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Calcium-binding protein immunoreactivity delineates the intralaminar nuclei of the thalamus in the human brain
Pergamon
PII:
Neuroscience Vol. 90, No. 2, pp. 485–491, 1999 Copyright 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/99 $19.00+0.00 S0306-4522(98)00444-8
CALCIUM-BINDING PROTEIN IMMUNOREACTIVITY DELINEATES THE INTRALAMINAR NUCLEI OF THE THALAMUS IN THE HUMAN BRAIN M. C. MU } NKLE, H. J. WALDVOGEL and R. L. M. FAULL* Department of Anatomy with Radiology, Faculty of Medicine and Health Science, University of Auckland, Private Bag 92019, Auckland, New Zealand Abstract––Immunohistochemical studies have shown that the three calcium-binding proteins (calbindinD28k, calretinin and parvalbumin) are heterogeneously distributed in the mammalian brain and are useful for delineating nuclear boundaries.2,8,9,16 We have investigated the distribution of the three calciumbinding proteins in the human thalamus in order to assist in the delineation of the equivocal nuclear boundaries of the intralaminar nuclei of the thalamus. The results show that each of the ‘‘functional’’ nuclear complexes in the human thalamus demonstrates a characteristic pattern of calcium-binding protein immunoreactivity. In particular, the intralaminar nuclei are characterized by a unique combination of calcium-binding protein staining which clearly delineates the component nuclei in this complex from the other nuclei of the human thalamus. The anterior group of intralaminar nuclei (central lateral nucleus, paracentral nucleus and central medial nucleus) showed intense staining for both calbindin-D28k and calretinin. By contrast, the posterior group of intralaminar nuclei (centre me´dian nucleus and parafascicular nucleus) showed a complementary pattern of staining; the centre me´dian nucleus showed immunoreactivity only for one calcium-binding protein, parvalbumin, while the parafascicular nucleus showed immunoreactivity for both calbindin-D28k and calretinin. No other nucleus in the human thalamus showed these particular combinations of calcium-binding protein staining. Since the intralaminar nuclei also have unique topographically organized connectional affiliations with both the cerebral cortex and the basal ganglia, these results suggest that the calcium-binding proteins may play an important role in the influence of the intralaminar nuclei on interactions between the cerebral cortex and the basal ganglia. 1999 IBRO. Published by Elsevier Science Ltd. Key words: calbindin-D28k, calretinin, parvalbumin, intralaminar, thalamus, human.
The calcium-binding proteins calbindin-D28k, calretinin and parvalbumin all belong to the EF-hand family of calcium-binding proteins.1,3 Because of their calcium-buffering properties7 they are thought to play an important role in maintaining the calcium homeostasis in cells and may provide a neuroprotective function against neurotoxic insults.4,14,22 Immunohistochemical studies in the mammalian brain2,5,6,8,9,11,16–20 have shown that all three calcium-binding proteins are distributed heterogeneously which has often helped to delineate nuclear boundaries not obvious using other criteria.11 For example, a recent detailed study on the distribution of calretinin in the human thalamus8 has shown that calretinin immunoreactivity is a useful marker for delineating some of the thalamic nuclei. In the present study, we have therefore investigated the immunohistochemical distribution of all three calcium-binding proteins—calbindin-D28k, calretinin and parvalbumin—in the human thalamus in order to investigate their usefulness in identifying thalamic nuclear subdivisions. Because the intralaminar nuclei have been especially difficult to pre*To whom correspondence should be addressed. 485
cisely delineate in the mammalian thalamus we have particularly concentrated on the identification of the component nuclei of the intralaminar complex. EXPERIMENTAL PROCEDURES
The human brain tissue was obtained from the New Zealand Neurological Foundation Human Brain Bank and the study was approved by the University of Auckland Human Subjects Ethics Committee. Tissue was obtained from eight post mortem brains of cases with no known history of neurological disease (see Table 1); the average age of the eight cases was 58.5 years and the average post mortem delay was 13 h. The brains were perfusion fixed via the internal carotid and basilar arteries using 0.1 M phosphate-buffered saline with 1% sodium nitrite for approximately 10 min followed by 5 l of fixative (15% formalin in 0.1 M phosphate buffer pH 7.4 over 1 h). The brains were sectioned and blocks of the thalamus were taken and immersion fixed for a further 24 h in the same fixative. The blocks of tissue were immersed for up to three days in 20% and then 30% sucrose in 0.1 M phosphate buffer at pH 7.4 with 0.1% sodium azide. Coronal sections were cut on a freezing microtome at a thickness of 70 µm and serial sections were taken from the anterior, middle and posterior thirds of the thalamus. The sections were stained free floating using a rabbit anti-calbindin-D28k antibody (Emson, Cambridge, U.K.)21 at a dilution of 1:5000– 1:10,000, a rabbit anti-calretinin antibody (SWANT) at a dilution of 1:5000, and a mouse anti-parvalbumin antibody
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Fig. 1A–F.
