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Calbindin D28k-containing neurons are restricted to the medial substantia nigra in humans
Neuroscience Vol. 65, No. 1, pp. 87-91, 1995
~
Pergamon
Elsevier ScienceLtd Copyright © 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00
0306-4522(94)00483-8
CALBINDIN D28k-CONTAINING N E U R O N S ARE RESTRICTED TO THE MEDIAL SUBSTANTIA NIGRA IN H U M A N S D. A. M c R I T C H I E * and G. M. H A L L I D A Y Prince of Wales Medical Research Institute, Prince of Wales Hospital, Randwick, NSW 2031, Australia Abstract--A controversy exists in the literature as to whether neurons containing the calcium binding protein calbindin-D28 k are located within the human substantia nigra. The point of variance between reports, however, is not the anatomical distribution of these neurons, but rather the delineation of the dorsal border of the substantia nigra. It has been suggested that the dense substance P striatonigral innervation delimits the substantia nigra in the human. The aim of the present study is to re-examine the distribution of calbindin-D28k-positive neurons throughout the substantia nigra using substance P to delimit its borders. Although a few calbindin-D28k-positive neurons were found in the medial cell group of the substantia nigra, the vast majority of positive neurons were located in the adjacent A8 and AI0 dopaminergic cell groups. This anatomical location of calbindin-D28k-positive neurons is consistent with previous reports, though our results indicate that when the striatonigral projection is used to define the substantia nigra, calbindin-D28 k is not a notable feature of these neurons. This questions the neuroprotective role of this protein in Parkinson's disease.
Calbindin-D28k is a m e m b e r o f the E F - h a n d family o f calcium binding proteins. 48 The precise functional role o f these proteins is not yet known, t h o u g h they are believed to have some calcium "buffering" properties. 3'46 As such, they may be i m p o r t a n t in buffering high levels o f intracellular calcium, which would otherwise lead to excitotoxic cell damage. While m a n y studies s u p p o r t this putative neuroprotective role for the calcium binding proteins, 1926'28'4a'42'51'53'54'59
The present study aims to reassess the distribution o f calbindin-D28k-containing neurons in the midbrain using the striatonigrat projection to demarcate the SN.
EXPERIMENTAP LROCEDURES Case selection
Brains were collected at autopsy from eight patients (see Table 1 for patient details) with no clinical or pathological evidence of nervous system abnormality. Written consent for autopsy was obtained and the study was approved by the Human Ethics Committee of The University of Sydney.
the concept is not unchallenged and several groups suggest that neuronal populations containing these proteins are differentially vulnerable to neurodegeneration. 2°'29,31'37'58 In some instances the presence o f these proteins seem to predispose neurons to damage. 1,50
Tissue collection
The brains were immersion fixed in 15% buffered formalin for two weeks. The brainstem was removed from the cerebrum at the level of the mamillary bodies (to ensure that the entire rostrocaudal extent of the SN could be sampled) and detached from the cerebellum. The cerebrum and brainstem were embedded separately in 3% agarose before cutting on a rotary slicer. The cerebrum was cut into 3 mm-coronal slices and the brainstem was cut into 3 mm-blocks in the transverse plane, allowing detailed macroscopic ex~lmination of cases. Standard blocks of the premotor, motor, inferior temporal and parahippocampal cortices, the hippocampus and the cerebellar vermis were paraffin-embedded, sectioned at 7/~m and the sections stained with either haematoxylin and eosin or silver to discount pathology. The midbrain blocks were cryoprotected in 30% sucrose in 0.1 M Tris-HC1 buffer, pH 7.4 for two to four days and serially sectioned on a Bright cryostat at 50/lm intervals. Parallel 1 in 10 series of sections were stained with buffered Cresyl Violet (0.5% in acetate buffer, pH 5.3), haematoxylin and eosin, luxol fast blue, the Garvey silver stain ~3 and processed for immunohistochemistry with substance P and
