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Purkinje-like cells in the cochlear nucleus of the Common Tree Shrew (Tupaia glis) identified by calbindin immunohistochemistry
Brain Research 983 (2003) 230–232 www.elsevier.com / locate / brainres
Short communication
Purkinje-like cells in the cochlear nucleus of the Common Tree Shrew (Tupaia glis) identified by calbindin immunohistochemistry W.B. Spatz* ¨ Freiburg, Hansastrasse 9, 79104 Freiburg, Morphologische Hirnforschung, HNO-Klinik, und AG Hirnforschung, Anatomisches Institut, Universitat Germany Accepted 21 May 2003
Abstract The dorsal cochlear nucleus (DCN) of Tree Shrews (Tupaia glis; n52) was examined by calbindin (CB) immunohistochemistry for the presence of Purkinje-like cells (PLCs), detected previously in only four different mammals. We found up to eight CB-immunoreactive PLCs in the left and right DCN, and a few axons, likely of PLC origin, that appeared to leave the DCN. These findings suggest that PLCs may have a wider distribution through mammalian species, and may represent more than just misrouted cells. 2003 Elsevier B.V. All rights reserved. Theme: Other systems of the CNS Topic: Comparative neuroanatomy Keywords: Auditory; Dorsal cochlear nucleus; Comparative anatomy; Molecular identification
Purkinje-like cells (PLCs) are distinctive neurons consistently found, though in very small numbers, in the cochlear nuclei (CN) of the mouse [1], rat [3–5,7,10], guinea pig [10], and marmoset (Callithrix [9,10], preliminary reports). For other mammals, PLCs have not previously been described. In the rodents, the PLCs reside almost exclusively in the outer layer of the dorsal cochlear nucleus (DCN). Similar cells (‘displaced’ or ‘ectopic’ Purkinje cells (PCs), not further addressed here) have been found in white matter regions of the brainstem close to the cerebellum [3]. Immunohistochemical studies indicated that the PLCs are characterized by their co-expression of a variety of molecules all expressed by the cerebellar PCs as well, but, in this combination, not by any other neuron of the CN (e.g. the L7 protein is expressed by the PCs, and in the brainstem exclusively by the PLCs [1]). Furthermore, the PLCs receive climbing fiber input [7] and mossy fiber input via the parallel fibers originating from CN granule cells [2], similar as the PCs. This, and their morphological characteristics, identify them as closely related to the PCs. A basic question is, whether the PLCs are to be seen *Tel.: 149-761-203-9555; fax: 149-761-203-9500. E-mail address: [email protected] (W.B. Spatz). 0006-8993 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)03050-6
simply as ‘displaced PCs’ misrouted during developmental migration, or whether they have a place in the circuitry of the DCN [3,5,10]. More information on these neurons, including data on their existence in other mammals, might be useful for further discussions. Therefore, we took advantage of the opportunity to examine the brainstem of tree shrews (Tupaia glis), a representative of the distant mammalian order Scandentia [8], using sections through the CN immunostained for the calcium-binding protein calbindin D28k (CB), well known to be expressed by PCs and PLCs [4,5,7,10]. The brainstems of two young adult Tupaia (Tup. I, Tup. II) were available. The animals, sacrificed for nonneuronal studies, were perfused transcardially with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7,4). The cryoprotected and frozen brainstems were transversely cut at 30 mm. Series of sections (one of five) were processed with standard immunohistochemical methods for the visualization of CB. Briefly, floating sections were pretreated in hydrogen peroxide (H 2 O 2 ), in a solution of defatted dry milk, and in PB containing 1% bovine serum albumin and 0.05% Triton X-100. Sections were then incubated for 48 h at 4 8C with a monoclonal mouse antibody directed against CB (Sigma, clone CL-300;
W.B. Spatz / Brain Research 983 (2003) 230–232
diluted 1:10 000), and transfered to a biotinylated species specific secondary antibody for 30 min, followed by the avidin–biotin–peroxidase complex (ABC Vectastain Elite kit). The bound peroxidase was visualized with diaminobenzidine tetrahydrochloride and H 2 O 2 . Control sections processed without the primary antibody did not show specific immunostaining. Transverse sections through the Tupaia brainstem showed the DCN in a conspicuously lateral position, with
231
a remarkable steep orientation of the long axis of the profiles along the entire rostro-caudal extent of the nucleus (Fig. 1A; see also Ref. [11]); in the rat and guinea pig, the DCN is dorsally positioned, with the long axis oriented more horizontally [10]. CB-like immunoreactivity (CB-IR) revealed a very light general immunostaining of the neurophil containing axonal processes and fields of CB-IR puncta, and few lightly and moderately labeled small and medium-sized neurons (Fig. 1A).