Calcium-binding proteins in the human thalamus (Sigma) at a dilution of 1:10,000 using standard immunohistochemical techniques with biotinylated secondary antibodies and a streptavidin–horseradish peroxidase
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conjugate.23 The rabbit anti-calbindin-D28k antibody was raised in chick and its specificity was tested by incubating sections either with serum or with the primary antibody that had been pre-incubated with the antigen.21 The distribution of the label was visualized using a 3,3 -diaminobenzidene reaction. RESULTS
All the nuclei in the human thalamus showed immunoreactivity for one or more of the calciumbinding proteins in various combinations (Fig. 1). The anterior group of nuclei (nomenclature of Hirai and Jones13,15) (Fig. 1A–C) and the lateral dorsal nucleus (Fig. 1D–F) showed immunoreactivity for all three calcium-binding proteins except that the anterior dorsal nucleus showed no parvalbumin immunoreactivity. In contrast, the ventral nuclei (ventral anterior, ventral lateral, ventral medial and ventral posterior nuclei) (Fig. 1), the pulvinar nuclei, the lateral posterior nucleus (Fig. 1G–I) and the mediodorsal nucleus (Fig. 1A–F) showed immunoreactivity mainly for calbindin-D28k and parvalbumin, although the mediodorsal nucleus additionally showed patches of calretinin-immunoreactive cells and fibres (Fig. 1E). The medial and lateral geniculate nuclei showed moderately dense parvalbumin staining (Fig. 1I), light neuropil staining for calretinin (Fig. 1H) and weak to moderate calbindin-D28k staining in the cell bodies (Fig. 1G). The reticular nucleus was the only thalamic nucleus which showed immunoreactivity for both calretinin and parvalbumin, but it was immunonegative for calbindin-D28k (Fig. 1). The intralaminar nuclei showed a very distinctive pattern of calcium-binding immunoreactivity which served to precisely delineate this often poorly
Fig. 1G–I.
Fig. 1. The distribution of calcium-binding immunoreactivity in the human thalamus. Serial sections of the human thalamus at the level of the anterior third (A–C), the middle third (D–F) and the posterior third (G–I) of the thalamus stained for calbindin-D28k (A, D, G), calretinin (B, E, H) and parvalbumin (C, F, I). The intralaminar nuclei of the human thalamus are distinguished by their pattern of immunostaining for the three calcium-binding proteins calbindin-D28k, calretinin and parvalbumin. The central lateral, central medial and paracentral intralaminar nuclei stained positively for calbindin-D28k (A, D, G) and for calretinin (B, E, H), but were immunonegative for parvalbumin (C, F, I). By contrast, the centre me´dian nucleus was immunopositive for parvalbumin (I) but immunonegative for calbindin-D28k (G) and calretinin (H). Scale bars=1 cm. CeM, central medial nucleus; CL, central lateral nucleus; CM, centre me´dian nucleus; CN, caudate nucleus; GPe, globus pallidus external part; GPi, globus pallidus internal part; IC, internal capsule; LD, lateral dorsal nucleus; LGN, dorsal lateral geniculate nucleus; LP, lateral posterior nucleus; MD, mediodorsal nucleus; OT, optic tract; P, putamen; Pg, pregeniculate nucleus; R, reticular nucleus; Sg-L, suprageniculate-limitans nucleus; SN, substantia nigra; STN, subthalamic nucleus; VLa, ventral lateral anterior nucleus; VLp, ventral lateral posterior nucleus; VPL, ventral posterior lateral nucleus; ZI, zona incerta.
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M. C. Mu¨nkle et al. Table 1. Sources of human brain tissue Case no.