Past reports o f the distribution o f calbindinD28k-containing neurons in the ventral midbrain suggest that this protein in particular may offer some protection in the pathogenesis o f P a r k i n s o n ' s disease. 16'59 Calbindin-D28k-containing neurons have been reported to be present in the dorsal tier o f the substantia nigra (SN) 19'59 in humans, which is the region o f the SN selectively spared in P a r k i n s o n ' s disease. 4'I1'18"19'2°'21'57'59 However, this pattern o f distribution has been disputed and several studies report that calbindin-D2sk-containing neurons are not f o u n d within the SN at all. 12'2°'33 Recent studies suggest that the SN is delimited by its dense substance P innervation 6A°'15'23'32'39'4°'43'49'52'57 from striatal afferents. 2'9'38 *To whom correspondence should be addressed. Abbreviations: CB-LI, calbindin-D28k-like immunoreactive;
SN, substantia nigra. 87
88
D . A . McRitchie and G. M. Halliday
Fig. 1. Camera lucida drawings and photomicrographs of the distribution of CB-LI neurons in the h u m a n midbrain. (A) Photomicrograph of substance P-like immunoreactive structures in the ventral midbrain around the level of the exiting third nerve (3n). The dense afferent input to the SN was used to delimit this nucleus, as it labelled both pars compacta and the reticulata (SNr). The substance P-like immunoreactivity often diminished around the pigmented cell clusters of the compacta; dorsal (SNd) and ventral (SNv) tiers, medial cell group (SNm) and the pars lateralis (SNI). In contrast the adjacent A I0 parabrachial pigmented nucleus (PBP) and A8 retrorubral fields ( R R F ) display very sparse immunoreactivity for this tachykinin. The red nucleus (R) and the cerebral peduncle (cp) are also indicated. Scale bar = 1 ram. (B) Photomicrograph of the adjacent section stained for Cresyl Violet. The dense pigmented cell clusters are seen in contrast to the relatively cell sparse A10 PBP. Additional structures indicated and scale are as for A. (C) Photomicrograph of CB-LI immunoreactive structures in the ventral midbrain. Dense fibre labelling is seen in the dorsomedial region of the SN, though diminishes around the pigmented cell clusters. The CB-LI neurons in the PBP are seen to arch around the circumference of the R. The CB-LI fibre labelling in the A8 R R F and the A I 0 PBP is m u c h more sparse than that in the SN. Additional structures labelled and scale are as for A. (D) Line drawing of the ventral midbrain at the level of the exiting oculomotor nerve. The section outline is provided in the inset with the box indicating the region enlarged in A~2. Each asterix represents one CB-LI neuron from a single 50 ~tm-section. A few CB-LI neurons were located in SNm, though never present in the SNd or SNv, SN1 or SNr. In contrast they were present in considerable numbers in the A10 PBP, paranigral (PN) and rostral linear (RLi) nuclei, and in the A8 R R F . Additional structures labelled are as for A. Scale bar - 2 m m . (E) Photomicrograph depicting the border between the PBP and SNd. The diagonally aligned CB-LI neurons and fibres in the PBP are clearly separate from the SNd with its non-aligned, unlabelled pigmented neurons. The paucity of fibre labelling around the pigmented cell clusters in the SNd also helps distinguish the border between the SNd and the PBP. The exiting fibres of the oculomotor nerve pierce both of these nuclei. Scale bar = 500 # m .
Calbindin D28k in the human nigra Table 1. Patient details Postmortem Case Age Sex delay 1 2 3 4 5 6 7 8
71 46 58 61 37 85 88 47
F F M F M M F M
18 24 26 19 28.5 21 32 23
Cause of Death Pulmonary Embolism Ischemic Heart Disease Ischemic Heart Disease Metastatic Carcinoma Aspiration Pneumonia Metastatic Carcinoma Cardiorespiratory Arrest Pulmonary Embolism
calbindin-D28 k antibodies. Remaining sections were stored for later use.
