Differential survival patterns among midbrain dopaminergic cells of MPTP-treated monkeys and 6OHDA-lesioned rats
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- Fitzpatrick, E., Ashkan, K., Wallace, B.A. et al. Anat Embryol (2005) 210: 101. doi:10.1007/s00429-005-0003-y
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We explore the patterns of survival among dopaminergic cells of the midbrain in MPTP-treated macaque monkeys and 6OHDA-lesioned Sprague-Dawley rats. For the monkeys, animals were injected intramuscularly with MPTP for 8 days consecutively and then allowed to survive for 21 days. For the rats, 6OHDA was injected into the midbrain and then allowed to survive for either 7, 28 or 84 days. Brains were processed for tyrosine hydroxylase (TH) and calbindin immunocytochemistry to label populations in the ventral and dorsal tiers of midbrain dopaminergic cells. In monkeys, while there was a decrease in the TH+ cell number in the ventral tier of MPTP-treated cases (65%), there was an overall increase (22%) in the TH+ and calbindin+ cell number in the dorsal tier. Double labelling studies indicate that ∼50% of TH+ cells of the dorsal tier contain calbindin also. In rats, there was a decrease in TH+ cell number in the ventral tier of 6OHDA-lesioned cases (97%), and to a lesser extent, in the TH+ and calbindin+ cell number in the dorsal tier (∼40%). In conclusion, we show a surprising increase in TH+ and calbindin+ cell number in the dorsal tier in response to MPTP insult; such an increase was not evident after 6OHDA insult. We suggest that the increase in antigen expression relates to the dopaminergic reinnervation of the striatum in MPTP-treated cases. We also suggest that the greater loss of dopaminergic cells in the ventral tier when compared to the dorsal tier relates to glutamate toxicity.
KeywordsCalbindinTyrosine hydroxylaseMPTPParkinson disease6OHDA
Avidin biotin peroxidase complex
Calbindin D28 k
Dorsal substantia nigra pars compacta
Lateral geniculate nucleus
Medial geniculate nucleus
Substantia nigra pars compacta
Substantia nigra pars reticulata
Ventral lateral nucleus of thalamus
Ventral posterior nucleus of thalamus
Ventral substantia nigra pars compacta
Ventral tegmental area
The mammalian midbrain houses several groups of dopaminergic cells that differ in topographical location, expression of various neurochemicals and in patterns of projections with other parts of the brain. In addition, there are reports that these cells differ in their patterns of survival in Parkinson disease.
Classically, the dopaminergic cells of the midbrain have been divided into three main areas, the substantia nigra (ventral and dorsal sectors; A9), the ventral tegmental area (A10) and the retrorubral field (A8) (Björklund and Lindvall 1984; Domburg and Donkelaar 1991). Recently, on the basis of particular neurochemical identities and various patterns of connections, these midbrain dopaminergic areas have been divided further into two distinct ’‘tiers‘’, ventral and dorsal (rat: Fallon and Moore 1978; Gerfen et al. 1987a, 1987b; monkey: Lynd-Balta and Haber 1994; Haber et al. 1995; Francois et al. 1999; human: McRitchie et al. 1997). The ventral tier is made up entirely by the densely packed cells of the ventral sector of the substantia nigra (vSNc). The dorsal tier is made up mainly by cells of the dorsal sector of the substantia nigra (dSNc), the ventral tegmental area (together with many smaller assorted nuclei, for example, the parabrachial pigmented nucleus, the paranigral, parapeduncular, interfascicular, caudal and rostral linear nuclei) and the retrorubral field. In the present study, focus will be on the vSNc of the ventral tier, together with the dSNc, ventral tegmental area and retrorubral field areas of the dorsal tier. Collectively, these form the major dopaminergic cell groups in the midbrain.