Sex
Age (years)
Post mortem delay (h)
Cause of death
3176 H103 3392 3212 H104 3446 4164 4168
M M M M M M M F
47 70 25 47 69 44 82 84
13.25 5 15 13.25 14 8 21 14.5
ischaemic heart disease myocardial infarct asphyxia ischaemic heart disease ischaemic heart disease myocardial infarct ischaemic heart disease ruptured myocardial infarct
demarcated group of nuclei from the remaining nuclear complexes of the human thalamus. The anterior group of intralaminar nuclei (central lateral nucleus, paracentral nucleus, central medial nucleus) showed a characteristic intense staining for both calbindin-D28k and calretinin (Fig. 1A, B, D, E). All of the component cell clusters comprising the central lateral nucleus were selectively highlighted by intense staining with calretinin (Fig. 1E, H) and calbindin-D28k (Fig. 1D, G). In the anterior two thirds of the thalamus the densely stained immunoreactive cell bodies for calretinin (Fig. 2B) and calbindin-D28k (Fig. 2A) in the central lateral nucleus (Fig. 1D, E) formed a well demarcated, thin, peri-nuclear shell immediately lateral to the spherical mediodorsal nucleus (Fig. 1D, E). At posterior thalamic levels the central lateral nucleus comprises a series of isolated cell clusters which are dispersed within the medial region of the thalamus and these groups of cells are conspicuously delineated with calbindin-D28k (Fig. 1G) and calretinin (Fig. 1H) immunoreactivity. The central lateral nucleus was immunonegative for parvalbumin except for a few scattered fibre terminals (Fig. 1F, I). The paracentral nucleus was clearly delineated in sections stained for both calbindin-D28k and calretinin (Fig. 1A, B); it showed very intense immunostaining for calretinin of both the cell bodies and fibres (Fig. 2C) and the traversing immunonegative fibre bundles tended to disperse the clusters of immunopositive cells thus giving the nucleus a distinctive reticulated appearance (Fig. 2C). The central medial nucleus showed intense staining for both calbindin-D28k (Fig. 1A, D) and calretinin (Fig. 1B, E); in particular it showed numerous cell bodies intensely immunoreactive for calretinin which lay in a very dense and strongly stained neuropil (Fig. 2D) containing fewer calbindin-D28k-positive neurons. No staining for parvalbumin was observed in the central medial nucleus (Fig. 1C, F). The posterior group of intralaminar nuclei (centre me´dian nucleus, parafascicular nucleus) showed a complementary pattern of staining for the three calcium-binding proteins. The centre me´dian nucleus (Fig. 1G–I) was distinctively different from all the other nuclear complexes in the human thalamus by
displaying immunoreactivity for only one calciumbinding protein, parvalbumin (Fig. 1I). The dorsomedial region of the centre me´dian nucleus showed moderately stained elongated cell bodies (Fig. 2E), while the ventrolateral portion of the nucleus showed moderately stained polygonal multipolar cell bodies set against a moderately stained neuropil (Fig. 2F). In contrast, the parafascicular nucleus showed moderate immunoreactivity for both calbindin-D28k and calretinin. Most immunoreactive cells in the parafascicular nucleus were medium-sized and oval to round in shape; however, a few large, round calbindin-D28k-positive neurons were also observed.
DISCUSSION
The results of this study show that the nuclei of the human thalamus demonstrate a distinctive heterogeneous pattern of calcium-binding protein immunoreactivity and that the three calcium-binding proteins provide very useful markers for identifying and delineating the various thalamic nuclei. In general terms, each of the ‘‘functional’’ nuclear complexes of the human thalamus demonstrated a characteristic pattern of calcium-binding protein immunoreactivity, suggesting that the various combinations of calcium-binding proteins may contribute to the functional characteristics associated with each of the thalamic nuclear groups. The detailed staining pattern of the thalamic nuclei demonstrated here agrees in general terms with previous findings in the monkey9,16 and human thalamus.8,18 However, our results on the distribution of calbindin-D28k in the human thalamus differ from those of Morel et al.,18 which may be due to differences in the method of tissue fixation. Of special interest and importance in the present study is the demonstration that the intralaminar nuclei of the human thalamus show a unique and characteristic pattern of staining for the three calcium-binding proteins. The anterior intralaminar nuclei and the parafascicular nucleus showed immunoreactivity for both calbindin-D28k and calretinin whereas the centre me´dian nucleus showed immunoreactivity only for parvalbumin. No other nucleus in
Calcium-binding proteins in the human thalamus
Fig. 2. Photomicrographs of the intralaminar nuclei of the human thalamus stained for the three calcium-binding proteins calbindin-D28k (A), calretinin (B–D), and parvalbumin (E, F). (A, B) Photomicrographs of corresponding regions of the central lateral nucleus stained for calbindin-D28k (A) and calretinin (B) showing patches of intensely stained cell bodies. (C) The paracentral nucleus shows intense immunoreactivity for calretinin in both the cell bodies and fibres; immunonegative fibre bundles traversing the paracentral nucleus give it a very distinctive reticulated appearance in calretinin-immunoreactive sections. (D) The central medial nucleus shows very intense staining for calretinin in both the cell bodies and the neuropil. (E, F) Parvalbumin-positive cell bodies in the dorsal (E) and ventral (F) parts of the centre me´dian nucleus. The cell bodies in the dorsal part (E) are more dispersed and show a different cytoarchitecture to those in the ventral part (F). Scale bars=50 µm.