Irnrnunohistochernistry Sections chosen for immunohistochemistry were first pretreated in 0.01% hydrogen peroxide in 50% alcohol to abolish endogenous peroxidase activity and incubated in 1% normal horse serum prior to incubation in either substance P (rat monoclonal, 1:1000, Seralab) or calbindinD28k (mouse monoclonal, I : 2000, Sigma) primary antibody. The antibody was localized using the avidin-biotinperoxidase technique 3° with biotinylated anti-rat or antimouse IgG diluted 1:200 and the ABC reagent diluted I:100. The peroxidase reaction was visualized using diaminobenzidine tetrachloride (0.05%, Sigma) as the chromogen. RESULTS
Calbindin-D28k-like immunoreactive (CB-LI) structures were clearly visualized in the ventral midbrain. Neurons were considered to be positive only if the immunoreaction clearly delineated the cell soma and proximal processes (see inset in Fig. IC). Filamentous staining was interpreted to be fibres and punctate structures were interpreted to be terminals. There was little variation in the staining pattern and intensity of CB-LI structures throughout the midbrain indicating that this protein is preserved over post mortem delays of up to 36 h and a wide range of ages. Substance P-like immunoreactive fibres were used to demarcate the SN (Fig. 1A). The pigmented neurons in the SN were organized into discrete clusters (Fig. 1B) which were labelled based on position; medial cell cluster, lateral cell cluster (SN pars lateralis), and dorsal and ventral tiers of the SN pars compacta (Fig. 1B). The most striking feature of the staining within the SN was the extensive CB-LI fibre plexus. In the medial part of the pars compacta the CB-LI fibre labelling was more dense than it was laterally with light fibre labelling seen in the pars reticulata (Fig. 1C). Only a small population of CB-LI neurons were found in the SN restricted to the caudal part of the medial cell cluster (Fig. 1D). As illustrated, less than 10 CB-LI neurons per 50/zm section were found over the caudal one third of the SN on serial section analyses of eight cases. This distribution was quite consistent, with CB-LI neurons localised near the dorsal border of the medial cell cluster. N o CB-LI neurons were found in the dorsal or ventral tiers, pars lateralis or pars reticulata of the SN at any level of the midbrain in any case (Fig. 1D),
89
though CB-LI fibre labelling was diminished around these pigmented cell clusters (Fig. 1C,E). In contrast to the SN, abundant CB-LI neurons were observed in the A10 and A8 dopaminergic cell groups (Fig. 1D). The CB-LI neurons in the A8 group in the midbrain reticular formation had randomly arranged dendrites, while those situated more medially in the A10 region had aligned dendrites. For example, the dendrites of CB-LI neurons in the caudal and rostral linear nuclei were aligned mediolaterally, while those in the parabrachial pigmented nucleus were aligned parallel to the circumference of the red nucleus, dorsal to the dorsal tier of the SN pars compacta. A clear distinction could be made between these aligned CB-LI neurons in the parabrachial pigmented nucleus and the unlabelled pigmented neurons in the adjacent dorsal tier of the SN (Fig. 1C,E). Again in contrast to the SN, sparse CB-LI fibre labelling was present in these A8 and A I 0 cell regions. DISCUSSION
The distribution of CB-LI neurons throughout the ventral midbrain in the rat has been elegantly documented and corroborated. 8,16'~7 In the human, however, there is little consensus on the distribution of CB-LI neurons in this region of the brain. 12'w'2°'33'59 Given that the substantia nigra is the site of the hallmark pathology for Parkinson's disease 22'34and is affected in a variety of other pathologies, 35'45"55'56an accurate account of the chemistry of the SN seems essential. The reason for discrepancies in reports of CB-LI throughout the human ventral midbrain appears to be purely anatomical. O f the studies which report CB-LI neurons in the human S N , 19'59 the majority utilize the brainstem atlas of Olszewski and Baxter 44 which has rostrocaudal inconsistencies in the parcellation of the SN (caudally located parabrachial pigmented nucleus and rostrally located SN pars 7 in the same position). By utilizing the striatal innervation of the A9 dopaminergic neurons, a clear demarcation of these functional groups can be achieved. Thus the parabrachial pigmented nucleus is found ventral to the red nucleus over the entire midbrain as has been previously published. 7'14'18'2°'25'36'47 When reconstructed in three dimensions, the parabrachial pigmented nucleus is continuous with more medial A10 neuronsfl 5 The separation of this region from the SN has been recently proposed by Gibb 2° (though he retains the pars y terminology) using the same criteria. Thus it would seem that previous reports of CB-LI neurons in the dorsal tier of the human SN have mistakenly identified this region. The differentiation of midbrain dopaminergic neurons is not trivial or esoteric, as many groups report a differential loss of dopaminergic neurons within the SN in Parkinson's disease. The most severely affected regions are the ventral tier cell
