell counts and atlas of the catecholaminergic cell populations provided here enable the comparison of the larval populations to the adult counterparts. MPP+ may be a more useful tool than MPTP in larval zebrafish to model PD, since it seems more specific than MPTP. The results also suggest that the diencephalic population 5,6,11 is involved in the control of locomotion in larval zebrafish and is the target of both MPTP and MPP. Additionally, one 5-HT cell population was also affected by MPTP and MPTP decreased 5-HT levels, a finding similar to that of human PD patients (Kish et al. 2008).