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Identification of Cajal-Retzius cells in immature rodent cerebral cortex: A combined Golgi-EM study
Neuroscience Letters, 27 (1981) 225 -229
225
Elsevier/North-Holland Scientific Publishers Ltd.
IDENTIFICATION OF CAJAL-RETZIUS CELLS IN IMMATURE RODENT CEREBRAL CORTEX: A COMBINED G O L G I - E M STUDY§
NORBERT KONIG, JEAN-PIERRE HORNUNG*,** and HENDRIK VAN DER LOOS*
Laboratory of Neurophysiology, University of Montpellier 11, 34060 Montpellier Cedex (France) and
*Institute of Anatomy, University of Lausanne, Rue du Bugnon 9, 1011 Lausanne (Switzerland) (Received September 7th, 1981; Revised version received and accepted October 16th, 1981)
In an attempt at establishing the ultrastructural characteristics of Cajal-Retzius cells defined classically by Golgi methods, neonatal mouse dorso-occipital cortex was prepared for a combined Golgi-EM investigation including 3-D computer reconstructions. All analyzed Cajal-Retzius cells had a well developed rough endoplasmic reticulum whose cisterns, stacked in parallel arrays, had a relatively dark content. Furthermore, they had axons and specialized contacts with other cells.
Cajal [1, 2] described in the first cortical layer of immature small mammals 'special cells" having, in Golgi-impregnated material, unique morphological features. Retzius [11 ] confirmed the existence of these cells and included in that class a cell type he had discovered earlier in human foetal cortex and considered to be glial in nature [I0]. The cells of this class are called Cajal-Retzius cells (CRs). Recently, several attempts at characterizing their ultrastructure have been made. There was no.general agreement about their identity (refs. 6, 8 and 12 vs. 14). The present study provides direct evidence concerning the ultrastructure of typical Golgi-impregnated CRs in neonatal mouse cerebral cortex. Dorso-occipital cortex of 8 Swiss albino mice (ICR, housebred; original stock obtained from the Institut for Zuchthygiene of the University of Zurich, 8057 Zurich, Switzerland) at postnatal days (PD) 2 or 5 (day of birth equals day 0) was prepared for combined Golgi-EM study [4]. Blocks were impregnated with dichromate, coated with blood [3] in order to avoid excessive accumulation of precipitate, and immersed in silver nitrate. Seven Golgi-impregnated cells (2 from PD 2; 5 from PD 5) most closely resembling the classical drawing published by Cajal [2] and Retzius [11] were selected from a large number of 100 #m thick coronally cut **Current address: Department of Neurobiology, Harvard Medical School, Boston, MA 02115, U.S.A. §Part of this work has been represented at the Fifth European Neuroscience Congress (September, 1981)
ISJ 0304-3940/81/0000-0000/$ 02.75 © Elsevier/North-Holland Scientific Publishers Ltd.
226
slices. The selected slices were embedded in plastic and processed for 3-D electron microscopy using serial 0.25 ~m sections as described previously [7]. Cell contours of electron micrographs were digitized with a Tektronix 4956 graphics tablet, and 3D reconstructions (orthographic projection) were made with a Tektronix 4051 (32K) desk-computer. The most distinctive feature of the analyzed CRs was a characteristic electrondense rough endoplasmic reticulum (RER) whose cisterns were arranged in parallel arrays and concentrated mainly at the periphery of the thick principal dendrite (Figs. lc and 2b, c). This finding confirms preliminary electron microscopic observations of conventionally Golgi-impregnated cells of rat temporal cortex (KOnig, unpublished). It also supports the conclusion of Raedler and Sievers [12] and K0nig and co-workers [6, 8], that cells with this type of RER indeed are CRs. This conclusion had been contested by Wolff and Rickmann [14] who called such cells 'quiescent astroblasts'. This latter interpretation is not supported by a recent immu-
Fig. 1. Two-day old mouse. This and the following images are from the first layer of dorso-occipital cortex, a: typical Golgi-impregnated Cajal-Retzius cell (CR). One of the ascending branchlets leaving the principal dendrite (arrow) is shown in b. x 550. b: computer-reconstruction of a part of the principal dendrite and of the initial part of an ascending branchlet, based upon electron micrographs of 5 adjacent serial 0.25 ~m sections. The 3-D image has been rotated (30 °) until the viewing axis was normal to the horizontal bifurcated process (not visible by light microscopy). The calibration cross represents two perpendicular 5 ~tm bars, rotated in the same way as the reconstruction, c: electron micrograph of a 0.25 t~m section through the body of the same cell. × 5400.