There are distinct differences in the neurochemical expression and connectivity patterns between dopaminergic cells located in the two tiers. To a lesser extent, some of these differences are evident among cells located within the individual nuclei of the dorsal tier. Perhaps the most striking neurochemical difference between dopaminergic cells of the ventral and dorsal tiers lies in their expression of the calcium-binding protein, calbindin D28 k. Many studies have shown that there are few calbindin+ dopaminergic cells within the ventral tier, but many within the dorsal tier (mouse: Liang et al. 1996a, 1996b; Ng et al. 1996; Airaksinen 1997; rat: Roderiguez et al. 2001; monkey: Lavioe and Parent 1991; Lynd-Balta and Haber 1994; Haber et al. 1995; Parent et al. 1996; Francois et al. 1999; human: German et al. 1989, 1990, 1992; Gibb 1992; McRitchie and Halliday 1995). There is also a higher level of dopamine transporter molecule mRNA within cells of the ventral tier, than within those in the dorsal tier (mouse: Sanghera et al. 1997; rat: Blanchard et al. 1994; Mengod et al 1989; Augood et al 1993; Cerruti et al 1993; monkey: Haber et al. 1995; human: Fernandez et al 1994). The two tiers differ in their patterns of connections with other centres of the brain, most notably with the striatum (caudate and putamen) and the cerebral cortex. For the ventral tier, the dopaminergic cells project mainly to the dorsal striatum, to the striosomes and to a lesser extent the matrix (rat: Gerfen et al. 1987a, 1987b; Prensa and Parent 2001; monkey: François et al. 1999; human: Gibb and Lees 1991). These dopaminergic cells have no, or at best a small, projection to the cortex (eg, monkey; Francois et al. 1999). For the dorsal tier, the dopaminergic cells of the ventral tegmental area project heavily to the ventral striatum, to the matrix and to a lesser extent the striosomes, together with the nucleus accumbens. The dopaminergic cells of the dSNc and retrorubral field also have heavier projections to matrix, but these cells tend to project to the dorsal rather than the ventral striatum (rat: Fallon and Moore 1978; Gerfen et al. 1987a, 1987b; Brog et al. 1993; Prensa and Parent 2001; cat: Vandermaelen et al. 1978; Jimenez-Castellanos and Graybiel 1987; monkey: Francois et al. 1999; human: Gibb and Lees 1991). The dorsal tier dopaminergic cells project to most, if not all areas of the cortex, in particular, to motor and prefrontal areas (rat: Swanson 1982; Albanese and Minciacchi 1983; Mantz et al. 1988; Seroogy et al. 1989; Deutch et al. 1991; Ohara et al. 2003; cat: Scheibner and Törk 1987; monkey: Porrino and Goldman-Rakic 1982; Levitt et al. 1984; Berger et al. 1986; Lewis et al. 1987; Gaspar et al. 1992; Gaspar et al. 1993; Francois et al. 1999).
Previous studies have reported that the ventral and dorsal tiers differ in their survival patterns in Parkinsonian patients and animals. In Parkinsonian patients (Bernheimer 1973; Waters et al. 1988; German et al. 1989; Yamada et al. 1990; Fearnley and Lees 1991; Gibb and Lees 1991; German et al. 1992; Gibb 1992; Mouatt-Prigent et al. 1994; Hirsch et al. 1997; McRitchie et al 1997; Damier et al. 1999; Liang et al. 2004), and in MPTP-treated (mouse: German et al. 1992; Iacopino et al. 1992; Muthane et al. 1994; Liang et al. 1996b; Ng et al. 1996; German et al. 1997; monkey: Schneider et al. 1987; German et al. 1988, 1992; Lavoie and Parent 1991; Francois et al. 1999) and 6OHDA-lesioned (rat: Rodriguez et al. 2001) animals, the dopaminergic cells of the ventral tier suffer more degeneration than those in the dorsal tier. In general, these studies show a 70–90% loss of dopaminergic cells in the ventral tier, compared to a 25–70% loss in the dorsal tier.
The major aim of this study was to examine the patterns of dopaminergic cell loss in the midbrain of MPTP-treated monkeys and 6OHDA-lesioned rats. Our approach and experimental paradigm was somewhat different to those of previous studies. We used a low dose, subacute model of MPTP administration in monkeys, while previous studies employed a higher dose and more chronic model (eg Schneider et al. 1987; German et al. 1988). For the 6OHDA lesions in rats, we made injections of the toxin directly into the midbrain, while previous studies used either striatal (in neonates; Gerfen et al. 1987a, 1987b) or interventricular (Rodriguez et al. 2001) injections. Using these methods, we explore if there are comparable cell losses in the same dopaminergic cell groups (dorsal tier; dSNc, ventral tegmental area, retrorubral field: ventral tier; vSNc) within these models of Parkinson disease. We used tyrosine hydroxylase (TH) and calbindin immunocytochemistry to label the dopaminergic cells.