90
D. A. McRitchie and G. M. Halliday
clusters, 4'H'18'21'57'59 although some dorsal tier involvement is noted. In comparison, the A10 dopaminergic cell clusters are relatively well preserved in Parkinson's disease. 5'24'2v'59It is therefore difficult to attribute a neuroprotective role to calbindin-D28k with the argument that it is present within the A9 SN neurons spared in P a r k i n s o n ' s disease. It is also unlikely that this protein alone accounts for the preservation o f other midbrain dopaminergic neurons, as only a p r o p o r t i o n o f A8 and A 10 neurons contain calbindinD28k. The selective vulnerability o f particular A9
neurons in patients with P a r k i n s o n ' s disease requires further experimentation. As it appears that a major factor differentiating the midbrain dopaminergic cell groups is their striatal innervation, a better understanding o f this pathway may provide the answers to the selective vulnerability o f particular A9 neurons. We thank Drs Pamphlett and Kril for the diagnostic evaluation of the cases and Mr Hardman for technical assistance. This work was funded by the National Medical and Health Research Council of Australia and the Australian Brain Foundation. Acknowledgements
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27. Hirsch E. C., Graybiel A. M. and Agid Y. A. (1988) Melanized dopaminergic neurons are differentially susceptible to degeneration in Parkinson's disease. Nature 334, 345-348. 28. Hof P. R., Cox K., Young W. G., Celio M. R., Rogers J. and Morrison J. H. (1991) Parvalbumin-immunoreactive neurons in the neocortex are resistant to degeneration in Alzheimer's disease. J. Neuropath. exp. Neurol. 511,451-462. 29. HofP. R. and Morrison J. H. (1991) Neocortical subpopulations labeled by a monoclonal antibody to calbindin exhibit differential vulnerability in Alzheimer's disease. Expl Neurol. I11, 293-301. 30. Hsu S. M., Raine L. and Fanger H. (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29, 577-580. 31. Iacopino A. M. and Christakos S. (1990) Specific reduction of calcium-binding protein (28-kitodalton calbindin-D) gene expression in aging and neurodegenerative diseases. Proc. natn. Acad. Sci. U.S.A. 87, 4078-4082. 32. Inagaki S., Sakanaka M., Shiosaka S., Senba E., Takatsuki K., Takagi H., Kawai Y., Minagawa H. and Tohyama M. (1982) Ontogeny of substance P-containing neuron system of the rat: immunohistochemical analysis I. Forebrain and upper brain stem. Neuroscience 7, 251-277. 33. Ince P., Stout N., Shaw P., Slade J., Hunziker W., Heizmann C. W. and Baimbridge K. G. (1993) Parvalbumin and calbindin D-28k in the human motor system and in motor neuron disease Neuropath. appl. Neurobiol. 19, 291-299. 34. Jellinger K. (1986) Overview of the morphological changes in Parkinson's disease. In Advances in Neurology (eds Yahr M. and Bergmann K. K. J.), Vol. 45, pp. 1-18. Raven Press, New York. 35. Kato S., Oda M. and Tanabe H. (1993) Diminution of dopaminergic neurons in the substantia nigra of sporadic amyotrophic lateral sclerosis. Neuropath. appl. Neurobiol. 19, 300-304. 36. Kitahama K., Denoroy L., Goldstein M., Jouvet M. and Pearson J. (1988) Immunohistochemistry of tyrosine hydroxylase and phenylethanolamine N-methyltransferase in the human brain stem: description of adrenergic perikarya and characterization of longitudinal catecholaminergic pathways. Neuroscience 25, 97 111. 37. Kiyama H., Seto-Ohshima A. and Emson P. C. (1990) Calbindin-D2s k as a marker for the degeneration of the striatonigral pathway in Huntington's disease. Brain Res. 525, 209 214. 38. Kohno J., Shiosaka S., Shinoda K., Inagaki S. and Tohyama M. 0984) Two distinct strio-nigral substance P pathways in the rat: an experimental immunohistochemical study. Brain Res. 3118, 309 317. 39. Ljungdahl /~., Hrkfelt T. and Nilsson G. (1978) Distribution of substance P-like immunoreactivity in the central nervous system. Neuroscience 3, 861-943. 40. Mai J. K., Stephens P. H., HopfA. and Cuello A. C. (1986) Substance P in the human brain. Neuroscience 17, 709 739. 41. Mattson M. P., Rychlik B., Chu C. and Christakos S. (1991) Evidence for calcium-reducing and excito-protective roles for the calcium-binding protein calbindin-D28 k in cultured hippocampal neurons. Neuron 6, 41-51. 42. Nitsch C., Scotti A., Sommacal A. and Kalt G. (1989) GABAergic hippocampal neurons resistant to ischemia-induced neuronal death contain the Ca2+-binding protein parvalbumin. Neurosci. Lett. 105, 263-268. 