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the human thalamus showed these particular combinations of calcium-binding protein staining. Furthermore, in addition to their distinctive calcium-binding protein staining characteristics, the intralaminar nuclei are the only thalamic nuclear groups which have major projections to both the cerebral cortex and to the basal ganglia.12,15 In a recent review of the overall pattern of connections of the intralaminar nuclei, Groenewegen and Berendse10 stated that ‘‘the rostral intralaminar nuclei project to prefrontal association cortical areas and the posterior part of the parietal cortex, whereas the caudal intralaminar nuclei project to motor and premotor cortical areas in the frontal lobe and to the anterior part of the parietal cortex’’;10 in particular, they described10 that the organization of the intralaminar thalamocortical projection seemed to be such that individual intralaminar nuclei project to cortical areas that are in turn interconnected by corticocortical connections and that have convergent projections to the striatum of the basal ganglia. They also emphasized that the direct thalamostriate projections of the intralaminar nuclei on to the striatum (caudate–putamen) of the basal ganglia are organized in a very strict topographical manner such that the lateral-to-medial co-ordinate in the intralaminar nuclei projects onto the dorsolateral-to-ventromedial co-ordinate of the striatum.10 Finally, concerning the
overall pattern of connections of the intralaminar nuclei with the cerebral cortex and the basal ganglia, it is especially interesting that the targets of the thalamocortical and thalamostriatal projections of a given intralaminar nucleus are interconnected through the corticostriatal projection.10 CONCLUSION
This corresponding topographical pattern of the interconnections between the intralaminar nuclei, the cerebral cortex and the basal ganglia shows that the intralaminar nuclei are therefore well placed to selectively modulate the activity of the various functionally segregated basal ganglia-thalamocortical circuits. Thus, the distinctive connectional affiliations of these nuclei and their characteristic calcium-binding protein properties suggest that the calcium-binding proteins may play a critical role in the influence of these nuclei on their interactions with the cerebral cortex and the basal ganglia. Acknowledgements—This study was supported by grants from the Health Research Council of New Zealand, the New Zealand Neurological Foundation and the New Zealand Lottery Board. M.C.M. was supported by a scholarship from the German Academic Exchange Service (Doktorandenstipendium HSPIII).
REFERENCES
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Andressen C., Blu¨mcke I. and Celio M. R. (1993) Calcium-binding proteins: selective markers of nerve cells. Cell Tiss. Res. 271, 181–208. Arai R., Jacobowitz D. M. and Deura S. (1994) Distribution of calretinin, calbindin-D28k, and parvalbumin in the rat thalamus. Brain Res. Bull. 33, 595–614. Baimbridge K. G., Celio M. R. and Rogers J. H. (1992) Calcium-binding proteins in the nervous system. Trends Neurosci. 15, 303–308. Batini C., Guegan M., Palestini M., Thomasset M. and Vigot R. (1997) Upregulation of calbindin-D-28k immunoreactivity by excitatory amino acids. Archs ital. Biol. 135, 385–397. Celio M. R. (1990) Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience 35, 375–475. De Biasi S., Arcelli P. and Spreafico R. (1994) Parvalbumin immunoreactivity on the thalamus of guinea pig: light and electron microscopic correlation with gamma-aminobutyric acid immunoreactivity. J. comp. Neurol. 348, 556–569. Fierro L. and Llano I. (1996) High endogenous calcium buffering in Purkinje cells from rat cerebellar slices. J. Physiol. 496, 617–625. Fortin M., Asselin M.-C., Gould P. V. and Parent A. (1998) Calretinin-immunoreactive neurons in the human thalamus. Neuroscience 84, 537–548. Fortin M., Asselin M.-C. and Parent A. (1996) Calretinin immunoreactivity in the thalamus of the squirrel monkey. J. chem. Neuroanat. 10, 101–117. Groenewegen H. J. and Berendse H. W. (1994) The specificity of the ‘‘nonspecific’’ midline and intralaminar thalamic nuclei. Trends Neurosci. 17, 52–57. Gutierrez C., Yaun A. and Cusick C. G. (1995) Neurochemical subdivisions of the inferior pulvinar in macaque monkeys. J. comp. Neurol. 363, 545–562. Herkenham M. (1980) Laminar organisation of thalamic projections to the rat neocortex. Science 207, 532–535. Hirai T. and Jones E. G. (1989) A new parcellation of the human thalamus on the basis of histochemical staining. Brain Res. Rev. 14, 1–34. Isaacs K. R., Wolpoe M. E. and Jacobowitz D. M. (1997) Calretinin-immunoreactive dopaminergic neurons from embryonic rat mesencephalon are resistant to levodopa-induced neurotoxicity. Expl Neurol. 146, 25–32. Jones E. G. (1985) The Thalamus. Plenum, New York. Jones E. G. and Hendry S. H. C. (1989) Differential calcium binding protein immunoreactivity distinguishes classes of relay neurons in monkey thalamic nuclei. Eur. J. Neurosci. 1, 222–246. Molinari M., Leggio M. G., Dell’Anna M. E., Giannetti S. and Macchi G. (1994) Chemical compartmentation and relationships between calcium-binding protein immunoreactivity and layer-specific cortical and caudate-projecting cells in the anterior intralaminar nuclei of the cat. Eur. J. Neurosci. 6, 299–312. Morel A., Magnin M. and Jeanmonod D. (1997) Multiarchitectonic and stereotactic atlas of the human thalamus. J. comp. Neurol. 387, 588–630.