Fig. 1. Transverse sections through the DCN of Tupaia processed for the visualization of CB-IR. (A) (Tup. II, right DCN): Overview. Note the lateral position of the DCN relative to the brainstem, with the long axis of the profile oriented almost vertically, and the pale general immunostain of the DCN showing granular fields and very few labeled cells (small arrow). The arrowhead points on heavily stained portions of the dendritic tree of a single PLC. Scale bar5500 mm. (B) (Tup I, right DCN): Two CB-ir PLCs reside in layer I of the DCN some distance from the surface, with their dendrites extending towards the surface and into the depth of layer I. Portions of the dendritic tree of a 3rd PLC are seen to the right. Scale bar5100 mm. (C) (Tup. II, left DCN): At high magnification, numerous spines are visible at the CB-ir dendritic branches. Scale bar520 mm. (D) (Tup. II, left DCN): A group of seven CB-ir PLCs (four out of focus) residing in the most rostral and dorsal portion of the left DCN. Note the bundle of CB-ir axons (arrow head) passing below a cut blood vessel and running dorsally (at right) towards the brainstem. Scale bar550 mm.
232
W.B. Spatz / Brain Research 983 (2003) 230–232
Occasionally, a few large neurons intensely immunostained for CB were encountered that compared favorable to the PLCs of rodents. Their large somata resided in layer I of the DCN, mostly at some distance from the surface, and emitted one or more thick, richly branched and heavily spined dendrites into the depth and towards the surface of the layer (Fig. 1B–D). PLCs were detected in the DCN of both sides of the brainstem in both Tupaia, but in very small numbers (one (left) and three (right) PLCs in Tup. I, and eight (left) and three (right) in Tup. II). In view of the spacing of the sections available, these countings might be slight underestimates. However, since the PLC dendrites clearly span more than a single section, and since even isolated pieces of dendrites were easily identified, the error should be small. The PLCs showed a preference for the anterior DCN (Fig. 1D), but single cells were found more posteriorly as well. Occasionally, a few thick, smooth CB-IR axons were detected close to the PLCs running in a rostrodorsal direction towards the junction of the DCN with the brainstem where they may eventually leave the DCN (Fig. 1D). The number of these axons equaled that of the PLCs, but their origin from the PLCs was not immediately seen in the present materials. This study presents evidence for the DCN of Tupaia housing a small number of PLCs. The existence of PLCs, thus, is now documented for five different mammals: three rodents belonging to two suborders (mouse, rat: Myomorpha; guinea pig: Hystricomorpha), a primate (Callithrix), and Tupaia. The PLCs of Tupaia strikingly resemble the rodent PLCs by their small number, the position of their soma in layer I, the restriction of their dendrites to that layer, and by their intense CB-IR. In both, the rat [10] and Tupaia, we noticed CB-IR axons in close apposition to the CB-IR PLCs. Although the origin of these axons from the PLCs was not immediately seen, observations in the marmoset CN encourage the interpretation that these are PLC axons that may leave the CN, possibly to meet the nearby mass of the PC axons heading for the cerebellar nuclei [10]. Given that we detected PLCs in all three species we examined (guinea pig, marmoset, Tupaia), it appears somewhat curious that PLCs have not been described in other mammals except for the mouse and rat. Possibly, PLCs do not exist in some species, as a matter of fact. But in others, the few PLCs may only have been overlooked or ignored. The healthy appearance of these neurons, their presumed connectivity, and their constant occurrence in the five mammals described, may encourage the interpretation that the PLCs are not merely accidentally misrouted neurons without any significance, but play their part in the
CN, similar as other neurons residing in both the cerebellum and the CN, e.g. granule cells and unipolar brush cells [6].
Acknowledgements The author wishes to express his gratefulness to M. Seibert for technical assistance, and to Professor Dr M. ¨ Frotscher (Anatomie I), Professor Dr J. Kruger (AG Hirnforschung), and Professor Dr R. Laszig (HNO-Klinik), for generous support. I thank Professor Dr M. Nassal (Medizinische Klinik) for the Tupaia brains. The author was recipient of a grant from the Deutsche Forschungsgemeinschaft (Sp 70 / 5-3).
