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Transient expression of calretinin during development of chick cerebellum Comparison with calbindin-D28k
Neuroscience Research, 17 (1993) 53-61 © 1993 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved 0168-0102/93/$06.00
53
NSR 00648
Transient expression of calretinin during development of chick cerebellum Comparison with calbindin-D28k E. B a s t i a n e l l i a n d R. P o c h e t Laboratoire d'Histologie, Facult~ de M~decine, UniversitJ Libre de Bruxelles, Route de Lennik 808, B-1070 Brussels, Belgium
(Received 8 March 1993; accepted 3 April 1993)
Key words: Calcium binding proteins; Immunohistochemistry; Development; Avian; Brain; Purkinje cell Summary Calcium ions play a critical role in neural development. Insights into the ontogeny of Ca 2+ homeostasis were gained by investigating the developmental expression of two E-F hand calcium-binding proteins. Calretinin and calbindin were monitored through their immunoreactivity in the developing chick cerebellum (from E6 to E20). Calbindin was detected from El3 and in Purkinje cells only. Intensity of labelling increased with Purkinje cell development. Calretinin presented a transitory immunoreactivity between E11 and E20 in the internal granular cell layer. This cell layer contains ceils which will differentiate into Golgi and granular cells which are calretinin-negative in adult chick cerebellum. Calretinin immunoreactivity presented a peak (both in number of cells and in intensity) at El5 and fell dramatically after E20 while calbindin immunoreactivity was restricted to the Purkinje cells and increased with the development of these cells.
Introduction Changes in intracellular C a 2+ levels [Ca2+]i play a major role in the modulation of many key cellular processes, especially in the central nervous system. Regulation of cellular metabolism, cell division, cell mobility, protein synthesis, exocytosis are [Ca2+]i-dependent (Brostom et al., 1976; Marcum et al., 1978; Dedman et al., 1979). The different steps (calcium mobilization, mediator calcium binding and activation, cellular cascade activation by CaZ+-mediator) necessary for a calcium-coupled response involve that the calcium concentration is very precisely regulated (Heiz-
Correspondence to: Dr. R. Pochet, Laboratoire d'Histologie, Facult6 de M6decine, Universit6 Libre de Bruxelles, Route de Lennik 808, B-1070 Brussels, Belgium. Tel.: 32 2 5556374; Fax: 32 2 5556285. Abbreviations: CaBP, calbindin; Calret, calretinin; CN, cerebellar nuclei; EG, external granular layer; IG, internal granular layer; Ir, immunoreactivity; M, molecular layer; Md, medullary zone; P, Purkinje cell layer.
mann and Schaefer, 1990). Intracellular calcium binding proteins such as calbindin and calretinin may play a role in this [Ca2+]i control, although a precise physiological function, besides binding Ca 2÷, has not yet been demonstrated. Recently, it has been proposed that calbindin may act as a cytosolic calcium buffer protecting neurons against excitotoxic damage caused by calcium influx (Mattson et al., 1991). The highly conserved structure of calbindin and calretinin throughout evolution (Parmentier et al., 1987; Parmentier, 1990) suggests that those proteins should have fundamental functions. These calcium binding proteins might also serve to redistribute calcium within the neurons. But assuming that the neuroanatomical distribution of these proteins is highly selective (Jande et al., 1981; GarciaSegura et al., 1984; Rogers, 1987; Pochet et al., 1991; REsibois and Rogers, 1992), it appears that, at cell maturity, these proteins are probably not involved in general regulator mechanisms, but rather in more specific physiological functions shared by restricted neuronal cell population. They might act like calmodulin
54 in affecting directly Ca2+-dependent regulation of different proteins. Calbindin and calretinin appearance during development of the chick retina has recently been studied (Ellis et al., 1991) and seems to be linked with synaptogenesis. Ontogenesis of calbindin in cerebellum has been investigated to some extent and is limited to rat (Legrand et al., 1983; Enderlin et al., 1987) and mouse (Iacopino et al., 1990). Some authors think that calbindin could act as atrophic factor during neuronal development (Enderlin et al., 1987). Legrand et al. (1983) did not find calbindin before El7 whereas Enderlin et al. (1987) found it at El5. Interestingly, the latter authors have noticed that calbindin was transiently expressed (from El5 to E21) in the deep nuclei of the cerebellum (cerebellar "anlage") and in non neuronal cells. They also concluded that in rat, calbindin was expressed in neurons long before the beginning of synapse formation contrasting with the conclusion drawn by Ellis et al. (1991) studying the developing chick retina. Calbindin in mouse cerebellum was only studied at birth and after. The present study was undertaken in an attempt to obtain information about possible functions for calbindin and calretinin by examining their developmental profile using immunohistochemistry and comparing it with known events in the histogenesis of a well-characterized tissue: the cerebellum. The cerebellum offers the most distinctive cellular architecture in the brain (Ram6n y Cajal, 1911; Palay and Chan-Palay, 1974) with only a few well-known cell types. Chick embryos of known ages are easily available and the intrinsic organization of the avian cerebellum cortex is comparable to that of mammals.
Materials and methods
Embryos of chicks (Gallus gallus) from 6, 8, 11, 13, 15, 17, and 20 days of incubation (denoted E6, E8, E l l , etc.) were used. Six embryos of each age were dissected and immersed in Helly's fixative (68 mM potassium bichromate, 70 mM sodium sulfate, 180 mM HgC12, and 4% paraformaldehyde). Fixed embryos were routinely dehydrated, embedded in paraffin, and sectioned at 5-10/zm.