Calcium-binding proteins in the human thalamus 19. 20. 21. 22. 23.
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Rausell E. and Jones E. G. (1991) Histochemical and immunocytochemical compartments of the thalamic VPM nucleus in monkeys and their relationship to the representational map. J. Neurosci. 11, 210–225. Rausell E., Bae C. S., Vin˜uela A., Huntley G. W. and Jones E. G. (1992) Calbindin and parvalbumin cell in monkey VPL thalamic nucleus: distribution, laminar cortical projections, and relations to spinothalamic terminations. J. Neurosci. 12, 4088–4111. Seto-Ohshima A., Emson P. C., Lawson E., Mountjoy C. Q. and Carrasco L. H. (1988) Loss of matrix calcium-binding protein-containing neurons in Huntington’s disease. Lancet 1, 1252–1255. Sloviter R. (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. 280, 183–196. Waldvogel H. J., Kubota Y., Trevallyan S. C., Kawaguchi Y., Fritschy J.-M., Mohler H. and Faull R. L. M. (1997) The morphological and chemical characteristics of striatal neurons immunoreactive for the á1-subunit of the GABAA receptor in the rat. Neuroscience 80, 775–792. (Accepted 15 July 1998)
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Neuroscience Vol. 90, No. 2, pp. 485–491, 1999 Copyright 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/99 $19.00+0.00 S0306-4522(98)00444-8
CALCIUM-BINDING PROTEIN IMMUNOREACTIVITY DELINEATES THE INTRALAMINAR NUCLEI OF THE THALAMUS IN THE HUMAN BRAIN M. C. MU } NKLE, H. J. WALDVOGEL and R. L. M. FAULL* Department of Anatomy with Radiology, Faculty of Medicine and Health Science, University of Auckland, Private Bag 92019, Auckland, New Zealand Abstract––Immunohistochemical studies have shown that the three calcium-binding proteins (calbindinD28k, calretinin and parvalbumin) are heterogeneously distributed in the mammalian brain and are useful for delineating nuclear boundaries.2,8,9,16 We have investigated the distribution of the three calciumbinding proteins in the human thalamus in order to assist in the delineation of the equivocal nuclear boundaries of the intralaminar nuclei of the thalamus. The results show that each of the ‘‘functional’’ nuclear complexes in the human thalamus demonstrates a characteristic pattern of calcium-binding protein immunoreactivity. In particular, the intralaminar nuclei are characterized by a unique combination of calcium-binding protein staining which clearly delineates the component nuclei in this complex from the other nuclei of the human thalamus. The anterior group of intralaminar nuclei (central lateral nucleus, paracentral nucleus and central medial nucleus) showed intense staining for both calbindin-D28k and calretinin. By contrast, the posterior group of intralaminar nuclei (centre me´dian nucleus and parafascicular nucleus) showed a complementary pattern of staining; the centre me´dian nucleus showed immunoreactivity only for one calcium-binding protein, parvalbumin, while the parafascicular nucleus showed immunoreactivity for both calbindin-D28k and calretinin. No other nucleus in the human thalamus showed these particular combinations of calcium-binding protein staining. Since the intralaminar nuclei also have unique topographically organized connectional affiliations with both the cerebral cortex and the basal ganglia, these results suggest that the calcium-binding proteins may play an important role in the influence of the intralaminar nuclei on interactions between the cerebral cortex and the basal ganglia. 1999 IBRO. Published by Elsevier Science Ltd. Key words: calbindin-D28k, calretinin, parvalbumin, intralaminar, thalamus, human.