Materials and methods
Results were obtained from 5 adult male macaque monkeys (Macaca fascicularis, CRP, Port Louis, Mauritius) weighing 4.8–6.6 kg. Animals were maintained in individual primate cages under controlled conditions of temperature (25±1°C) and light (12 h light/dark cycles). They were fed regularly on a diet of fruit and biscuits, and had free access to water. The Grenoble lab is authorised by the French Ministry of Environment and all experiments were performed in accordance with the European Communities Council Directive of Nov 24 1986 (86/609/EEC) for care of lab animals. Results were also obtained from 10 male (∼8 weeks old) Sprague-Dawley albino rats (250–300 g). Animals were kept in a 12-h light-dark cycle and had unrestricted access to food and water. All of the experiments on rats were approved by the Animal Ethics Committee of the University of Sydney.
MPTP administration in monkeys
In three cases (E1, E2, E3), 0.2 mg/kg/day of MPTP (Sigma) was administered intramuscularly for 8 consecutive days. In two cases, no MPTP injection was given (controls; C1, C2). Twenty one days after the last MPTP injection, animals were processed for immunocytochemistry. Some of these animals (E2,E3) were used as part of a study examining neuroprotection of the substantia nigra after deep brain stimulation of the subthalamus (Ashkan et al. 2005; Wallace et al. 2005). They in no way affected the analysis of the present study on the midbrain dopaminergic cells (subthalamic stimulations were on right hand side of brain, while the results presented here are from the left hand side).
6 hydroxydopamine (6OHDA) administration in rats
Rats were anaesthetised after an intraperitoneal injection of ketamine (100 mg/kg) and rompun (10 mg/kg). Thirty minutes prior to surgery, rats were given intraperitoneal injections of desipramine hydrochloride (25 mg/kg; to protect noradrenergic cells) and pargyline (50 mg/kg; to help stop peripheral breakdown of 6OHDA). Rats were placed in a stereotactic apparatus and had 6OHDA (hydrochloride; 4 μg/μl saline with addition of 0.1–1% ascorbic acid) injected by pressure (12–16 μl injected total) into the medial forebrain bundle (2 separate injections; coordinates 4.5–4.8 mm caudal to bregma, 1.1–1.2 mm lateral, 8.2–8.4 mm ventral; Paxinos and Watson 1986). The right hand side was injected with 6OHDA, while the left hand side was injected with saline (with addition of 0.1–1% ascorbic acid; controls). Rats were allowed to recover for either 7 days (n=3), 28 days (n=3) or 84 days (n=4).
During the study period, each monkey underwent behavioural assessment. Full details of the procedure and protocol are outlined in other publications (Ashkan et al. 2005; Wallace et al. 2005). Briefly, animals were assessed in the home cages starting four weeks after initial arrival. Assessments were performed on five occasions before the start of MPTP to establish the baseline. Thereafter, assessment occurred on a daily basis. On each occasion, animals were scored independently by two observers over a 30 min period. Video taping was used to facilitate this. A modified Benazzouz scale (Benazzouz et al. 1995) was applied and the following parameters were rated: tremor (0–3), bradykinesia (0–3), change in posture (0–3), vocalisation (0–1), frequency of arm movements (0–3 for each arm) and the general level of activity (0–3). A total maximum score possible was 19; a minimum score was 0. Parkinsonian symptoms were considered present with scores of ≥4. For the rats, analysis of 6OHDA lesions was carried out by either apomorphine (dopaminergic agonist; Sigma) injections (0.05 mg/kg) and by post-mortem tyrosine hydroxylase (TH) immunohistochemistry (see below). Rats were given a subcutaneous injection of apomorphine (5 days post-lesion) and they were monitored for any rotational behaviour. If the lesion was successful, rats would start rotating contralateral to the lesion soon after each injection. If rats rotated more that 20 times per 5 min, the lesion was deemed successful.