Antibodies Two rabbit polyclonal antisera against calretinin were used at dilutions of 1:1000 or 1:2000. The first antiserum (kindly donated by J. Rogers, Cambridge, UK) was raised against /3-galactosidase-calretinin fusion protein which contained either two or four do-
TABLE
1
DISTRIBUTION
OF
EG
Calret-lr
M
E 8
.
.
E11
-
-
El3
-
-
IN DEVELOPING
P .
IG
-
CN
+
. . . .
+-
+
El5
-
-
-
+
-
-
-
+
E20
-
+
-
+
indicate
Md
.
El7
Symbols
CEREBELLUM
positive
l a y e r s ( + ), n e g a t i v e
-
+ +
-
+
layers(-
).
mains of the chick calretinin (Rogers, 1987, 1989b). Rabbit antiserum against the tw0-domain fusion protein was used unless otherwise stated. This antiserum was previously characterized and no cross-reactivity between calbindin and the antiserum could be detected (Pasteels et al., 1990). The second antiserum (#7696) was obtained commercially (SWant, Bellinzona, Switzerland) and made from human recombinant calretinin. Controls included preincubation of both antibodies with 1 /xg/ml of human recombinant calretinin (SWant, Bellinzona, Switzerland). Rabbit antiserum raised against chick duodenal calbindin-D28k was prepared as described by Spencer et al. (1976) and routinely used at a dilution of 1 : 6000.
Immunohistochemical staining Routinely dewaxed and hydrated sections were processed for immunohistochemistry according to a peroxidase-antiperoxidase (PAP) procedure modified from Vacca et al. (1980). Serum dilutions were made up in Coon's veronal-buffered saline (CVBS: 10 mM 5,5-diethylbarbituric acid, sodium salt (sodium veronal), pH 7.2, 150 mM NaCl) supplemented with 1% (v/v) normal sheep serum. The immunostaining sequence comprised the following steps: (1) rinse in CVBS; (2) preincubation in 5% (v/v) normal sheep serum; (3) incubation with antibodies for 48 h at 4°C in a moist chamber;
TABLE
2
DISTRIBUTION
EG
OF
CaBP-Ir
IN DEVELOPING
M
P
E 8
m
-
-
Ell
-
-
-
El3
-
-
+
El5
-
-
+
El7
-
-
+
E20
-
-
+
Symbols
indicate
positive
IG
CEREBELLUM
Md
CN
m
l a y e r s ( + ), n e g a t i v e
layers(-
).
55 TABLE 3 T I M E O F A P P E A R A N C E O F D I F F E R E N T I A T E D C E L L * A N D O F T H E I R L A B E L L I N G IN C H I C K C E R E B E L L U M Purkinje:
-
-
-
Granule cell:
-
-
-
Stellate cell:
Differentiation takes place between E l 0 and E l 2 First synapses on Purkinje cells appear at E12 Labelling: since E13 with CaBP Differentiation takes place between E l l and E13 First synapses of mossy fibers on granule cells are scarce until E15 Transient labelling between E13 and E20 with Calret
Differentiation takes place after E15 First synapses on stellate cells appear at E19 - Labelling with Calret at E20 (scarce) -
-
Basket cell:
- Differentiation takes place at E15-EI6 and is not completed before hatching - The synaptic contacts by parallel fibers on basket cells begin at E19 - Labelling with Calret at E20 (scarce)
Golgi cell:
Differentiation takes place at E16 - The synaptic connections between mossy fibers on Golgi cells are a very late processus and begin at E19 Labelling with Calret at E17 -
-
*
According to Mugnaini (1969).
(4) incubation with sheep anti-rabbit immunoglobulin G (IgG) serum (1 : 100 dilution) (Laboratoire d'Hormonologie et Immunologie, Marloie, Belgium); (5) incuba-
tion with soluble rabbit PAP complex 1:300 dilution (DAKO, Denmark). Between each step, sections were thoroughly rinsed for 10 min in CVBS. After the last
Fig. 1. Immunohistochemistry of E l 3 chick cerebellum using A: anti-calretinin antibody (1 : 2000); B: anti-calbindin antibody (1 : 6000) in sagittal section. Bar = 40/zm.
56 rinse, staining was performed in citrate phosphate buffer, p H 6.2, containing 16 mM 3,3'-diaminobenzidine-HC1 (DAB; Sigma, St. Louis, MO) and 0.01% H 2 0 2. Each of these steps was performed at room temperature, except step 3 which was performed at 4°C. Controls for specificity of staining included use of normal rabbit serum and immune serum preincubated with either 1 / z g / m l human recombinant calretinin or calbindin purified by affinity chromatography (Pasteels et al., 1990). Under these conditions, no labelling was observed (Fig. 4A,B).
Results
The structural differentiation of lobules in developing chick cerebellum was similar to the findings of previous studies (Larsell, 1948; Mugnaini, 1969; Okado et al., 1978). We therefore adopted the same classification as in LarseU (1948). The distribution of calretinin-Ir and calbindin-Ir cerebellar layers during development is summarized in
Tables 1 and 2. The time of appearance of differentiated cerebellar cells and their immunoreactivity is shown in Table 3.
Days 06-08 (E6-E8) No immunoreactivity for calbindin and calretinin could be detected on sections.