The calcium-binding proteins calbindin-D28k, calretinin and parvalbumin all belong to the EF-hand family of calcium-binding proteins.1,3 Because of their calcium-buffering properties7 they are thought to play an important role in maintaining the calcium homeostasis in cells and may provide a neuroprotective function against neurotoxic insults.4,14,22 Immunohistochemical studies in the mammalian brain2,5,6,8,9,11,16–20 have shown that all three calcium-binding proteins are distributed heterogeneously which has often helped to delineate nuclear boundaries not obvious using other criteria.11 For example, a recent detailed study on the distribution of calretinin in the human thalamus8 has shown that calretinin immunoreactivity is a useful marker for delineating some of the thalamic nuclei. In the present study, we have therefore investigated the immunohistochemical distribution of all three calcium-binding proteins—calbindin-D28k, calretinin and parvalbumin—in the human thalamus in order to investigate their usefulness in identifying thalamic nuclear subdivisions. Because the intralaminar nuclei have been especially difficult to pre*To whom correspondence should be addressed. 485
cisely delineate in the mammalian thalamus we have particularly concentrated on the identification of the component nuclei of the intralaminar complex. EXPERIMENTAL PROCEDURES
The human brain tissue was obtained from the New Zealand Neurological Foundation Human Brain Bank and the study was approved by the University of Auckland Human Subjects Ethics Committee. Tissue was obtained from eight post mortem brains of cases with no known history of neurological disease (see Table 1); the average age of the eight cases was 58.5 years and the average post mortem delay was 13 h. The brains were perfusion fixed via the internal carotid and basilar arteries using 0.1 M phosphate-buffered saline with 1% sodium nitrite for approximately 10 min followed by 5 l of fixative (15% formalin in 0.1 M phosphate buffer pH 7.4 over 1 h). The brains were sectioned and blocks of the thalamus were taken and immersion fixed for a further 24 h in the same fixative. The blocks of tissue were immersed for up to three days in 20% and then 30% sucrose in 0.1 M phosphate buffer at pH 7.4 with 0.1% sodium azide. Coronal sections were cut on a freezing microtome at a thickness of 70 µm and serial sections were taken from the anterior, middle and posterior thirds of the thalamus. The sections were stained free floating using a rabbit anti-calbindin-D28k antibody (Emson, Cambridge, U.K.)21 at a dilution of 1:5000– 1:10,000, a rabbit anti-calretinin antibody (SWANT) at a dilution of 1:5000, and a mouse anti-parvalbumin antibody
486
M. C. Mu¨nkle et al.
Fig. 1A–F.
Calcium-binding proteins in the human thalamus (Sigma) at a dilution of 1:10,000 using standard immunohistochemical techniques with biotinylated secondary antibodies and a streptavidin–horseradish peroxidase
487
conjugate.23 The rabbit anti-calbindin-D28k antibody was raised in chick and its specificity was tested by incubating sections either with serum or with the primary antibody that had been pre-incubated with the antigen.21 The distribution of the label was visualized using a 3,3 -diaminobenzidene reaction. RESULTS
All the nuclei in the human thalamus showed immunoreactivity for one or more of the calciumbinding proteins in various combinations (Fig. 1). The anterior group of nuclei (nomenclature of Hirai and Jones13,15) (Fig. 1A–C) and the lateral dorsal nucleus (Fig. 1D–F) showed immunoreactivity for all three calcium-binding proteins except that the anterior dorsal nucleus showed no parvalbumin immunoreactivity. In contrast, the ventral nuclei (ventral anterior, ventral lateral, ventral medial and ventral posterior nuclei) (Fig. 1), the pulvinar nuclei, the lateral posterior nucleus (Fig. 1G–I) and the mediodorsal nucleus (Fig. 1A–F) showed immunoreactivity mainly for calbindin-D28k and parvalbumin, although the mediodorsal nucleus additionally showed patches of calretinin-immunoreactive cells and fibres (Fig. 1E). The medial and lateral geniculate nuclei showed moderately dense parvalbumin staining (Fig. 1I), light neuropil staining for calretinin (Fig. 1H) and weak to moderate calbindin-D28k staining in the cell bodies (Fig. 1G). The reticular nucleus was the only thalamic nucleus which showed immunoreactivity for both calretinin and parvalbumin, but it was immunonegative for calbindin-D28k (Fig. 1). The intralaminar nuclei showed a very distinctive pattern of calcium-binding immunoreactivity which served to precisely delineate this often poorly
Fig. 1G–I.