References [1] A.S. Berrebi, J.I. Morgan, E. Mugnaini, The Purkinje cell class may extend beyond the cerebellum, J. Neurocytol. 19 (1990) 643–654. [2] A.S. Berrebi, E. Mugnaini, Alterations in the dorsal cochlear nucleus ´ J.M. Juiz, D.A. of cerebellar mutant mice, in: M.A. Merchan, Godfrey, E. Mugnaini (Eds.), The Mammalian Cochlear Nuclei. Organization and Function, Plenum Press, New York, 1993, pp. 107–119. [3] P. De Camilli, P.E. Miller, P. Levitt, U. Walter, P. Greengard, Anatomy of cerebellar Purkinje cells in the rat determined by a specific immunohistochemical marker, Neuroscience 11 (1984) 761–817. [4] E. Friauf, Distribution of calcium-binding protein calbindin-D-28k in the auditory system of adult and developing rats, J. Comp. Neurol. 349 (1994) 193–211. [5] L.B. Hurd, M.L. Feldman, Purkinje-like cells in rat cochlear nucleus, Hear. Res. 72 (1994) 143–158. ˜ D. Jaarsma, The unipolar brush cells of the [6] E. Mugnaini, M.R. Dino, mammalian cerebellum and cochlear nucleus: Cytology and microcircuitry, in: C.I. de Zeeuw, P. Strata, J. Voogd (Eds.), Progress in Brain Research, The Cerebellum: From Structure To Control, Vol. 114, Elsevier, Amsterdam, 1997, pp. 131–150. [7] F. Rossi, T. Borsello, Ectopic Purkinje cells in the adult rat: olivary innervation and different capabilities of migration and development after grafting, J. Comp. Neurol. 337 (1993) 70–82. ¨ [8] J. Schmitz, M. Ohme, H. Zischler, The complete mitochondrial genome of Tupaia belangeri and the phylogenetic affiliation of scandentia to other eutherian orders, Mol. Biol. Evol. 17 (2000) 1334–1343. [9] W.B. Spatz, Purkinje-like cells in the dorsal cochlear nucleus of the marmoset (Callithrix), Eur. J. Neurosci. Suppl. 9 (1996) 90. [10] W.B. Spatz, Differences between guinea pig and rat in the dorsal cochlear nucleus: expression of calcium-binding proteins by cartwheel and Purkinje-like cells, Hear. Res. 107 (1997) 136–146. [11] J. Tigges, T.R. Shantha, A Stereotaxic Brain Atlas of the Tree Shrew (Tupaia glis), Williams and Wilkins, Baltimore, 1969, 149 pp.
Short communication
Purkinje-like cells in the cochlear nucleus of the Common Tree Shrew (Tupaia glis) identified by calbindin immunohistochemistry W.B. Spatz* ¨ Freiburg, Hansastrasse 9, 79104 Freiburg, Morphologische Hirnforschung, HNO-Klinik, und AG Hirnforschung, Anatomisches Institut, Universitat Germany Accepted 21 May 2003
Abstract The dorsal cochlear nucleus (DCN) of Tree Shrews (Tupaia glis; n52) was examined by calbindin (CB) immunohistochemistry for the presence of Purkinje-like cells (PLCs), detected previously in only four different mammals. We found up to eight CB-immunoreactive PLCs in the left and right DCN, and a few axons, likely of PLC origin, that appeared to leave the DCN. These findings suggest that PLCs may have a wider distribution through mammalian species, and may represent more than just misrouted cells. 2003 Elsevier B.V. All rights reserved. Theme: Other systems of the CNS Topic: Comparative neuroanatomy Keywords: Auditory; Dorsal cochlear nucleus; Comparative anatomy; Molecular identification
Purkinje-like cells (PLCs) are distinctive neurons consistently found, though in very small numbers, in the cochlear nuclei (CN) of the mouse [1], rat [3–5,7,10], guinea pig [10], and marmoset (Callithrix [9,10], preliminary reports). For other mammals, PLCs have not previously been described. In the rodents, the PLCs reside almost exclusively in the outer layer of the dorsal cochlear nucleus (DCN). Similar cells (‘displaced’ or ‘ectopic’ Purkinje cells (PCs), not further addressed here) have been found in white matter regions of the brainstem close to the cerebellum [3]. Immunohistochemical studies indicated that the PLCs are characterized by their co-expression of a variety of molecules all expressed by the cerebellar PCs as well, but, in this combination, not by any other neuron of the CN (e.g. the L7 protein is expressed by the PCs, and in the brainstem exclusively by the PLCs [1]). Furthermore, the PLCs receive climbing fiber input [7] and mossy fiber input via the parallel fibers originating from CN granule cells [2], similar as the PCs. This, and their morphological characteristics, identify them as closely related to the PCs. A basic question is, whether the PLCs are to be seen *Tel.: 149-761-203-9555; fax: 149-761-203-9500. E-mail address: [email protected] (W.B. Spatz). 0006-8993 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0006-8993(03)03050-6