43. Nomura H., Shiosaka S. and Tohyama M. (1987) Distribution of substance P-like immunoreactive structures in the brainstem of the adult human brain: an immunohistochemical study. Brain Res. 404, 365-370. 44. Olszewski J. and Baxter D. (1954) Cytoarchitecture o f the Human Brain Stem. Karger, Basel. 45. Oyanagi K., Takeda S., Takahashi H., Ohama E. and Ikuta F. (1989) A quantitative investigation of the substantia nigra in Huntington's disease. Ann. Neurol. 26, 13 19. 46. Pasteels J. L., Pochet R., Surdat L., Hubeau C., Chirnoaga M., Parmentier M. and Lawson D. E. M. (1986) Ultrastructural localization of brain "'vitamin D-dependent" calcium binding proteins. Brain Res. 384, 294-303. 47. Pearson J., Halliday G., Sakamoto N. and Michel J.-P. (1990) Catecholaminergic neurons. In The Human Nervous System (ed. Paxinos G.), pp. 1023-1049. Academic Press, San Diego. 48. Persechini A., Moncrief N. D. and Kretsinger R. H. (1989) The EF-hand family of calcium modulating proteins. Trends Neurosci. 12, 462-467. 49. Pioro E. P., Mai J. K. and Cuello C. A. (1990) Distribution of substance P- and enkephalin-immunoreactive neurons and fibres. In The Human Nervous System (ed. Paxinos G.), pp. 1051 1094. Academic Press, San Diego. 50. Satoh J., Tabira T., Sano M., Nakayama H. and Tateishi J. (1991) Parvalbumin-immunoreactive neurons in the human central nervous system are decreased in Alzheimer's disease. Acta neuropath. 81, 388-395. 51. Scharfman H. E. and Schwartzkroin P. A. (1989) Protection of dentate hilar cells from prolonged stimulation by intracellular calcium chelation. Science 246, 257-260. 52. Shults C. W., Quirion R., Chronwall B., Chase T. N. and O'Donohue T. L. (1984) A comparison of the anatomical distribution of substance P and substance P receptors in the rat central nervous system. Peptides 5, 1097-I 128. 53. Sloviter R. S. (1989) Calcium-binding protein (calbindin-D28k) and parvalbumin immunocytochemistry: localization in the rat hippocampus with specific reference to the selective vulnerability of hippocampal neurons to seizure activity. J. comp. Neurol. 2811, 183 196. 54. Sloviter R. S., Sollas A, L., Barbaro N. M. and Laxer K. D. (1991) Calcium-binding protein (calbindin-D28k) and parvalbumin immunocytochemistry in the normal and epileptic human hippocampus. J. comp. Neurol. 308, 381-396. 55. Uchihara T., Kondo H., Kosaka K. and Tsukagoshi H. (1992) Selective loss of nigral neurons in Alzheimer's disease: a morphometric study. Acta neuropath. 83, 271 276. 56. Uchihara T., Tsuchiya K. and Kosaka K. (1990) Selective loss of nigral neurons in Pick's disease: a morphometric study. Acta Neuropath. 81, 155-161. 57. Waters C. M., Peck R., Rossor M., Reyolds G. P. and Hunt S. P. (1988) Immunocytochemical studies on the basal ganglia and substantia nigra in Parkinson's disease and Huntington's chorea. Neuroscience 25, 419-438. 58. Weiss J. H., Koh J.-Y., Baimbridge K. G. and Choi D. W. (1990) Cortical neurons containing somatostatin- or parvalbumin-like immunoreactivity are atypically vulnerable to excitotoxic injury in vitro. Neurology 40, 1288-1292. 59. Yamada T., McGeer P. L., Baimbridge K. G. and McGeer E. G. (1990) Relative sparing in Parkinson's disease of substantia nigra dopamine neurons containing calbindin-D2s k. Brain Res. 526, 303 307. (Accepted 14 September 1994)
~
Pergamon
Elsevier ScienceLtd Copyright © 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00
0306-4522(94)00483-8
CALBINDIN D28k-CONTAINING N E U R O N S ARE RESTRICTED TO THE MEDIAL SUBSTANTIA NIGRA IN H U M A N S D. A. M c R I T C H I E * and G. M. H A L L I D A Y Prince of Wales Medical Research Institute, Prince of Wales Hospital, Randwick, NSW 2031, Australia Abstract--A controversy exists in the literature as to whether neurons containing the calcium binding protein calbindin-D28 k are located within the human substantia nigra. The point of variance between reports, however, is not the anatomical distribution of these neurons, but rather the delineation of the dorsal border of the substantia nigra. It has been suggested that the dense substance P striatonigral innervation delimits the substantia nigra in the human. The aim of the present study is to re-examine the distribution of calbindin-D28k-positive neurons throughout the substantia nigra using substance P to delimit its borders. Although a few calbindin-D28k-positive neurons were found in the medial cell group of the substantia nigra, the vast majority of positive neurons were located in the adjacent A8 and AI0 dopaminergic cell groups. This anatomical location of calbindin-D28k-positive neurons is consistent with previous reports, though our results indicate that when the striatonigral projection is used to define the substantia nigra, calbindin-D28 k is not a notable feature of these neurons. This questions the neuroprotective role of this protein in Parkinson's disease.