227
Fig. 2. Five-day old mouse, a: Golgi impregnated CR. x 550. b: electron micrograph of a 0.25 #m section through the body of the same cell. x 5400. c: electron micrograph of another 0.25 #m section through the body of the same cell, showing part of its rough endoplasmic reticulum and Golgi-complex, and one of the nucleoli, x 22,000.
n o h i s t o l o g i c a l s t u d y [9] i n d i c a t i n g the n e u r o n a l n a t u r e o f CRs. Besides, all CRs a n a l y z e d in the p r e s e n t s t u d y h a d a process that on the basis o f its G o l g i - a p p e a r a n c e was j u d g e d to be an a x o n (see, f o r e x a m p l e , Figs. 1a a n d 2a). T h e c o n t i n u i t y o f these processes with the i m p r e g n a t e d s o m a t a was c o n f i r m e d by inspection o f serial 0.25 /~m sections in the electron m i c r o s c o p e . A l s o , in t h o s e cases in which the s o m a o f a C R was l o c a t e d n e a r the pia, electron m i c r o s c o p y s h o w e d a glial sheet s e p a r a t i n g it f r o m the b a s e m e n t m e m b r a n e . A n o t h e r c h a r a c t e r i s t i c f e a t u r e o f CRs were the a s c e n d i n g b r a n c h l e t s going o f f r o u g h l y at right angles f r o m the h o r i z o n t a l p a r t o f the p r i n c i p a l d e n d r i t e . In s o m e
228
cases, these branchlets had tiny bifurcated lateral processes visible only in the electron microscope (Fig. lb). Specialized junctions were frequently encountered upon dendrites and somata of CRs. They had marked symmetrical or asymmetrical densities, but few - if any - were associated with synaptic vesicles (Fig. 4). Nonspecialized contact areas between CR dendrites and dendrites of other CRs (Fig. 3) or different cell types were very extensive; these areas may well have an ephaptic function (cf. ref. 13). We emphasize that our observations pertain to a limited period in cortical development, and have been made in two species of rodents. It remains to be clarified whether the same or similar ultrastructural characteristics of CRs can be found at other developmental stages and in other species. While we may conclude that CRs have a characteristic RER, we cannot yet state that any cell with that RER
Fig. 3. Computer reconstruction based upon electron micrographs of 12 adjacent 0.25 ~m sections showing, when viewed with a stereo-loupe, the close relationship between the main dendrite of the CR represented in Fig. 2a (solid lines) and 2 dendrites of non-impregnated CRs (dotted lines). Tilt angle at left, 40°; at right, 30 ° . Calibration as in Fig. lb. Fig. 4. Stereographs of an asymmetric specialized contact upon the main dendrite of a non-impregnated CR. Tilt angle between the images is 6 °. × 53,000.