Day 11 (Ell) The cerebellum is thick and has lost its plate-like appearance. The cerebellar nuclei are present in the central region of the cerebellum. The external granular layer is now well formed. The molecular layer is located just beneath the external granular layer. The Purkinje cell layer, internal granular cell layer, and the medullary zone which corresponds to the adult white matter are recognizable. Calretinin. Neurons from the internal granular cell layer (IG) were weakly but definitely positive. Scattered neurons from the cerebellar nuclei were also positive (not shown). Calbindin. No labelling could be detected.
Fig. 2. Immunohistochemistryof chick cerebellum (lobe IV) using anti-calretinin antibody (1 : 2000). Sagittal section. A: El5. B: El7. C: E20. Arrowheads indicate granule cells, arrow indicates basket cell. Bar = 40 izm.
57
Days 13-20 All the lobules can be clearly recognized. The general appearance of the cerebellar cortex is similar to the cerebeUar cortex of the posthatching chick. But several structures characteristic of an immature cerebellum are still present. The outer granular layer remains thick up to day 17. At day 13, the Purkinje cell layer is still pluristratified and the molecular layer thin. These embryonic characteristics disappear progressively until day 17 and reach a mature architecture at day 20.
Day 13 (E13) (Fig. 1A,B) Calretinin. Some cells in IG layer are strongly positive (Fig. 1A) and in the CN many neurons are also positive (not shown). Calbindin. Large cells are labelled in P and IG layers. The strongest labelling is seen in lobes I - I V (Fig. 1B).
Day 15 (El5) (Figs. 2A, 3A) Calretinin. There is a strong positive reaction in the IG cell layer (Fig. 2A). These cells could either be granules or Golgi cells (see Discussion). The strongest labelling was seen in lobes VI, VII, and VIII. CN neurons were strongly positive (not shown). Calbindin. The double layer of Purkinje cells is strongly positive (Fig. 3A).
Day 17 (E17) (Figs. 2B and 3B) Calretinin. The positive reaction in the IG layer is still visible but fewer cells are labelled as compared to E15 (Fig. 2B). The labelling is also weaker. These cells could be either granules or Golgi cells (see Discussion). The strongest labelling is seen in lobes VI, VII, VIII. CN neurons are still strongly positive (not shown). Calbindin. Branching of Purkinje cells in the molecular layer is becoming apparent. No axons are labelled yet with the exception of a few fibers in the medulla (Fig. 3B).
Day 20 (E20) (Figs. 2C and 3C) Calretinin. Labelling is confined to only a few cells of the IG and M layer. In the latter, only a few stellate cells are positive. Few basket cells are also labelled in the P layer (Fig. 2C). In the IG layer, positive Golgi cells are recognizable. The CN is still labelled. Calbindin. Purkinje cells, including their dendritic and axonal branching, are strongly labelled (Fig. 3C). Nearly all P cells are positive. In lobes I and X, labelling is less intense (not shown).
Discussion
Neuronal differentiation not only entails the acquisition of various molecular and cellular characteristics but also involves the loss of characteristics that appear only during certain stages of development (Oppenheim, 1991). Moreover, calcium ions have been implicated in the regulation of neuronal development (Kater et al., 1990). Therefore it was decided to investigate the developmental expression of two E-F hand calcium binding proteins, calretinin and calbindin. Their appearance was monitored through their immunoreactivity in the developing chick cerebellum. In chick cerebellum, Purkinje cells differentiate between El0 and El2. They present at this stage an apical and a basal process. The first true synaptic connections on Purkinje cells are recognizable only after El2 (Mugnaini, 1969; Bertossi et al., 1986), coinciding with the appearance of CaBP-Ir. At this stage large CaBP D-28k-positive cells seen in the IG layer could be migrating Purkinje cells. During Purkinje cell development, accompanied by dendritic growth and increase in synapse number, labelling increases regularly. It might therefore be speculated that calbindin function is rather specific, and may be associated with the process of dendritic synaptic formation and stabilization. Two studies (Iacopino et al., 1990; Ellis et al., 1991) are in favor of this hypothesis. In the first, Ellis et al. (1991) showed that in chick developing retina appearance of calbindin coincides, at least approximately, with the establishment of functional synapses; in the second, Iacopino et al. (1990) found that the peak of calbindin gene expression is correlated with cessation of migration and peak synapse formation of Purkinje cells in mouse cerebellum. Synaptogenesis may result in a more complex regulation of Ca 2÷ fluxes, perhaps requiring increased intraneuronal levels of calbindin. Besides many arguments for a buffering role of calcium in neurons (Baimbridge and Miller, 1984; Johansen et al., 1990; Freund et al., 1992), no specific function for calbindin has yet been demonstrated, although it represents one of the most abundant cerebellar proteins. Why calbindin should be restricted to Purkinje cells in the cerebellum might be explained if we take into account that: (1) a typical Purkinje cell may form as many as 200000 synapses with afferent fibers, more than any other cell in the CNS (Llinas and Walton, 1989); the majority of these fibers come from granular cells using glutamate as neurotransmitter which increases intracellular calcium; (2) calbindin may play a neuroprotective role important
OC)
59
Fig. 4. Comparison between immunolabeiling using (A) anti-calretinin antibody (1 : 2000) and (B) anti-calretinin antibody (1 : 2000) pre-incubated with calretinin (1/zg/ml) in El5 chick cerebellum in sagittal section.
<.Fig.
3. Immunohistochemistry of chick cerebellum using anti-calbindin antibody (1 : 6000). Sagittal section. A: El5. B: El7. C: E20. Bar = 20/.Lm.