Fig. 1. The distribution of calcium-binding immunoreactivity in the human thalamus. Serial sections of the human thalamus at the level of the anterior third (A–C), the middle third (D–F) and the posterior third (G–I) of the thalamus stained for calbindin-D28k (A, D, G), calretinin (B, E, H) and parvalbumin (C, F, I). The intralaminar nuclei of the human thalamus are distinguished by their pattern of immunostaining for the three calcium-binding proteins calbindin-D28k, calretinin and parvalbumin. The central lateral, central medial and paracentral intralaminar nuclei stained positively for calbindin-D28k (A, D, G) and for calretinin (B, E, H), but were immunonegative for parvalbumin (C, F, I). By contrast, the centre me´dian nucleus was immunopositive for parvalbumin (I) but immunonegative for calbindin-D28k (G) and calretinin (H). Scale bars=1 cm. CeM, central medial nucleus; CL, central lateral nucleus; CM, centre me´dian nucleus; CN, caudate nucleus; GPe, globus pallidus external part; GPi, globus pallidus internal part; IC, internal capsule; LD, lateral dorsal nucleus; LGN, dorsal lateral geniculate nucleus; LP, lateral posterior nucleus; MD, mediodorsal nucleus; OT, optic tract; P, putamen; Pg, pregeniculate nucleus; R, reticular nucleus; Sg-L, suprageniculate-limitans nucleus; SN, substantia nigra; STN, subthalamic nucleus; VLa, ventral lateral anterior nucleus; VLp, ventral lateral posterior nucleus; VPL, ventral posterior lateral nucleus; ZI, zona incerta.
488
M. C. Mu¨nkle et al. Table 1. Sources of human brain tissue Case no.
Sex
Age (years)
Post mortem delay (h)
Cause of death
3176 H103 3392 3212 H104 3446 4164 4168
M M M M M M M F
47 70 25 47 69 44 82 84
13.25 5 15 13.25 14 8 21 14.5
ischaemic heart disease myocardial infarct asphyxia ischaemic heart disease ischaemic heart disease myocardial infarct ischaemic heart disease ruptured myocardial infarct
demarcated group of nuclei from the remaining nuclear complexes of the human thalamus. The anterior group of intralaminar nuclei (central lateral nucleus, paracentral nucleus, central medial nucleus) showed a characteristic intense staining for both calbindin-D28k and calretinin (Fig. 1A, B, D, E). All of the component cell clusters comprising the central lateral nucleus were selectively highlighted by intense staining with calretinin (Fig. 1E, H) and calbindin-D28k (Fig. 1D, G). In the anterior two thirds of the thalamus the densely stained immunoreactive cell bodies for calretinin (Fig. 2B) and calbindin-D28k (Fig. 2A) in the central lateral nucleus (Fig. 1D, E) formed a well demarcated, thin, peri-nuclear shell immediately lateral to the spherical mediodorsal nucleus (Fig. 1D, E). At posterior thalamic levels the central lateral nucleus comprises a series of isolated cell clusters which are dispersed within the medial region of the thalamus and these groups of cells are conspicuously delineated with calbindin-D28k (Fig. 1G) and calretinin (Fig. 1H) immunoreactivity. The central lateral nucleus was immunonegative for parvalbumin except for a few scattered fibre terminals (Fig. 1F, I). The paracentral nucleus was clearly delineated in sections stained for both calbindin-D28k and calretinin (Fig. 1A, B); it showed very intense immunostaining for calretinin of both the cell bodies and fibres (Fig. 2C) and the traversing immunonegative fibre bundles tended to disperse the clusters of immunopositive cells thus giving the nucleus a distinctive reticulated appearance (Fig. 2C). The central medial nucleus showed intense staining for both calbindin-D28k (Fig. 1A, D) and calretinin (Fig. 1B, E); in particular it showed numerous cell bodies intensely immunoreactive for calretinin which lay in a very dense and strongly stained neuropil (Fig. 2D) containing fewer calbindin-D28k-positive neurons. No staining for parvalbumin was observed in the central medial nucleus (Fig. 1C, F). The posterior group of intralaminar nuclei (centre me´dian nucleus, parafascicular nucleus) showed a complementary pattern of staining for the three calcium-binding proteins. The centre me´dian nucleus (Fig. 1G–I) was distinctively different from all the other nuclear complexes in the human thalamus by
displaying immunoreactivity for only one calciumbinding protein, parvalbumin (Fig. 1I). The dorsomedial region of the centre me´dian nucleus showed moderately stained elongated cell bodies (Fig. 2E), while the ventrolateral portion of the nucleus showed moderately stained polygonal multipolar cell bodies set against a moderately stained neuropil (Fig. 2F). In contrast, the parafascicular nucleus showed moderate immunoreactivity for both calbindin-D28k and calretinin. Most immunoreactive cells in the parafascicular nucleus were medium-sized and oval to round in shape; however, a few large, round calbindin-D28k-positive neurons were also observed.