229
is a classical Cajal-Retzius cell. Only 3-D reconstruction of a number of those cells in conventionally prepared EM material could answer this point, important for any analysis of fate, function and connectivity of these interesting elements of cerebral cortex. This work was supported by the Swiss National Science Foundation (3.516), CNRS (ERA 187) and DGRST (DN 79.7. 1076). Collaboration between our laboratories was greatly facilitated by a Twinning Grant from the European Training Programme in Brain and Behaviour Research. We thank Mr. Pierre Sibleyras and Mr. Bernard Arnaud for excellent photographical work. 1 Cajal, S. Ram6n, y, Sobre la existencia de c~lulas nerviosas especiales en la primera capa de las circunvoluciones cerebrales, Gaceta m6dica Catalana, 15 de diciembre (1890) 625-628. 2 Cajal, S. Ram6n y, Nuevo concepto de la histologia de los centros nerviosos, Rev. Ciencias Mbdicas de Cataluna, 18 (1893) 5-68. 3 Cajal, S. Ram6n, y and De Castro, F., Elementos de T6cnica Micrografia del Systema Nervioso, Tipografia artistica, Madrid, 1933, p. 119. 4 Fair6n, A., Peters, A. and Saldanha, J., A new procedure for examining Golgi impregnated neurons by light and electron microscopy, J. Neurocytol., 6 (1977) 311-337. 5 K6nig, N., Hornung, J.P., Schachner, M. and Van der Loos, H., Identification of 'special cells' in immature rodent cerebral cortex: a combined Golgi-EM study and immunofluorescence observations, Neurosci. Lett.,.Suppl. 7 (1981) $35. 6 KOnig, N., Valat, J. Fulcrand, J. and Marty R., The time of origin of Cajal-Retzius cells in the rat temporal cortex. An autoradiographic study, Neurosci. Lett., 4 (1977) 21-26. 7 KOnig N. and Van der Loos, H., Two useful techniques in three-dimensional electron microscopy: quarter-micron serial sectioning and stereoscopy, J. Neurosci. Meth., 2 (1980) 79-86. 8 KOnig, N. and Marty, R., Early neurogenesis and synaptogenesis in cerebral cortex, Bibl. anat., 19 (1981) 152-160. 9 K6nig, N. and Schachner, M., Neuronal and glial cells in the superficial layers of early postnatal mouse neocortex" immunofluorescence observations, Neurosci. Lett., 26 (1981) 227-231. 10 Retzius, G., Ueber den Bau der Oberfleichenschicht der Grosshirnrinde beim Menschen und bei den Seiugethieren, Vortr. Biol. Vereins, 3 (1891) 90-102. 11 Retzius, G., Die Cajal'schen Zellen der Grosshirnrinde beim Menschen und bei S~iugethieren, Biol. Untersuch., 5 (1893) 1-8. 12 Raedler, A. and Sievers, J., Light and electron microscopical studies on specific cells of the marginal zone in the developing rat cerebral cortex, Anat. Embryol., 149 (1976) 173-181. 13 Van der Loos, H., Anatomic and physiologic considerations. In R.E. Cood (Ed.), The Biologic Basis of Pediatric Practice, Sect. 16, The Regulating Systems, McGraw-Hill, New York, 1968, pp. 1177-1200. 14 Wolff, J.R. and Rickmann, M., Cytological characteristics of early stages of glial differentiation in the neocortex, Folia morph., 25 (1977) 235-237.
225
Elsevier/North-Holland Scientific Publishers Ltd.
IDENTIFICATION OF CAJAL-RETZIUS CELLS IN IMMATURE RODENT CEREBRAL CORTEX: A COMBINED G O L G I - E M STUDY§
NORBERT KONIG, JEAN-PIERRE HORNUNG*,** and HENDRIK VAN DER LOOS*
Laboratory of Neurophysiology, University of Montpellier 11, 34060 Montpellier Cedex (France) and
*Institute of Anatomy, University of Lausanne, Rue du Bugnon 9, 1011 Lausanne (Switzerland) (Received September 7th, 1981; Revised version received and accepted October 16th, 1981)
In an attempt at establishing the ultrastructural characteristics of Cajal-Retzius cells defined classically by Golgi methods, neonatal mouse dorso-occipital cortex was prepared for a combined Golgi-EM investigation including 3-D computer reconstructions. All analyzed Cajal-Retzius cells had a well developed rough endoplasmic reticulum whose cisterns, stacked in parallel arrays, had a relatively dark content. Furthermore, they had axons and specialized contacts with other cells.