60 for its survival (Mattson et al., 1991) or atrophic role in synapse formation. Calretinin-Ir was first detected at E l l . Calretininpositive cells are located in the IG layer. This labelling increased from E l l to El3. At El5 the peak of calretinin immunoreactivity is reached in the IG layer containing granule and Golgi cells. The positive cells are small and might correspond to granule cells. Indeed, granule cells are fully differentiated at El3 whereas Golgi cells are larger and begin to differentiate at El6. From E15 to El7 the number of calretinin-Ir cells decreases progressively followed by a sharp decline until E20. Two hypotheses might explain this decrease in calretinin-positive cells: (1) a dilution effect due to an increase in the cell number of the IG layer, and (2) a loss of positive cells. The first hypothesis is unlikely because in chick cerebellum all IG cells have terminated both their mitosis and their migration since El5 (Fujita, 1969). In contrast, the second hypothesis is validated by the known decrease of neurons along development in the brain (Oppenheim, 1991) and more specifically in the cerebellum (Janowsky and Finlay, 1983) reinforcing our statement that calretinin was transiently expressed between E l l and E20. From El4 the organization of mossy fibers which connect granule cells changes considerably. Density of mossy fibers in the IG decreases by El6 until hatching. Loss of synaptic connection can produce cellular death by absence of stimulation which induces the blockage of cellular synthesis. But neuronal death could also be an active process in which, following the loss of trophic support, new gene expression is initiated, which acts to trigger a cascade of specific biosynthetic events that actively induce degeneration and death (Oppenheim et al., 1990). Calretinin might be expressed by such events. Its immunoreactivity in granular cells may be linked to this new gene expression inducing cellular death. At E20 calretinin-IR is almost restricted to stellate cells, basket cells and cerebellar nuclei; immunoreactivity in granular cells has almost completely disappeared. These results are in agreement with the distribution of calretinin in the adult chick cerebellum (Rogers, 1989a). It is becoming more and more evident that neurodegeneration is closely linked to a calcium-dependent mechanism (Choi, 1988). Rises in Ca 2+ can lead to cell death (Clarke, 1990; Kater et al., 1990). Pyknosis occurs by activation of Ca 2+- and MgZ+-dependent endonucleases (Oppenheim, 1991). Contrary to this, excessively low levels of free intracellular Ca 2+ concentration may also favor cell death (Oppenheim et al., 1990). Could calretinin expression be linked to this type of cellular phenomenon? It also remains to be
determined whether the induction and repression of calretinin production are influenced by epigenetic variables such as neuronal activity and neurotrophic agents or whether it stems from an immutable genetic program. In any case, the acquisition and loss of a calcium-binding protein by these neurons impart a developmental regulation that may alter their sensitivity to Ca 2+ signals.
Acknowledgments
We thank Professor Annette R6sibois for stimulating discussions and advice, L6on Surardt for excellent technical assistance and Paulette Miroir for typing the manuscript. This work was supported by FRSM (grant no. 3.4517.92).
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61 binding protein (calbindin-D28k) gene expression in the developing and aging mouse cerebellum, Mol. Brain Res., 8: 283-290. Jande, S.S., Maler, L. and Lawson, D.E.M. (1981) Immunohistochemical mapping of vitamin D-dependent calcium-binding protein in brain, Nature, 294: 765-767. Janowsky, J.S. and Finlay, B.L. (1983) Cell degeneration in early development of the forebrain and cerebellum. Anat. Embryol., 167: 439-447. Johansen, F.F., Tonder, N., Zimmer, J., Baimbridge, K.G. and Diemer, N.H. (1990) Short-term changes of parvalbumin and calbindin immunoreactivity in the rat hippocampus following cerebral ischemia, Neurosci. Lett., 120: 171-174. Kater, S.B., Mattson, M.P. and Guthrie, P.B. (1990) Calcium-induced neuronal degeneration, Ann. N.Y. Acad. Sci., 568: 252-261. Larsell, O. (1948) The development and subdivisions of the cerebellum of birds, J. Comp. Neurol., 89: 123-190. Legrand, C., Thomasset, M., Parkes, C.O., Clavel, M.C. and Rabie, A. (1983) Calcium-binding protein in the developing rat cerebellum. An immunocytochemical study, Cell Tissue Res., 233: 389402. Llinas, R. and Walton, K.D. (1989) Cerebellum. In G.M. Shepherd (Ed.), The Synaptic Organization of the Brain, 3rd edn., Oxford Univ. Press, Oxford, pp. 214-245. Marcum, J.M., Dedman, J.R. and Means, A.R. (1978) Control of microtubules assembly-disassembly by calcium-dependent regulated protein, Proc. Natl..&cad. Sci. USA, 75: 3771-3775. 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-D28k in cultured hippocampal neurons, Neuron, 6: 41-51. Mugnaini, E. (1969) Ultrastructural studies on the cerebellar histogenesis, lI. Maturation of nerve cell populations and establishment of synaptic connections in the cerebellar cortex of the chick. In R. Llinas (Ed.), Neurobiology of Cerebellar Evolution and Development, Am. Med. Assoc. Educ. Res. Fund, Chicago, IL, pp. 749-782. Okado, N., Yoshimoto, M. and Furber, S.E. (1987) Pathway formation and the terminal distribution pattern of the spinocerebellar projection in the chick embryo, Anat. Embryol., 176: 165-174.
