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.2014 Dec 3:8:146.
doi: 10.3389/fnana.2014.00146. eCollection 2014.

Calbindin content and differential vulnerability of midbrain efferent dopaminergic neurons in macaques

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Calbindin content and differential vulnerability of midbrain efferent dopaminergic neurons in macaques

Iria G Dopeso-Reyes et al. Front Neuroanat..

Abstract

Calbindin (CB) is a calcium binding protein reported to protect dopaminergic neurons from degeneration. Although a direct link between CB content and differential vulnerability of dopaminergic neurons has long been accepted, factors other than CB have also been suggested, particularly those related to the dopamine transporter. Indeed, several studies have reported that CB levels are not causally related to the differential vulnerability of dopaminergic neurons against neurotoxins. Here we have used dual stains for tyrosine hydroxylase (TH) and CB in 3 control and 3 MPTP-treated monkeys to visualize dopaminergic neurons in the ventral tegmental area (VTA) and in the dorsal and ventral tiers of the substantia nigra pars compacta (SNcd and SNcv) co-expressing TH and CB. In control animals, the highest percentages of co-localization were found in VTA (58.2%), followed by neurons located in the SNcd (34.7%). As expected, SNcv neurons lacked CB expression. In MPTP-treated animals, the percentage of CB-ir/TH-ir neurons in the VTA was similar to control monkeys (62.1%), whereas most of the few surviving neurons in the SNcd were CB-ir/TH-ir (88.6%). Next, we have elucidated the presence of CB within identified nigrostriatal and nigroextrastriatal midbrain dopaminergic projection neurons. For this purpose, two control monkeys received one injection of Fluoro-Gold into the caudate nucleus and one injection of cholera toxin (CTB) into the postcommissural putamen, whereas two more monkeys were injected with CTB into the internal division of the globus pallidus (GPi). As expected, all the nigrocaudate- and nigroputamen-projecting neurons were TH-ir, although surprisingly, all of these nigrostriatal-projecting neurons were negative for CB. Furthermore, all the nigropallidal-projecting neurons co-expressed both TH and CB. In summary, although CB-ir dopaminergic neurons seem to be less prone to MPTP-induced degeneration, our data clearly demonstrated that these neurons are not giving rise to nigrostriatal projections and indeed CB-ir/TH-ir neurons only originate nigroextrastriatal projections.

Keywords: MPTP; Parkinson’s disease; calbindin; neuronal tracers; neuroprotection; nigroextrastriatal pathway.