simply as ‘displaced PCs’ misrouted during developmental migration, or whether they have a place in the circuitry of the DCN [3,5,10]. More information on these neurons, including data on their existence in other mammals, might be useful for further discussions. Therefore, we took advantage of the opportunity to examine the brainstem of tree shrews (Tupaia glis), a representative of the distant mammalian order Scandentia [8], using sections through the CN immunostained for the calcium-binding protein calbindin D28k (CB), well known to be expressed by PCs and PLCs [4,5,7,10]. The brainstems of two young adult Tupaia (Tup. I, Tup. II) were available. The animals, sacrificed for nonneuronal studies, were perfused transcardially with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7,4). The cryoprotected and frozen brainstems were transversely cut at 30 mm. Series of sections (one of five) were processed with standard immunohistochemical methods for the visualization of CB. Briefly, floating sections were pretreated in hydrogen peroxide (H 2 O 2 ), in a solution of defatted dry milk, and in PB containing 1% bovine serum albumin and 0.05% Triton X-100. Sections were then incubated for 48 h at 4 8C with a monoclonal mouse antibody directed against CB (Sigma, clone CL-300;
W.B. Spatz / Brain Research 983 (2003) 230–232
diluted 1:10 000), and transfered to a biotinylated species specific secondary antibody for 30 min, followed by the avidin–biotin–peroxidase complex (ABC Vectastain Elite kit). The bound peroxidase was visualized with diaminobenzidine tetrahydrochloride and H 2 O 2 . Control sections processed without the primary antibody did not show specific immunostaining. Transverse sections through the Tupaia brainstem showed the DCN in a conspicuously lateral position, with
231
a remarkable steep orientation of the long axis of the profiles along the entire rostro-caudal extent of the nucleus (Fig. 1A; see also Ref. [11]); in the rat and guinea pig, the DCN is dorsally positioned, with the long axis oriented more horizontally [10]. CB-like immunoreactivity (CB-IR) revealed a very light general immunostaining of the neurophil containing axonal processes and fields of CB-IR puncta, and few lightly and moderately labeled small and medium-sized neurons (Fig. 1A).
Fig. 1. Transverse sections through the DCN of Tupaia processed for the visualization of CB-IR. (A) (Tup. II, right DCN): Overview. Note the lateral position of the DCN relative to the brainstem, with the long axis of the profile oriented almost vertically, and the pale general immunostain of the DCN showing granular fields and very few labeled cells (small arrow). The arrowhead points on heavily stained portions of the dendritic tree of a single PLC. Scale bar5500 mm. (B) (Tup I, right DCN): Two CB-ir PLCs reside in layer I of the DCN some distance from the surface, with their dendrites extending towards the surface and into the depth of layer I. Portions of the dendritic tree of a 3rd PLC are seen to the right. Scale bar5100 mm. (C) (Tup. II, left DCN): At high magnification, numerous spines are visible at the CB-ir dendritic branches. Scale bar520 mm. (D) (Tup. II, left DCN): A group of seven CB-ir PLCs (four out of focus) residing in the most rostral and dorsal portion of the left DCN. Note the bundle of CB-ir axons (arrow head) passing below a cut blood vessel and running dorsally (at right) towards the brainstem. Scale bar550 mm.