Oppenheim, R.W. (1991) Cell death during development of the nervous system, Annu. Rev. Neurosci., 14: 453-501. Oppenheim, R.W., Prevette, D., Tytell, M. and Homma, S. (1990) Naturally occurring and induced neuronal death in the chick embryo in vivo requires protein and rna synthesis, Dev. Biol., 138: 104-113. Palay, S.L. and Chan-Palay, V. (1974) Cerebellar Cortex, SpringerVerlag, Berlin. Parmentier, M. (1990) The human calbindins: cDNA and gene cloning, Adv. Exp. Med. Biol., 255: 233-240. Parmentier, M., Ghysens, M., Rypens, F., Lawson, D.E.M., Pasteels, J.L. and Pochet, R. (1987) Calbindin in vertebrate classes: Immunohistochemical localization and Western blot analysis, Gen. Comp. Endocrinol., 65: 399-407. Pasteels, B., Rogers, J., Blachier, F. and Pochet, R. (1990) Calbindin and calretinin localization in retina from different species, Visual Neurosci,, 5: 1-16. Pochet, R., Pasteels, B., Seto-Ohshima, A., Bastianelli, E., Kitajima, S. and Van Eldik, L.J. (1991) Calmodulin and Calbindin localization in retina from six vertebrate species, J. Comp. Neurol., 314: 750-762. Ram6n y Cajal, S. (1911) Histologie du Syst~me Nerveux de l'Homme et des Vert6br6s, vol. 2, Instituto Ram6n y Cajal, Madrid. R6sibois, A. and Rogers, J.H. (1992) Calretinin in rat brain: an imrnunohistochemical study, Neuroscience, 46: 101-134. Rogers, J.H. (1987) Calretinin: a gene for a novel calcium-binding protein expressed principally by neurons, J. Cell Biol., 105: 13431353. Rogers, J.H. (1989a) Immunoreactivity for calretinin and other calcium-binding proteins in cerebellum, Neuroscience, 31: 711-721. Rogers, J.H. (1989b) Two calcium-binding proteins mark many chick sensory neurons, Neuroscience, 31: 697-709. Spencer, R., Charman, M., Emtage, J.S. and Lawson, D.E.M. (1976) Production and properties of vitamin-D-induced mRNA for chick calcium-binding protein, Eur. J. Biochem., 71: 399-409. Vacca, L.L., Abrahams, S.J. and Naftchi, N.E. (1980) A modified peroxidase-antiperoxidase procedure for improved localization of substance P in rat spinal cord, J. Histochem. Cytochem., 28: 297-304.
53
NSR 00648
Transient expression of calretinin during development of chick cerebellum Comparison with calbindin-D28k E. B a s t i a n e l l i a n d R. P o c h e t Laboratoire d'Histologie, Facult~ de M~decine, UniversitJ Libre de Bruxelles, Route de Lennik 808, B-1070 Brussels, Belgium
(Received 8 March 1993; accepted 3 April 1993)
Key words: Calcium binding proteins; Immunohistochemistry; Development; Avian; Brain; Purkinje cell Summary Calcium ions play a critical role in neural development. Insights into the ontogeny of Ca 2+ homeostasis were gained by investigating the developmental expression of two E-F hand calcium-binding proteins. Calretinin and calbindin were monitored through their immunoreactivity in the developing chick cerebellum (from E6 to E20). Calbindin was detected from El3 and in Purkinje cells only. Intensity of labelling increased with Purkinje cell development. Calretinin presented a transitory immunoreactivity between E11 and E20 in the internal granular cell layer. This cell layer contains ceils which will differentiate into Golgi and granular cells which are calretinin-negative in adult chick cerebellum. Calretinin immunoreactivity presented a peak (both in number of cells and in intensity) at El5 and fell dramatically after E20 while calbindin immunoreactivity was restricted to the Purkinje cells and increased with the development of these cells.
Introduction Changes in intracellular C a 2+ levels [Ca2+]i play a major role in the modulation of many key cellular processes, especially in the central nervous system. Regulation of cellular metabolism, cell division, cell mobility, protein synthesis, exocytosis are [Ca2+]i-dependent (Brostom et al., 1976; Marcum et al., 1978; Dedman et al., 1979). The different steps (calcium mobilization, mediator calcium binding and activation, cellular cascade activation by CaZ+-mediator) necessary for a calcium-coupled response involve that the calcium concentration is very precisely regulated (Heiz-
Correspondence to: Dr. R. Pochet, Laboratoire d'Histologie, Facult6 de M6decine, Universit6 Libre de Bruxelles, Route de Lennik 808, B-1070 Brussels, Belgium. Tel.: 32 2 5556374; Fax: 32 2 5556285. Abbreviations: CaBP, calbindin; Calret, calretinin; CN, cerebellar nuclei; EG, external granular layer; IG, internal granular layer; Ir, immunoreactivity; M, molecular layer; Md, medullary zone; P, Purkinje cell layer.