Animals were anaesthetised after intravenous or intraperitoneal injection of sodium pentobarbital (50–60 mg/kg) and perfused transcardially with 0.9% saline followed by 4% buffered formaldehyde. Brains were removed, blocked, immersed in the same fixative for 24 h, and then placed in saline with the addition of 30% sucrose until the block sank. They were then sectioned coronally on a cryostat at a thickness of 50–55 μm. Every section was collected in sequence; one series was collected onto gelatinised slides and processed for routine cresyl violet staining while five series were collected in phosphate-buffered saline (PBS; free floating) for immunocytochemistry. Sections were immersed in a solution of 10% normal goat serum and 1% bovine serum albumin (made up with PBS) for 1 h. In some cases, sections were placed in 10% H2O2 in 70% ethanol for 10 min in order to block endogenous peroxidase activity. Alternate sections were then incubated in either anti-TH or anti-calbindin for 48 h at 4°C. Both antibodies were diluted 1:500 (in PBS) and were purchased from Chemicon. The sections were then incubated with biotinylated anti-mouse (IgG, Sigma; 1:200 in PBS) for 2 h at room temperature. Finally sections were incubated in the avidin-biotin-peroxidase complex (ABC; Vectastain; 1:50 in PBS) for 1 h at room temperature. The bound peroxidase molecule was visualised using 3,3-diaminobenzidine (DAB; Sigma; sections reacted for ∼5 mins). In between each incubation, the sections were washed several times with PBS. Sections were mounted on gelatinised slides, dried overnight, dehydrated in ascending alcohols, cleared in Histoclear and coverslipped with DPX. For control experiments, the primary and/or secondary antibodies were replaced by PBS and then reacted as above. Control sections were immunonegative. There were no differences in the labelling patterns of the non-H2O2 pretreated compared to the H2O2 pretreated sections, indicating minimal endogenous peroxidase activity. Double labelling experiments were undertaken also. For these, sections of monkey midbrain were processed as above for TH immunostaining, except for the last step when they were incubated in avidinCy2 complex (Vector Labs; 1:100), instead of ABC, for 2–3 h. After washing in PBS, these sections were then processed for calbindin immunostaining except for the last step again, when the sections were incubated in avidinCy3 complex (Vector Labs; 1:100) (instead of ABC) for 2–3 h. Thereafter, sections were washed in PBS, mounted on slides, left until almost dry and coverslipped with glycerol. Sections were then viewed under a fluorescence microscope. The TH and calbindin immunolabelling patterns with the fluorescence tags were no different to the immunolabelling patterns with the peroxidase tag. Hence, we are confident that the DAB method did not produce any false positive cells due to overstaining.
The results generated on the midbrain dopaminergic cells in monkeys and rats will be considered in separate sections. In each section, the clinical and behavioural outcome after treatment/lesion, together with the patterns of TH and calbindin immunolabelling in the midbrain will be examined.
A subacute model of MPTP treatment in monkeys, designed to include many of the essential elements of an effective model for Parkinson disease, was used in this study (Bezard et al. 1997; Ashkan et al. 2005; Wallace et al. 2005). The monkeys were injected with MPTP for 8 consecutive days and then allowed to survive for 21 days thereafter. Due to the rareness of these animals for scientific use, this was the only survival period and regime that could be undertaken in this study. Clinically, the MPTP-treated monkeys (E1, E2, E3) developed parkinsonian symptoms including akinesia, bradykinesia, rigidity, postural instability, mask-like faces, reduced vocalisation and dysphagia. Tremor was less common and when it occurred, it was of the intention rather than of the rest type. This is in keeping with previous studies suggesting that to induce tremor, additional mechanisms are required to those responsible for akinesia and rigidity. These include lesions to the ventral tegmental area and red nucleus (Larochelle et al. 1971; Ohye et al. 1988). MPTP-treated monkeys (E1, E2, E3) typically showed these behavioural changes after three doses of MPTP, reaching the peak score on or shortly after the last dosing date (scores of 11 to 14; maximum possible 19). Thereafter animals had improvements in the scores reaching stability around the 15th post MPTP day. The behavioural score for the control monkeys (C1, C2) remained zero throughout testing. Thus, in behavioural terms, the three MPTP-treated monkeys examined in this series of experiments were severely Parkinsonian.
General organisation of dopaminergic cells in midbrain
The morphology and number of TH+ and calbindin+ cells in the control and MPTP-treated monkey cases will be considered separately.
Figure 5 also shows graphs of the number of calbindin+ cells in the dSNc (Fig. 5e), ventral tegmental area (Fig. 5f) and retrorubral field (Fig. 5g) in the five monkey cases examined in this study. There was only the occasional calbindin+ cell seen in the vSNc, and hence, no analysis was performed in this nucleus. For the dorsal tier – dSNc (Fig. 5e), ventral tegmental area (Fig. 5f), retrorubral field (Fig. 5g) – the number of calbindin+ cells in the MPTP-treated cases (E1+E2+E3) was, on average, higher than the number of cells in the controls (C1+C2).