232
W.B. Spatz / Brain Research 983 (2003) 230–232
Occasionally, a few large neurons intensely immunostained for CB were encountered that compared favorable to the PLCs of rodents. Their large somata resided in layer I of the DCN, mostly at some distance from the surface, and emitted one or more thick, richly branched and heavily spined dendrites into the depth and towards the surface of the layer (Fig. 1B–D). PLCs were detected in the DCN of both sides of the brainstem in both Tupaia, but in very small numbers (one (left) and three (right) PLCs in Tup. I, and eight (left) and three (right) in Tup. II). In view of the spacing of the sections available, these countings might be slight underestimates. However, since the PLC dendrites clearly span more than a single section, and since even isolated pieces of dendrites were easily identified, the error should be small. The PLCs showed a preference for the anterior DCN (Fig. 1D), but single cells were found more posteriorly as well. Occasionally, a few thick, smooth CB-IR axons were detected close to the PLCs running in a rostrodorsal direction towards the junction of the DCN with the brainstem where they may eventually leave the DCN (Fig. 1D). The number of these axons equaled that of the PLCs, but their origin from the PLCs was not immediately seen in the present materials. This study presents evidence for the DCN of Tupaia housing a small number of PLCs. The existence of PLCs, thus, is now documented for five different mammals: three rodents belonging to two suborders (mouse, rat: Myomorpha; guinea pig: Hystricomorpha), a primate (Callithrix), and Tupaia. The PLCs of Tupaia strikingly resemble the rodent PLCs by their small number, the position of their soma in layer I, the restriction of their dendrites to that layer, and by their intense CB-IR. In both, the rat [10] and Tupaia, we noticed CB-IR axons in close apposition to the CB-IR PLCs. Although the origin of these axons from the PLCs was not immediately seen, observations in the marmoset CN encourage the interpretation that these are PLC axons that may leave the CN, possibly to meet the nearby mass of the PC axons heading for the cerebellar nuclei [10]. Given that we detected PLCs in all three species we examined (guinea pig, marmoset, Tupaia), it appears somewhat curious that PLCs have not been described in other mammals except for the mouse and rat. Possibly, PLCs do not exist in some species, as a matter of fact. But in others, the few PLCs may only have been overlooked or ignored. The healthy appearance of these neurons, their presumed connectivity, and their constant occurrence in the five mammals described, may encourage the interpretation that the PLCs are not merely accidentally misrouted neurons without any significance, but play their part in the
CN, similar as other neurons residing in both the cerebellum and the CN, e.g. granule cells and unipolar brush cells [6].
Acknowledgements The author wishes to express his gratefulness to M. Seibert for technical assistance, and to Professor Dr M. ¨ Frotscher (Anatomie I), Professor Dr J. Kruger (AG Hirnforschung), and Professor Dr R. Laszig (HNO-Klinik), for generous support. I thank Professor Dr M. Nassal (Medizinische Klinik) for the Tupaia brains. The author was recipient of a grant from the Deutsche Forschungsgemeinschaft (Sp 70 / 5-3).
References [1] A.S. Berrebi, J.I. Morgan, E. Mugnaini, The Purkinje cell class may extend beyond the cerebellum, J. Neurocytol. 19 (1990) 643–654. [2] A.S. Berrebi, E. Mugnaini, Alterations in the dorsal cochlear nucleus ´ J.M. Juiz, D.A. of cerebellar mutant mice, in: M.A. Merchan, Godfrey, E. Mugnaini (Eds.), The Mammalian Cochlear Nuclei. Organization and Function, Plenum Press, New York, 1993, pp. 107–119. [3] P. De Camilli, P.E. Miller, P. Levitt, U. Walter, P. Greengard, Anatomy of cerebellar Purkinje cells in the rat determined by a specific immunohistochemical marker, Neuroscience 11 (1984) 761–817. [4] E. Friauf, Distribution of calcium-binding protein calbindin-D-28k in the auditory system of adult and developing rats, J. Comp. Neurol. 349 (1994) 193–211. [5] L.B. Hurd, M.L. Feldman, Purkinje-like cells in rat cochlear nucleus, Hear. Res. 72 (1994) 143–158. ˜ D. Jaarsma, The unipolar brush cells of the [6] E. Mugnaini, M.R. Dino, mammalian cerebellum and cochlear nucleus: Cytology and microcircuitry, in: C.I. de Zeeuw, P. Strata, J. Voogd (Eds.), Progress in Brain Research, The Cerebellum: From Structure To Control, Vol. 114, Elsevier, Amsterdam, 1997, pp. 131–150. [7] F. Rossi, T. Borsello, Ectopic Purkinje cells in the adult rat: olivary innervation and different capabilities of migration and development after grafting, J. Comp. Neurol. 337 (1993) 70–82. ¨ [8] J. Schmitz, M. Ohme, H. Zischler, The complete mitochondrial genome of Tupaia belangeri and the phylogenetic affiliation of scandentia to other eutherian orders, Mol. Biol. Evol. 17 (2000) 1334–1343. [9] W.B. Spatz, Purkinje-like cells in the dorsal cochlear nucleus of the marmoset (Callithrix), Eur. J. Neurosci. Suppl. 9 (1996) 90. [10] W.B. Spatz, Differences between guinea pig and rat in the dorsal cochlear nucleus: expression of calcium-binding proteins by cartwheel and Purkinje-like cells, Hear. Res. 107 (1997) 136–146. [11] J. Tigges, T.R. Shantha, A Stereotaxic Brain Atlas of the Tree Shrew (Tupaia glis), Williams and Wilkins, Baltimore, 1969, 149 pp.