mann and Schaefer, 1990). Intracellular calcium binding proteins such as calbindin and calretinin may play a role in this [Ca2+]i control, although a precise physiological function, besides binding Ca 2÷, has not yet been demonstrated. Recently, it has been proposed that calbindin may act as a cytosolic calcium buffer protecting neurons against excitotoxic damage caused by calcium influx (Mattson et al., 1991). The highly conserved structure of calbindin and calretinin throughout evolution (Parmentier et al., 1987; Parmentier, 1990) suggests that those proteins should have fundamental functions. These calcium binding proteins might also serve to redistribute calcium within the neurons. But assuming that the neuroanatomical distribution of these proteins is highly selective (Jande et al., 1981; GarciaSegura et al., 1984; Rogers, 1987; Pochet et al., 1991; REsibois and Rogers, 1992), it appears that, at cell maturity, these proteins are probably not involved in general regulator mechanisms, but rather in more specific physiological functions shared by restricted neuronal cell population. They might act like calmodulin
54 in affecting directly Ca2+-dependent regulation of different proteins. Calbindin and calretinin appearance during development of the chick retina has recently been studied (Ellis et al., 1991) and seems to be linked with synaptogenesis. Ontogenesis of calbindin in cerebellum has been investigated to some extent and is limited to rat (Legrand et al., 1983; Enderlin et al., 1987) and mouse (Iacopino et al., 1990). Some authors think that calbindin could act as atrophic factor during neuronal development (Enderlin et al., 1987). Legrand et al. (1983) did not find calbindin before El7 whereas Enderlin et al. (1987) found it at El5. Interestingly, the latter authors have noticed that calbindin was transiently expressed (from El5 to E21) in the deep nuclei of the cerebellum (cerebellar "anlage") and in non neuronal cells. They also concluded that in rat, calbindin was expressed in neurons long before the beginning of synapse formation contrasting with the conclusion drawn by Ellis et al. (1991) studying the developing chick retina. Calbindin in mouse cerebellum was only studied at birth and after. The present study was undertaken in an attempt to obtain information about possible functions for calbindin and calretinin by examining their developmental profile using immunohistochemistry and comparing it with known events in the histogenesis of a well-characterized tissue: the cerebellum. The cerebellum offers the most distinctive cellular architecture in the brain (Ram6n y Cajal, 1911; Palay and Chan-Palay, 1974) with only a few well-known cell types. Chick embryos of known ages are easily available and the intrinsic organization of the avian cerebellum cortex is comparable to that of mammals.
Materials and methods
Embryos of chicks (Gallus gallus) from 6, 8, 11, 13, 15, 17, and 20 days of incubation (denoted E6, E8, E l l , etc.) were used. Six embryos of each age were dissected and immersed in Helly's fixative (68 mM potassium bichromate, 70 mM sodium sulfate, 180 mM HgC12, and 4% paraformaldehyde). Fixed embryos were routinely dehydrated, embedded in paraffin, and sectioned at 5-10/zm.
Antibodies Two rabbit polyclonal antisera against calretinin were used at dilutions of 1:1000 or 1:2000. The first antiserum (kindly donated by J. Rogers, Cambridge, UK) was raised against /3-galactosidase-calretinin fusion protein which contained either two or four do-
TABLE
1
DISTRIBUTION
OF
EG
Calret-lr
M
E 8
.
.
E11
-
-
El3
-
-
IN DEVELOPING
P .
IG
-
CN
+
. . . .
+-
+
El5
-
-
-
+
-
-
-
+
E20
-
+
-
+
indicate
Md
.
El7
Symbols
CEREBELLUM
positive
l a y e r s ( + ), n e g a t i v e
-
+ +
-
+
layers(-
).
mains of the chick calretinin (Rogers, 1987, 1989b). Rabbit antiserum against the tw0-domain fusion protein was used unless otherwise stated. This antiserum was previously characterized and no cross-reactivity between calbindin and the antiserum could be detected (Pasteels et al., 1990). The second antiserum (#7696) was obtained commercially (SWant, Bellinzona, Switzerland) and made from human recombinant calretinin. Controls included preincubation of both antibodies with 1 /xg/ml of human recombinant calretinin (SWant, Bellinzona, Switzerland). Rabbit antiserum raised against chick duodenal calbindin-D28k was prepared as described by Spencer et al. (1976) and routinely used at a dilution of 1 : 6000.
Immunohistochemical staining Routinely dewaxed and hydrated sections were processed for immunohistochemistry according to a peroxidase-antiperoxidase (PAP) procedure modified from Vacca et al. (1980). Serum dilutions were made up in Coon's veronal-buffered saline (CVBS: 10 mM 5,5-diethylbarbituric acid, sodium salt (sodium veronal), pH 7.2, 150 mM NaCl) supplemented with 1% (v/v) normal sheep serum. The immunostaining sequence comprised the following steps: (1) rinse in CVBS; (2) preincubation in 5% (v/v) normal sheep serum; (3) incubation with antibodies for 48 h at 4°C in a moist chamber;
TABLE
2
DISTRIBUTION
EG
OF
CaBP-Ir
IN DEVELOPING
M
P
E 8
m
-
-
Ell
-
-
-
El3
-
-
+
El5
-
-
+
El7
-
-
+
E20
-
-
+
Symbols
indicate
positive
IG
CEREBELLUM
Md
CN
m
l a y e r s ( + ), n e g a t i v e
layers(-
).