Double labelling patterns in the midbrain: TH+ /Calbindin+ cells
6 hydroxydopamine (6OHDA) lesion
In this series of experiments, the patterns of TH and calbindin immunoreactivity in the midbrain after 6OHDA lesion was examined. The main control used was saline injections into medial forebrain bundle of the side contralateral to the 6OHDA injections in the same animal (classical hemiparkinsonian model). This is a well-accepted control for these sorts of experiments because most nigral pathways are ipsilateral (Schober 2004; see also Heise and Mitrofanis 2005)
The survival period after 6OHDA lesion was staged, at 7, 28 and 84 days. This was done as to explore the acute and chronic effects of lesion on the expression of TH and calbindin. In most cases, there was a small region of gliosis marking the location of the injection site in the vicinity of the medial forebrain bundle. None of the 6OHDA-lesioned rats developed any observable motor and/or visceral deficit post-lesion and they were eating and grooming soon after surgery.
The efficacy of the lesion was measured in terms of apomorphine rotational behaviour and loss of TH+ cells from the vSNc. All rats that had a large loss of TH+ cells from the vSNc (>65–70%) after 6OHDA lesion (see below) displayed contralateral rotational behaviour after injection of apomorphine (>20 rotations per 5 minutes). Rats that did not have a successful 6OHDA lesion generally showed marked hyperkinetic movements, but no rotation. The brains of these rats were not analysed further in this study.
General organisation of dopaminergic cells in midbrain
The morphology and number of TH+ and calbindin+ cells in the control and 6OHDA-lesioned sides will be considered separately.
A very similar pattern was apparent in the number of calbindin+ cells at the different survival periods (Fig. 12). In the dSNc (Fig. 12e), the number of calbindin+ cells on the control side was higher than in the 6OHDA-lesioned side at all survival periods, but the differences did not reach significance (P=0.4 at 7d; P=0.07 at 28d; P=0.09 at 84d; Fig. 12e). The same patterns were apparent in the ventral tegmental area (P=0.6 at 7d; P=0.7 at 28d; P=0.6 at 84d; Fig. 12f) and the retrorubral field (P=0.3 at 7d; P=0.6 at 28d; P=0.1 at 84d; Fig. 12g).
Our results show a differential pattern of loss among dopaminergic cells of the midbrain in MPTP-treated monkeys and 6OHDA-lesioned rats. In general, the dopaminergic cells of the ventral tier suffer more degeneration than those of the dorsal tier. Indeed, in the case of MPTP-treated monkeys, the number of dopaminergic cells expressing TH and calbindin within the dorsal tier actually increases. These issues will form the focus of the discussion outlined below.
Comparisons with previous findings
Previous studies have reported that the midbrain dopaminergic cells of the ventral tier are more vulnerable to cell death after Parkinsonian insult compared to those of the dorsal tier. This has been shown in MPTP-treated mice (Iacopino et al 1992; Liang et al 1996b; Ng et al 1996; German et al 1997), rats (Iacopino et al 1992) and monkeys (Schneider et al 1987; German et al 1988; Lavoie and Parent 1991; German et al 1992; Francois et al 1999), as well 6OHDA-lesioned rats (Gerfen et al. 1987 ; Rodriguez et al. 2001) and Parkinson disease patients (Bernheimer 1973; Waters et al. 1988; German et al. 1989; Yamada et al. 1990; Fearnley and Lees 1991; Gibb and Lees 1991; German et al. 1992; Gibb 1992; Mouatt-Prigent et al. 1994; McRitchie et al. 1997; Damier et al. 1999; Liang et al. 2004). We extend these previous findings by providing a direct morphological and numeric comparison of midbrain dopaminergic cells within the two tiers in two animal models of Parkinson disease, namely MPTP-treated monkeys and 6OHDA-lesioned rats. Further, our experimental paradigm was different to those used previously. We used a low dose, subacute model of MPTP administration in monkeys, while previous studies employed a higher dose and more chronic model (eg Schneider et al. 1987; German et al. 1988). For the 6OHDA lesions in rats, we made injections of the toxin directly into the midbrain, while previous studies used either striatal (in neonates; Gerfen et al. 1987a, 1987b; Lee et al. 1996; Kirik et al. 1998) or interventricular (Rodriguez et al. 2001) injections.