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Figures

Figure 1
Figure 1
Distribution patterns of TH and CB immunoreactivity in caudate and putamen, SNc and VTA in control and MPTP-treated monkeys.(A–D) TH immunohistochemistry in control monkeys in striatum(A), rostral, medial, and caudal SNc(B–D) and VTA.(1–4) Insets taken at higher magnification of rostral(1), medial(2), and caudal(3) SNc and VTA(4) showing TH-ir profiles.(E–H) CB immunoreactivity in striatum(E), rostral, caudal, and medial(F–H) SNc and VTA.(5–8) Higher magnification insets of CB-ir cells in rostral(5), medial(6) and caudal(7) SNc, and VTA(8).(I–L) Extent of MPTP-induced dopaminergic depletion. The levels of TH in the striatum and SN that typically characterizes a control primate(A–D) are clearly reduced in the MPTP-treated monkeys(I–L).(9–12) Higher magnification insets of TH-ir cells in the rostral(9), medial(10) and caudal(11) SNc as well as in the VTA(12), showing a reduction of the TH immunoreactivity compared with control monkeys(1–4).(M–P) calbindin immunohistochemistry in striatum(M), rostral, medial, and caudal SNc(N–P) and VTA.(13–16) Insets taken a higher magnification from rostral(13), medial(14), and caudal SNc(15) and VTA(16) showing the presence of CB-ir cells in these areas. Unlike the TH expression, CB immunoreactivity did not changed between control and MPTP-treated monkeys. Scale bars: 1,000 µm in panels(A, E, I and M); 250 µm in panels(B–D, F–H, J–L and N–P); 25 µm in panels(1–16). Abbreviations: substantia nigra, pars compacta (SNc), ventral tegmental area (VTA), putamen (Put), caudate nucleus (CN), external division of the globus pallidus (GPe), internal capsule (ic), anterior commissure (ac).
Figure 2
Figure 2
Double immunofluorescent detection of TH and CB in VTA and SNc in control and MPTP-treated monkeys. At the level of the VTA in control macaques(A–A”), 58.2% of the CB-ir neurons are also TH-ir, and 62.1% in MPTP-treated animals(B–B”). When considering the dorsal tier of the SNc (SNcd, medial and lateral territories) of control animals(C–C” and E–E”) the percentage of co-localization is estimated as 34.7% (arrowheads). By contrast, in MPTP-treated macaques and besides a substantial loss of TH-ir neurons, most of the surviving dopaminergic neurons expressed CB (arrowheads inD–D” andF–F”). The percentage of TH-ir/CB-ir neurons increased up to 88.6%. In other words, dopaminergic neurons from the SNcd containing CB are less prone to degenerate following MPTP insult. At the level of the ventral tier of the SNc (SNcv) none of the TH+ neurons expressed CB in control animals(G–G”). Panels(H–H”) are taken from the boundaries between SNcd and SNcv, since most -if not all- of the TH-ir neurons in the SNcv degenerated following MPTP treatment because of the lack of CB in these cells. Scale bar is 25 µm in all panels.
Figure 3
Figure 3
Different types of nigrostriatal-projecting neurons identified following the delivery of retrograde tracers in the head of the caudate nucleus (A; injection of FG) and in dorsolateral territories of the postcommissural putamen (B; CTB deposit). Both injections are restricted to the targeted area, without any noticeable tracer leakage through the injection tract.(C–E) coronal sections through the substantia nigra taken from rostral(C), medial(D) and caudal(E) levels. Neurons innervating either the caudate nucleus (purple-stained; labeled with FG) or the putamen (brown-stained; labeled with CTB) are distributed in clusters containing both subtypes of neurons intermingled with each other.(F–K) Insets taken from(C–E) at higher magnification to better appreciate the cellular composition of the clusters containing projection neurons. It is worth noting that double-labeled cells, e.g., neurons innervating both the caudate and the putamen were never noticed. Scale bar is 2,000 µm in panels(A) and(B); 500 µm in panels(C–E), and 100 µm in panels(F–K). Abbreviations: putamen (Put), caudate nucleus (CN), external division of the globus pallidus (GPe), internal capsule (ic), anterior commissure (ac).
Figure 4
Figure 4
Nigrostriatal neurons innervating the caudate nucleus. Following the delivery of FG in the head of the caudate nucleus, retrogradely-labeled neurons (green channel) were found in the SNcd(C), in the SNcv(G) and to a lesser extent in the VTA(K). All these FG-ir neurons are TH-ir and negative for CB.(A’–C’) Insets taken from(A–D) showing one FG+/TH+ neuron, one CB-ir/TH-ir neuron as well as another neuron single-stained for TH only. It is worth noting that FG-ir/TH-ir/CB-ir neurons were never seen. Scale bar is 25 µm for panels(A–D, E–H and I–L) and 5 µm in panels(A’–D’).
Figure 5
Figure 5
Nigrostriatal neurons innervating the putamen nucleus. Following the delivery of CTB in dorsolateral territories of the postcommissural putamen, retrogradely-labeled neurons (green channel) were found in the SNcd(C), in the SNcv(G) and to a lesser extent in the VTA(K). All these CTB-ir neurons are TH-ir and negative for CB.(A’–C’) Insets taken from(A–D) showing one CTB-ir/TH-ir neuron, two CB-ir/TH-ir neurons as well as few more neurons single-stained for TH only. It is worth noting that FG-ir/TH-ir/CB-ir neurons were never seen. Scale bar is 25 µm for panels(A–D, E–H and I–L) and 5 µm in panels(A’–D’).
Figure 6
Figure 6
Nigroextrastriatal neurons innervating the internal division of the globus pallidus (GPi). Following the delivery of the retrograde tracer CTB(A, B), very few CTB-labeled neurons were found in the SNcd and in VTA(E, E’; I, I’). It is worth noting that nigropallidal-projecting neurons were never found in SNcv. Even at low magnification(panel A), a substantial number of CTB-labeled neurons were easily noticed in the caudate nucleus and to a lesser extent in medial territories of the putamen. At the level of the SNcd(panels C–F’), all CTB-ir neurons (green channel) were also positive for both TH and CB. The same holds true when considering the VTA area, since all the VTA neurons projecting to GPi co-expressed CTB, TH and CB(panels G–J’). Scale bar is 25 µm in panels(C–F and G–J) and 5 µm in panels(C’–F’ and G’–J’).
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