Cajal [1, 2] described in the first cortical layer of immature small mammals 'special cells" having, in Golgi-impregnated material, unique morphological features. Retzius [11 ] confirmed the existence of these cells and included in that class a cell type he had discovered earlier in human foetal cortex and considered to be glial in nature [I0]. The cells of this class are called Cajal-Retzius cells (CRs). Recently, several attempts at characterizing their ultrastructure have been made. There was no.general agreement about their identity (refs. 6, 8 and 12 vs. 14). The present study provides direct evidence concerning the ultrastructure of typical Golgi-impregnated CRs in neonatal mouse cerebral cortex. Dorso-occipital cortex of 8 Swiss albino mice (ICR, housebred; original stock obtained from the Institut for Zuchthygiene of the University of Zurich, 8057 Zurich, Switzerland) at postnatal days (PD) 2 or 5 (day of birth equals day 0) was prepared for combined Golgi-EM study [4]. Blocks were impregnated with dichromate, coated with blood [3] in order to avoid excessive accumulation of precipitate, and immersed in silver nitrate. Seven Golgi-impregnated cells (2 from PD 2; 5 from PD 5) most closely resembling the classical drawing published by Cajal [2] and Retzius [11] were selected from a large number of 100 #m thick coronally cut **Current address: Department of Neurobiology, Harvard Medical School, Boston, MA 02115, U.S.A. §Part of this work has been represented at the Fifth European Neuroscience Congress (September, 1981)
ISJ 0304-3940/81/0000-0000/$ 02.75 © Elsevier/North-Holland Scientific Publishers Ltd.
226
slices. The selected slices were embedded in plastic and processed for 3-D electron microscopy using serial 0.25 ~m sections as described previously [7]. Cell contours of electron micrographs were digitized with a Tektronix 4956 graphics tablet, and 3D reconstructions (orthographic projection) were made with a Tektronix 4051 (32K) desk-computer. The most distinctive feature of the analyzed CRs was a characteristic electrondense rough endoplasmic reticulum (RER) whose cisterns were arranged in parallel arrays and concentrated mainly at the periphery of the thick principal dendrite (Figs. lc and 2b, c). This finding confirms preliminary electron microscopic observations of conventionally Golgi-impregnated cells of rat temporal cortex (KOnig, unpublished). It also supports the conclusion of Raedler and Sievers [12] and K0nig and co-workers [6, 8], that cells with this type of RER indeed are CRs. This conclusion had been contested by Wolff and Rickmann [14] who called such cells 'quiescent astroblasts'. This latter interpretation is not supported by a recent immu-
Fig. 1. Two-day old mouse. This and the following images are from the first layer of dorso-occipital cortex, a: typical Golgi-impregnated Cajal-Retzius cell (CR). One of the ascending branchlets leaving the principal dendrite (arrow) is shown in b. x 550. b: computer-reconstruction of a part of the principal dendrite and of the initial part of an ascending branchlet, based upon electron micrographs of 5 adjacent serial 0.25 ~m sections. The 3-D image has been rotated (30 °) until the viewing axis was normal to the horizontal bifurcated process (not visible by light microscopy). The calibration cross represents two perpendicular 5 ~tm bars, rotated in the same way as the reconstruction, c: electron micrograph of a 0.25 t~m section through the body of the same cell. × 5400.
227
Fig. 2. Five-day old mouse, a: Golgi impregnated CR. x 550. b: electron micrograph of a 0.25 #m section through the body of the same cell. x 5400. c: electron micrograph of another 0.25 #m section through the body of the same cell, showing part of its rough endoplasmic reticulum and Golgi-complex, and one of the nucleoli, x 22,000.