Overall, our results compare favourably to those of previous studies, except in two notable ways. First, the overall reduction in cell number in the ventral tier of the MPTP-treated monkeys and in the ventral and dorsal tiers of the 6OHDA-lesioned rats reported here, is smaller than those recorded previously (∼30% smaller in most cases). For example, we report 65% cell loss in the vSNc in MPTP-treated monkeys, while other studies show ∼90% cell loss (eg Schneider et al. 1987; German et al. 1988). These differences are likely to reflect the lower doses of MPTP (1 mg compared to 2.4–5.6 mg [Schneider et al. 1987] and 1.8 mg [German et al. 1988]) and 6OHDA (eg, 48 μg compared to 300–500 μg; Rodriguez et al. 2001) used in this study compared to previous reports. In the case of the MPTP-treated monkeys, our shorter survival period may also contribute to the smaller reduction (21 days compared to 1–9 months [Schneider et al. 1987] and 23–45 days [German et al. 1988]). Second, for the dorsal tier of the MPTP-treated cases, we show a surprising increase (22%) in TH and calbindin expression among dopaminergic cells; previous studies have reported either no change or a small decrease in number. Again, these differences are likely to reflect, at least in part, the lower doses of MPTP used in this study.
Patterns in MPTP-treated monkeys: does the increase in antigen expression reflect dopaminergic reinnervation of the striatum?
Perhaps the most striking finding of this study is the small, but distinct increase in the number of dorsal tier dopaminergic cells expressing TH and calbindin in MPTP-treated monkeys compared to controls. Such an increase has not been reported by previous studies using higher dose/chronic models (see above), but has been shown in an acute MPTP model in mice, at least for calbindin expression (Ng et al. 1996). Taking these results together, there is an increase in the number of cells expressing TH and calbindin in the dorsal tier after a low dose MPTP insult; such an increase may not be evident after a higher dose and more extensive MPTP insult. Two issues arise regarding this increase in antigen expression among dorsal tier cells (i) the identity of the cell type newly expressing these antigens and (ii) the factor underlying its significance.
The increase in TH+ and calbindin+ cell number in the dorsal tier after MPTP treatment indicates that there is a population of cells in this region that does not normally have levels of TH or calbindin that are detectable by immunocytochemistry. Such cells may upregulate their expression of these antigens in response to MPTP insult. These cells have the same morphology as those of surrounding cells that express TH and calbindin normally, since there were no striking differences in the morphology of cells in the dorsal tier after MPTP treatment. Such cells are likely to contain other neurochemicals, such as γaminobutyric acid (GABA; rat: Steffenson et al 1998; Rodriguez and Gonzalez-Hernandez 1999) or neurotensin (rat: Uhl et al 1979; Palacios and Kuhar 1981; Jennes et al 1982 and Kalivas 1984), and be prompted to express TH and calbindin after MPTP insult. It should be noted that the dorsal tier is not the only neural cell group that newly expresses or increases TH expression after MPTP treatment. Betarbet and colleagues (1997) have reported that there are many more TH+ cells in the striatum of MPTP-treated monkeys than in the controls.
The increase in TH and calbindin expression in the midbrain dorsal tier cells after MPTP treatment is curious. On the one hand, there is mass degeneration in one type of midbrain dopaminergic cell (ventral tier), while on the other hand, there is an increase in antigen expression in another (dorsal tier). It is likely that these two features are linked, that there are axonal and/or dendritic interconnections between the two regions (Braak and Braak 1986; Deutch et al. 1988) and a loss of cells from the ventral tier prompts the changes in expression in the dorsal tier.
The significance of these changes in antigen expression is not clear, but it is tempting to speculate that they reflect the well-known striatal dopaminergic reinnervation seen in MPTP-treated rats (Blanchard et al 1996; Bezard et al. 2000) and mice (Ho and Blum 1998; Mitsumoto et al. 1998). These reinnervations have been shown to arise mainly from cells in the dorsal tier, in particular the ventral tegmental area (Ho and Blum 1998). From these previous studies, it was not clear whether the dopaminergic reinnervations were from dorsal tier or striatal cells newly expressing TH or whether surviving dorsal tier TH cells issue sprouts (Blanchard et al 1996; Betarbet et al. 1997; Ho and Blum 1998; Bezard et al. 2000). Our results here indicate that the dopaminergic reinnervations may be formed, at least in part, by newly TH expressing cells of the dorsal tier. Interestingly, previous studies have shown that with lower doses of MPTP, the reinnervations are apparent soon after insult (14 days; Ho and Blum 1998), while with higher doses, it occurs much later (5 months; Bezard et al. 2000). This would be consist with the present and other findings of TH+ cell number in the midbrain; no increase in cell number using higher dose/chronic MPTP models (eg Schneider et al. 1987; German et al. 1988), but a small increase using low dose/acute MPTP model (this study).