55 TABLE 3 T I M E O F A P P E A R A N C E O F D I F F E R E N T I A T E D C E L L * A N D O F T H E I R L A B E L L I N G IN C H I C K C E R E B E L L U M Purkinje:
-
-
-
Granule cell:
-
-
-
Stellate cell:
Differentiation takes place between E l 0 and E l 2 First synapses on Purkinje cells appear at E12 Labelling: since E13 with CaBP Differentiation takes place between E l l and E13 First synapses of mossy fibers on granule cells are scarce until E15 Transient labelling between E13 and E20 with Calret
Differentiation takes place after E15 First synapses on stellate cells appear at E19 - Labelling with Calret at E20 (scarce) -
-
Basket cell:
- Differentiation takes place at E15-EI6 and is not completed before hatching - The synaptic contacts by parallel fibers on basket cells begin at E19 - Labelling with Calret at E20 (scarce)
Golgi cell:
Differentiation takes place at E16 - The synaptic connections between mossy fibers on Golgi cells are a very late processus and begin at E19 Labelling with Calret at E17 -
-
*
According to Mugnaini (1969).
(4) incubation with sheep anti-rabbit immunoglobulin G (IgG) serum (1 : 100 dilution) (Laboratoire d'Hormonologie et Immunologie, Marloie, Belgium); (5) incuba-
tion with soluble rabbit PAP complex 1:300 dilution (DAKO, Denmark). Between each step, sections were thoroughly rinsed for 10 min in CVBS. After the last
Fig. 1. Immunohistochemistry of E l 3 chick cerebellum using A: anti-calretinin antibody (1 : 2000); B: anti-calbindin antibody (1 : 6000) in sagittal section. Bar = 40/zm.
56 rinse, staining was performed in citrate phosphate buffer, p H 6.2, containing 16 mM 3,3'-diaminobenzidine-HC1 (DAB; Sigma, St. Louis, MO) and 0.01% H 2 0 2. Each of these steps was performed at room temperature, except step 3 which was performed at 4°C. Controls for specificity of staining included use of normal rabbit serum and immune serum preincubated with either 1 / z g / m l human recombinant calretinin or calbindin purified by affinity chromatography (Pasteels et al., 1990). Under these conditions, no labelling was observed (Fig. 4A,B).
Results
The structural differentiation of lobules in developing chick cerebellum was similar to the findings of previous studies (Larsell, 1948; Mugnaini, 1969; Okado et al., 1978). We therefore adopted the same classification as in LarseU (1948). The distribution of calretinin-Ir and calbindin-Ir cerebellar layers during development is summarized in
Tables 1 and 2. The time of appearance of differentiated cerebellar cells and their immunoreactivity is shown in Table 3.
Days 06-08 (E6-E8) No immunoreactivity for calbindin and calretinin could be detected on sections.
Day 11 (Ell) The cerebellum is thick and has lost its plate-like appearance. The cerebellar nuclei are present in the central region of the cerebellum. The external granular layer is now well formed. The molecular layer is located just beneath the external granular layer. The Purkinje cell layer, internal granular cell layer, and the medullary zone which corresponds to the adult white matter are recognizable. Calretinin. Neurons from the internal granular cell layer (IG) were weakly but definitely positive. Scattered neurons from the cerebellar nuclei were also positive (not shown). Calbindin. No labelling could be detected.
Fig. 2. Immunohistochemistryof chick cerebellum (lobe IV) using anti-calretinin antibody (1 : 2000). Sagittal section. A: El5. B: El7. C: E20. Arrowheads indicate granule cells, arrow indicates basket cell. Bar = 40 izm.
57
Days 13-20 All the lobules can be clearly recognized. The general appearance of the cerebellar cortex is similar to the cerebeUar cortex of the posthatching chick. But several structures characteristic of an immature cerebellum are still present. The outer granular layer remains thick up to day 17. At day 13, the Purkinje cell layer is still pluristratified and the molecular layer thin. These embryonic characteristics disappear progressively until day 17 and reach a mature architecture at day 20.
Day 13 (E13) (Fig. 1A,B) Calretinin. Some cells in IG layer are strongly positive (Fig. 1A) and in the CN many neurons are also positive (not shown). Calbindin. Large cells are labelled in P and IG layers. The strongest labelling is seen in lobes I - I V (Fig. 1B).
Day 15 (El5) (Figs. 2A, 3A) Calretinin. There is a strong positive reaction in the IG cell layer (Fig. 2A). These cells could either be granules or Golgi cells (see Discussion). The strongest labelling was seen in lobes VI, VII, and VIII. CN neurons were strongly positive (not shown). Calbindin. The double layer of Purkinje cells is strongly positive (Fig. 3A).
Day 17 (E17) (Figs. 2B and 3B) Calretinin. The positive reaction in the IG layer is still visible but fewer cells are labelled as compared to E15 (Fig. 2B). The labelling is also weaker. These cells could be either granules or Golgi cells (see Discussion). The strongest labelling is seen in lobes VI, VII, VIII. CN neurons are still strongly positive (not shown). Calbindin. Branching of Purkinje cells in the molecular layer is becoming apparent. No axons are labelled yet with the exception of a few fibers in the medulla (Fig. 3B).
Day 20 (E20) (Figs. 2C and 3C) Calretinin. Labelling is confined to only a few cells of the IG and M layer. In the latter, only a few stellate cells are positive. Few basket cells are also labelled in the P layer (Fig. 2C). In the IG layer, positive Golgi cells are recognizable. The CN is still labelled. Calbindin. Purkinje cells, including their dendritic and axonal branching, are strongly labelled (Fig. 3C). Nearly all P cells are positive. In lobes I and X, labelling is less intense (not shown).