Calbindin-D28k is a m e m b e r o f the E F - h a n d family o f calcium binding proteins. 48 The precise functional role o f these proteins is not yet known, t h o u g h they are believed to have some calcium "buffering" properties. 3'46 As such, they may be i m p o r t a n t in buffering high levels o f intracellular calcium, which would otherwise lead to excitotoxic cell damage. While m a n y studies s u p p o r t this putative neuroprotective role for the calcium binding proteins, 1926'28'4a'42'51'53'54'59
The present study aims to reassess the distribution o f calbindin-D28k-containing neurons in the midbrain using the striatonigrat projection to demarcate the SN.
EXPERIMENTAP LROCEDURES Case selection
Brains were collected at autopsy from eight patients (see Table 1 for patient details) with no clinical or pathological evidence of nervous system abnormality. Written consent for autopsy was obtained and the study was approved by the Human Ethics Committee of The University of Sydney.
the concept is not unchallenged and several groups suggest that neuronal populations containing these proteins are differentially vulnerable to neurodegeneration. 2°'29,31'37'58 In some instances the presence o f these proteins seem to predispose neurons to damage. 1,50
Tissue collection
The brains were immersion fixed in 15% buffered formalin for two weeks. The brainstem was removed from the cerebrum at the level of the mamillary bodies (to ensure that the entire rostrocaudal extent of the SN could be sampled) and detached from the cerebellum. The cerebrum and brainstem were embedded separately in 3% agarose before cutting on a rotary slicer. The cerebrum was cut into 3 mm-coronal slices and the brainstem was cut into 3 mm-blocks in the transverse plane, allowing detailed macroscopic ex~lmination of cases. Standard blocks of the premotor, motor, inferior temporal and parahippocampal cortices, the hippocampus and the cerebellar vermis were paraffin-embedded, sectioned at 7/~m and the sections stained with either haematoxylin and eosin or silver to discount pathology. The midbrain blocks were cryoprotected in 30% sucrose in 0.1 M Tris-HC1 buffer, pH 7.4 for two to four days and serially sectioned on a Bright cryostat at 50/lm intervals. Parallel 1 in 10 series of sections were stained with buffered Cresyl Violet (0.5% in acetate buffer, pH 5.3), haematoxylin and eosin, luxol fast blue, the Garvey silver stain ~3 and processed for immunohistochemistry with substance P and
Past reports o f the distribution o f calbindinD28k-containing neurons in the ventral midbrain suggest that this protein in particular may offer some protection in the pathogenesis o f P a r k i n s o n ' s disease. 16'59 Calbindin-D28k-containing neurons have been reported to be present in the dorsal tier o f the substantia nigra (SN) 19'59 in humans, which is the region o f the SN selectively spared in P a r k i n s o n ' s disease. 4'I1'18"19'2°'21'57'59 However, this pattern o f distribution has been disputed and several studies report that calbindin-D2sk-containing neurons are not f o u n d within the SN at all. 12'2°'33 Recent studies suggest that the SN is delimited by its dense substance P innervation 6A°'15'23'32'39'4°'43'49'52'57 from striatal afferents. 2'9'38 *To whom correspondence should be addressed. Abbreviations: CB-LI, calbindin-D28k-like immunoreactive;
SN, substantia nigra. 87
88
D . A . McRitchie and G. M. Halliday
Fig. 1. Camera lucida drawings and photomicrographs of the distribution of CB-LI neurons in the h u m a n midbrain. (A) Photomicrograph of substance P-like immunoreactive structures in the ventral midbrain around the level of the exiting third nerve (3n). The dense afferent input to the SN was used to delimit this nucleus, as it labelled both pars compacta and the reticulata (SNr). The substance P-like immunoreactivity often diminished around the pigmented cell clusters of the compacta; dorsal (SNd) and ventral (SNv) tiers, medial cell group (SNm) and the pars lateralis (SNI). In contrast the adjacent A I0 parabrachial pigmented nucleus (PBP) and A8 retrorubral fields ( R R F ) display very sparse immunoreactivity for this tachykinin. The red nucleus (R) and the cerebral peduncle (cp) are also indicated. Scale bar = 1 ram. (B) Photomicrograph