DISCUSSION
The results of this study show that the nuclei of the human thalamus demonstrate a distinctive heterogeneous pattern of calcium-binding protein immunoreactivity and that the three calcium-binding proteins provide very useful markers for identifying and delineating the various thalamic nuclei. In general terms, each of the ‘‘functional’’ nuclear complexes of the human thalamus demonstrated a characteristic pattern of calcium-binding protein immunoreactivity, suggesting that the various combinations of calcium-binding proteins may contribute to the functional characteristics associated with each of the thalamic nuclear groups. The detailed staining pattern of the thalamic nuclei demonstrated here agrees in general terms with previous findings in the monkey9,16 and human thalamus.8,18 However, our results on the distribution of calbindin-D28k in the human thalamus differ from those of Morel et al.,18 which may be due to differences in the method of tissue fixation. Of special interest and importance in the present study is the demonstration that the intralaminar nuclei of the human thalamus show a unique and characteristic pattern of staining for the three calcium-binding proteins. The anterior intralaminar nuclei and the parafascicular nucleus showed immunoreactivity for both calbindin-D28k and calretinin whereas the centre me´dian nucleus showed immunoreactivity only for parvalbumin. No other nucleus in
Calcium-binding proteins in the human thalamus
Fig. 2. Photomicrographs of the intralaminar nuclei of the human thalamus stained for the three calcium-binding proteins calbindin-D28k (A), calretinin (B–D), and parvalbumin (E, F). (A, B) Photomicrographs of corresponding regions of the central lateral nucleus stained for calbindin-D28k (A) and calretinin (B) showing patches of intensely stained cell bodies. (C) The paracentral nucleus shows intense immunoreactivity for calretinin in both the cell bodies and fibres; immunonegative fibre bundles traversing the paracentral nucleus give it a very distinctive reticulated appearance in calretinin-immunoreactive sections. (D) The central medial nucleus shows very intense staining for calretinin in both the cell bodies and the neuropil. (E, F) Parvalbumin-positive cell bodies in the dorsal (E) and ventral (F) parts of the centre me´dian nucleus. The cell bodies in the dorsal part (E) are more dispersed and show a different cytoarchitecture to those in the ventral part (F). Scale bars=50 µm.
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the human thalamus showed these particular combinations of calcium-binding protein staining. Furthermore, in addition to their distinctive calcium-binding protein staining characteristics, the intralaminar nuclei are the only thalamic nuclear groups which have major projections to both the cerebral cortex and to the basal ganglia.12,15 In a recent review of the overall pattern of connections of the intralaminar nuclei, Groenewegen and Berendse10 stated that ‘‘the rostral intralaminar nuclei project to prefrontal association cortical areas and the posterior part of the parietal cortex, whereas the caudal intralaminar nuclei project to motor and premotor cortical areas in the frontal lobe and to the anterior part of the parietal cortex’’;10 in particular, they described10 that the organization of the intralaminar thalamocortical projection seemed to be such that individual intralaminar nuclei project to cortical areas that are in turn interconnected by corticocortical connections and that have convergent projections to the striatum of the basal ganglia. They also emphasized that the direct thalamostriate projections of the intralaminar nuclei on to the striatum (caudate–putamen) of the basal ganglia are organized in a very strict topographical manner such that the lateral-to-medial co-ordinate in the intralaminar nuclei projects onto the dorsolateral-to-ventromedial co-ordinate of the striatum.10 Finally, concerning the
overall pattern of connections of the intralaminar nuclei with the cerebral cortex and the basal ganglia, it is especially interesting that the targets of the thalamocortical and thalamostriatal projections of a given intralaminar nucleus are interconnected through the corticostriatal projection.10 CONCLUSION
This corresponding topographical pattern of the interconnections between the intralaminar nuclei, the cerebral cortex and the basal ganglia shows that the intralaminar nuclei are therefore well placed to selectively modulate the activity of the various functionally segregated basal ganglia-thalamocortical circuits. Thus, the distinctive connectional affiliations of these nuclei and their characteristic calcium-binding protein properties suggest that the calcium-binding proteins may play a critical role in the influence of these nuclei on their interactions with the cerebral cortex and the basal ganglia. Acknowledgements—This study was supported by grants from the Health Research Council of New Zealand, the New Zealand Neurological Foundation and the New Zealand Lottery Board. M.C.M. was supported by a scholarship from the German Academic Exchange Service (Doktorandenstipendium HSPIII).
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