Discussion
Neuronal differentiation not only entails the acquisition of various molecular and cellular characteristics but also involves the loss of characteristics that appear only during certain stages of development (Oppenheim, 1991). Moreover, calcium ions have been implicated in the regulation of neuronal development (Kater et al., 1990). Therefore it was decided to investigate the developmental expression of two E-F hand calcium binding proteins, calretinin and calbindin. Their appearance was monitored through their immunoreactivity in the developing chick cerebellum. In chick cerebellum, Purkinje cells differentiate between El0 and El2. They present at this stage an apical and a basal process. The first true synaptic connections on Purkinje cells are recognizable only after El2 (Mugnaini, 1969; Bertossi et al., 1986), coinciding with the appearance of CaBP-Ir. At this stage large CaBP D-28k-positive cells seen in the IG layer could be migrating Purkinje cells. During Purkinje cell development, accompanied by dendritic growth and increase in synapse number, labelling increases regularly. It might therefore be speculated that calbindin function is rather specific, and may be associated with the process of dendritic synaptic formation and stabilization. Two studies (Iacopino et al., 1990; Ellis et al., 1991) are in favor of this hypothesis. In the first, Ellis et al. (1991) showed that in chick developing retina appearance of calbindin coincides, at least approximately, with the establishment of functional synapses; in the second, Iacopino et al. (1990) found that the peak of calbindin gene expression is correlated with cessation of migration and peak synapse formation of Purkinje cells in mouse cerebellum. Synaptogenesis may result in a more complex regulation of Ca 2÷ fluxes, perhaps requiring increased intraneuronal levels of calbindin. Besides many arguments for a buffering role of calcium in neurons (Baimbridge and Miller, 1984; Johansen et al., 1990; Freund et al., 1992), no specific function for calbindin has yet been demonstrated, although it represents one of the most abundant cerebellar proteins. Why calbindin should be restricted to Purkinje cells in the cerebellum might be explained if we take into account that: (1) a typical Purkinje cell may form as many as 200000 synapses with afferent fibers, more than any other cell in the CNS (Llinas and Walton, 1989); the majority of these fibers come from granular cells using glutamate as neurotransmitter which increases intracellular calcium; (2) calbindin may play a neuroprotective role important
OC)
59
Fig. 4. Comparison between immunolabeiling using (A) anti-calretinin antibody (1 : 2000) and (B) anti-calretinin antibody (1 : 2000) pre-incubated with calretinin (1/zg/ml) in El5 chick cerebellum in sagittal section.
<.Fig.
3. Immunohistochemistry of chick cerebellum using anti-calbindin antibody (1 : 6000). Sagittal section. A: El5. B: El7. C: E20. Bar = 20/.Lm.
60 for its survival (Mattson et al., 1991) or atrophic role in synapse formation. Calretinin-Ir was first detected at E l l . Calretininpositive cells are located in the IG layer. This labelling increased from E l l to El3. At El5 the peak of calretinin immunoreactivity is reached in the IG layer containing granule and Golgi cells. The positive cells are small and might correspond to granule cells. Indeed, granule cells are fully differentiated at El3 whereas Golgi cells are larger and begin to differentiate at El6. From E15 to El7 the number of calretinin-Ir cells decreases progressively followed by a sharp decline until E20. Two hypotheses might explain this decrease in calretinin-positive cells: (1) a dilution effect due to an increase in the cell number of the IG layer, and (2) a loss of positive cells. The first hypothesis is unlikely because in chick cerebellum all IG cells have terminated both their mitosis and their migration since El5 (Fujita, 1969). In contrast, the second hypothesis is validated by the known decrease of neurons along development in the brain (Oppenheim, 1991) and more specifically in the cerebellum (Janowsky and Finlay, 1983) reinforcing our statement that calretinin was transiently expressed between E l l and E20. From El4 the organization of mossy fibers which connect granule cells changes considerably. Density of mossy fibers in the IG decreases by El6 until hatching. Loss of synaptic connection can produce cellular death by absence of stimulation which induces the blockage of cellular synthesis. But neuronal death could also be an active process in which, following the loss of trophic support, new gene expression is initiated, which acts to trigger a cascade of specific biosynthetic events that actively induce degeneration and death (Oppenheim et al., 1990). Calretinin might be expressed by such events. Its immunoreactivity in granular cells may be linked to this new gene expression inducing cellular death. At E20 calretinin-IR is almost restricted to stellate cells, basket cells and cerebellar nuclei; immunoreactivity in granular cells has almost completely disappeared. These results are in agreement with the distribution of calretinin in the adult chick cerebellum (Rogers, 1989a). It is becoming more and more evident that neurodegeneration is closely linked to a calcium-dependent mechanism (Choi, 1988). Rises in Ca 2+ can lead to cell death (Clarke, 1990; Kater et al., 1990). Pyknosis occurs by activation of Ca 2+- and MgZ+-dependent endonucleases (Oppenheim, 1991). Contrary to this, excessively low levels of free intracellular Ca 2+ concentration may also favor cell death (Oppenheim et al., 1990). Could calretinin expression be linked to this type of cellular phenomenon? It also remains to be
determined whether the induction and repression of calretinin production are influenced by epigenetic variables such as neuronal activity and neurotrophic agents or whether it stems from an immutable genetic program. In any case, the acquisition and loss of a calcium-binding protein by these neurons impart a developmental regulation that may alter their sensitivity to Ca 2+ signals.
Acknowledgments
We thank Professor Annette R6sibois for stimulating discussions and advice, L6on Surardt for excellent technical assistance and Paulette Miroir for typing the manuscript. This work was supported by FRSM (grant no. 3.4517.92).
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