of the adjacent section stained for Cresyl Violet. The dense pigmented cell clusters are seen in contrast to the relatively cell sparse A10 PBP. Additional structures indicated and scale are as for A. (C) Photomicrograph of CB-LI immunoreactive structures in the ventral midbrain. Dense fibre labelling is seen in the dorsomedial region of the SN, though diminishes around the pigmented cell clusters. The CB-LI neurons in the PBP are seen to arch around the circumference of the R. The CB-LI fibre labelling in the A8 R R F and the A I 0 PBP is m u c h more sparse than that in the SN. Additional structures labelled and scale are as for A. (D) Line drawing of the ventral midbrain at the level of the exiting oculomotor nerve. The section outline is provided in the inset with the box indicating the region enlarged in A~2. Each asterix represents one CB-LI neuron from a single 50 ~tm-section. A few CB-LI neurons were located in SNm, though never present in the SNd or SNv, SN1 or SNr. In contrast they were present in considerable numbers in the A10 PBP, paranigral (PN) and rostral linear (RLi) nuclei, and in the A8 R R F . Additional structures labelled are as for A. Scale bar - 2 m m . (E) Photomicrograph depicting the border between the PBP and SNd. The diagonally aligned CB-LI neurons and fibres in the PBP are clearly separate from the SNd with its non-aligned, unlabelled pigmented neurons. The paucity of fibre labelling around the pigmented cell clusters in the SNd also helps distinguish the border between the SNd and the PBP. The exiting fibres of the oculomotor nerve pierce both of these nuclei. Scale bar = 500 # m .
Calbindin D28k in the human nigra Table 1. Patient details Postmortem Case Age Sex delay 1 2 3 4 5 6 7 8
71 46 58 61 37 85 88 47
F F M F M M F M
18 24 26 19 28.5 21 32 23
Cause of Death Pulmonary Embolism Ischemic Heart Disease Ischemic Heart Disease Metastatic Carcinoma Aspiration Pneumonia Metastatic Carcinoma Cardiorespiratory Arrest Pulmonary Embolism
calbindin-D28 k antibodies. Remaining sections were stored for later use.
Irnrnunohistochernistry Sections chosen for immunohistochemistry were first pretreated in 0.01% hydrogen peroxide in 50% alcohol to abolish endogenous peroxidase activity and incubated in 1% normal horse serum prior to incubation in either substance P (rat monoclonal, 1:1000, Seralab) or calbindinD28k (mouse monoclonal, I : 2000, Sigma) primary antibody. The antibody was localized using the avidin-biotinperoxidase technique 3° with biotinylated anti-rat or antimouse IgG diluted 1:200 and the ABC reagent diluted I:100. The peroxidase reaction was visualized using diaminobenzidine tetrachloride (0.05%, Sigma) as the chromogen. RESULTS
Calbindin-D28k-like immunoreactive (CB-LI) structures were clearly visualized in the ventral midbrain. Neurons were considered to be positive only if the immunoreaction clearly delineated the cell soma and proximal processes (see inset in Fig. IC). Filamentous staining was interpreted to be fibres and punctate structures were interpreted to be terminals. There was little variation in the staining pattern and intensity of CB-LI structures throughout the midbrain indicating that this protein is preserved over post mortem delays of up to 36 h and a wide range of ages. Substance P-like immunoreactive fibres were used to demarcate the SN (Fig. 1A). The pigmented neurons in the SN were organized into discrete clusters (Fig. 1B) which were labelled based on position; medial cell cluster, lateral cell cluster (SN pars lateralis), and dorsal and ventral tiers of the SN pars compacta (Fig. 1B). The most striking feature of the staining within the SN was the extensive CB-LI fibre plexus. In the medial part of the pars compacta the CB-LI fibre labelling was more dense than it was laterally with light fibre labelling seen in the pars reticulata (Fig. 1C). Only a small population of CB-LI neurons were found in the SN restricted to the caudal part of the medial cell cluster (Fig. 1D). As illustrated, less than 10 CB-LI neurons per 50/zm section were found over the caudal one third of the SN on serial section analyses of eight cases. This distribution was quite consistent, with CB-LI neurons localised near the dorsal border of the medial cell cluster. N o CB-LI neurons were found in the dorsal or ventral tiers, pars lateralis or pars reticulata of the SN at any level of the midbrain in any case (Fig. 1D),