Patterns in 6OHDA-lesioned rats
The distinct increase in TH and calbindin expression is not seen in the 6OHDA-lesioned rats in both short (7 day) and long (28 and 84 days) survival periods. In these cases, the number of TH+ and calbindin+ cells in the dorsal tier decreases, but not to the extent seen in the ventral tier (for TH+ cells). For example, after 84 days post-lesion, there is a 97% decrease TH+ cell number in the ventral tier (vSNc), while the decrease in the dorsal tier was smaller, being 65% in the dSNc, 12% in the ventral tegmental area and 57% in the retrorubral field. These species differences in the patterns of antigen expression within the dorsal tier may well reflect the different cellular toxicity affected by MPTP and 6OHDA. Although both these toxins gain entry into the dopaminergic cells via the dopamine transporter molecule, there are subtle differences in the way by which they generate mitochondrial dysfunction and subsequent cell death (Schober 2004). Such differences may influence the expression of TH and/or calbindin. Further, in 6OHDA-lesioned animals there is no major dopaminergic sprouting within the striatum (eg, Touchon et al. 2004), except when animals were given injections of glial cell line derived neurotrophic factor (GDNF), for example (Georgievska et al. 2002). Hence the increase in antigen expression appears dependant on dopaminergic reinnervation of the striatum, of which does not occur after 6OHDA application, but does after MPTP treatment (see above).
The pattern of cell loss is not spread evenly among the different cell groups of the dorsal tier. The TH+ and calbindin+ cells of the ventral tegmental area suffers the least loss after 6OHDA insult; while cell loss in this area was ∼15%, the cell loss in the dSNc and retrorubral field was between 40–60%. Similar findings have been reported by other studies after 6OHDA-lesioned rats (eg, Rodriguez et al. 2001) and high dose chronic MPTP models (eg, Schneider et al. 1987; German et al. 1988). There is thus a factor that protects these cells from MPTP and 6OHDA toxicity. This issue will be discussed in the next section.
Factors that protect dorsal tier cells from MPTP and 6OHDA toxicity: does glutamate toxicity play a role?
Many factors are likely to contribute to the survival of dorsal tier cells in parkinsonian cases, including calbindin expression (eg, German et al 1992), dopamine transporter molecule levels (eg, Haber et al. 1995) and/or neuromelanin content (eg, Hirsch et al 1997). We will focus discussion on calbindin expression, the most relevant to the present findings. Next, we will consider the rather novel idea of glutamate toxicity and its possible role in the differential death of dopaminergic cells in the midbrain
The selective expression of the calcium-binding protein calbindin, that may buffer any intracellular increases in calcium prompted by MPTP or 6OHDA insult, could help protect the dorsal tier cells (Nicklas et al. 1987; Kass et al. 1988). Our results lend some support to this notion. In MPTP-treated monkeys, we reveal an increase in calbindin+ cell number, while in 6OHDA-lesioned rats, the cell groups that have many calbindin+ cells (dSNc, ventral tegmental area, retrorubral field) suffer less loss than those that do not (vSNc). These features indicate a putative "neuroprotective" role for calbindin. However, there is evidence suggesting that the expression of calbindin is not the sole factor that offers protection for these cells. For example, although there are many cells that are calbindin+ that survive in Parkinsonian cases, there are non-calbindin+ dopaminergic cells survivors as well (mouse: Liang et al. 1996b; rat: Rodriguez et al. 2001; this study; monkey: Parent et al. 1996; this study; human: Gibb 1992; Agid et al. 1993). For example, in this study, we show that approximately 50% of the cells that survive MPTP treatment are not calbindin+ . Further, in mice with a calbindin-deficient gene, there is no substantial increase in cell death within the dorsal tier after exposure to MPTP (Airaksinen et al. 1997).
We thank Medtronic and Tenix corp/Salteri family for their most generous funding of this work. Laurance Grotti and Sharon Spana provided invaluable technical assistance with the immunostaining and histology, while Rolande Gerbex and Louis Gonzales provided excellent assistance looking after the animal house.