89
though CB-LI fibre labelling was diminished around these pigmented cell clusters (Fig. 1C,E). In contrast to the SN, abundant CB-LI neurons were observed in the A10 and A8 dopaminergic cell groups (Fig. 1D). The CB-LI neurons in the A8 group in the midbrain reticular formation had randomly arranged dendrites, while those situated more medially in the A10 region had aligned dendrites. For example, the dendrites of CB-LI neurons in the caudal and rostral linear nuclei were aligned mediolaterally, while those in the parabrachial pigmented nucleus were aligned parallel to the circumference of the red nucleus, dorsal to the dorsal tier of the SN pars compacta. A clear distinction could be made between these aligned CB-LI neurons in the parabrachial pigmented nucleus and the unlabelled pigmented neurons in the adjacent dorsal tier of the SN (Fig. 1C,E). Again in contrast to the SN, sparse CB-LI fibre labelling was present in these A8 and A I 0 cell regions. DISCUSSION
The distribution of CB-LI neurons throughout the ventral midbrain in the rat has been elegantly documented and corroborated. 8,16'~7 In the human, however, there is little consensus on the distribution of CB-LI neurons in this region of the brain. 12'w'2°'33'59 Given that the substantia nigra is the site of the hallmark pathology for Parkinson's disease 22'34and is affected in a variety of other pathologies, 35'45"55'56an accurate account of the chemistry of the SN seems essential. The reason for discrepancies in reports of CB-LI throughout the human ventral midbrain appears to be purely anatomical. O f the studies which report CB-LI neurons in the human S N , 19'59 the majority utilize the brainstem atlas of Olszewski and Baxter 44 which has rostrocaudal inconsistencies in the parcellation of the SN (caudally located parabrachial pigmented nucleus and rostrally located SN pars 7 in the same position). By utilizing the striatal innervation of the A9 dopaminergic neurons, a clear demarcation of these functional groups can be achieved. Thus the parabrachial pigmented nucleus is found ventral to the red nucleus over the entire midbrain as has been previously published. 7'14'18'2°'25'36'47 When reconstructed in three dimensions, the parabrachial pigmented nucleus is continuous with more medial A10 neuronsfl 5 The separation of this region from the SN has been recently proposed by Gibb 2° (though he retains the pars y terminology) using the same criteria. Thus it would seem that previous reports of CB-LI neurons in the dorsal tier of the human SN have mistakenly identified this region. The differentiation of midbrain dopaminergic neurons is not trivial or esoteric, as many groups report a differential loss of dopaminergic neurons within the SN in Parkinson's disease. The most severely affected regions are the ventral tier cell
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D. A. McRitchie and G. M. Halliday
clusters, 4'H'18'21'57'59 although some dorsal tier involvement is noted. In comparison, the A10 dopaminergic cell clusters are relatively well preserved in Parkinson's disease. 5'24'2v'59It is therefore difficult to attribute a neuroprotective role to calbindin-D28k with the argument that it is present within the A9 SN neurons spared in P a r k i n s o n ' s disease. It is also unlikely that this protein alone accounts for the preservation o f other midbrain dopaminergic neurons, as only a p r o p o r t i o n o f A8 and A 10 neurons contain calbindinD28k. The selective vulnerability o f particular A9
neurons in patients with P a r k i n s o n ' s disease requires further experimentation. As it appears that a major factor differentiating the midbrain dopaminergic cell groups is their striatal innervation, a better understanding o f this pathway may provide the answers to the selective vulnerability o f particular A9 neurons. We thank Drs Pamphlett and Kril for the diagnostic evaluation of the cases and Mr Hardman for technical assistance. This work was funded by the National Medical and Health Research Council of Australia and the Australian Brain Foundation. Acknowledgements
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