n o h i s t o l o g i c a l s t u d y [9] i n d i c a t i n g the n e u r o n a l n a t u r e o f CRs. Besides, all CRs a n a l y z e d in the p r e s e n t s t u d y h a d a process that on the basis o f its G o l g i - a p p e a r a n c e was j u d g e d to be an a x o n (see, f o r e x a m p l e , Figs. 1a a n d 2a). T h e c o n t i n u i t y o f these processes with the i m p r e g n a t e d s o m a t a was c o n f i r m e d by inspection o f serial 0.25 /~m sections in the electron m i c r o s c o p e . A l s o , in t h o s e cases in which the s o m a o f a C R was l o c a t e d n e a r the pia, electron m i c r o s c o p y s h o w e d a glial sheet s e p a r a t i n g it f r o m the b a s e m e n t m e m b r a n e . A n o t h e r c h a r a c t e r i s t i c f e a t u r e o f CRs were the a s c e n d i n g b r a n c h l e t s going o f f r o u g h l y at right angles f r o m the h o r i z o n t a l p a r t o f the p r i n c i p a l d e n d r i t e . In s o m e
228
cases, these branchlets had tiny bifurcated lateral processes visible only in the electron microscope (Fig. lb). Specialized junctions were frequently encountered upon dendrites and somata of CRs. They had marked symmetrical or asymmetrical densities, but few - if any - were associated with synaptic vesicles (Fig. 4). Nonspecialized contact areas between CR dendrites and dendrites of other CRs (Fig. 3) or different cell types were very extensive; these areas may well have an ephaptic function (cf. ref. 13). We emphasize that our observations pertain to a limited period in cortical development, and have been made in two species of rodents. It remains to be clarified whether the same or similar ultrastructural characteristics of CRs can be found at other developmental stages and in other species. While we may conclude that CRs have a characteristic RER, we cannot yet state that any cell with that RER
Fig. 3. Computer reconstruction based upon electron micrographs of 12 adjacent 0.25 ~m sections showing, when viewed with a stereo-loupe, the close relationship between the main dendrite of the CR represented in Fig. 2a (solid lines) and 2 dendrites of non-impregnated CRs (dotted lines). Tilt angle at left, 40°; at right, 30 ° . Calibration as in Fig. lb. Fig. 4. Stereographs of an asymmetric specialized contact upon the main dendrite of a non-impregnated CR. Tilt angle between the images is 6 °. × 53,000.
229
is a classical Cajal-Retzius cell. Only 3-D reconstruction of a number of those cells in conventionally prepared EM material could answer this point, important for any analysis of fate, function and connectivity of these interesting elements of cerebral cortex. This work was supported by the Swiss National Science Foundation (3.516), CNRS (ERA 187) and DGRST (DN 79.7. 1076). Collaboration between our laboratories was greatly facilitated by a Twinning Grant from the European Training Programme in Brain and Behaviour Research. We thank Mr. Pierre Sibleyras and Mr. Bernard Arnaud for excellent photographical work. 1 Cajal, S. Ram6n, y, Sobre la existencia de c~lulas nerviosas especiales en la primera capa de las circunvoluciones cerebrales, Gaceta m6dica Catalana, 15 de diciembre (1890) 625-628. 2 Cajal, S. Ram6n y, Nuevo concepto de la histologia de los centros nerviosos, Rev. Ciencias Mbdicas de Cataluna, 18 (1893) 5-68. 3 Cajal, S. Ram6n, y and De Castro, F., Elementos de T6cnica Micrografia del Systema Nervioso, Tipografia artistica, Madrid, 1933, p. 119. 4 Fair6n, A., Peters, A. and Saldanha, J., A new procedure for examining Golgi impregnated neurons by light and electron microscopy, J. Neurocytol., 6 (1977) 311-337. 5 K6nig, N., Hornung, J.P., Schachner, M. and Van der Loos, H., Identification of 'special cells' in immature rodent cerebral cortex: a combined Golgi-EM study and immunofluorescence observations, Neurosci. Lett.,.Suppl. 7 (1981) $35. 6 KOnig, N., Valat, J. Fulcrand, J. and Marty R., The time of origin of Cajal-Retzius cells in the rat temporal cortex. An autoradiographic study, Neurosci. Lett., 4 (1977) 21-26. 7 KOnig N. and Van der Loos, H., Two useful techniques in three-dimensional electron microscopy: quarter-micron serial sectioning and stereoscopy, J. Neurosci. Meth., 2 (1980) 79-86. 8 KOnig, N. and Marty, R., Early neurogenesis and synaptogenesis in cerebral cortex, Bibl. anat., 19 (1981) 152-160. 9 K6nig, N. and Schachner, M., Neuronal and glial cells in the superficial layers of early postnatal mouse neocortex" immunofluorescence observations, Neurosci. Lett., 26 (1981) 227-231. 10 Retzius, G., Ueber den Bau der Oberfleichenschicht der Grosshirnrinde beim Menschen und bei den Seiugethieren, Vortr. Biol. Vereins, 3 (1891) 90-102. 11 Retzius, G., Die Cajal'schen Zellen der Grosshirnrinde beim Menschen und bei S~iugethieren, Biol. Untersuch., 5 (1893) 1-8. 12 Raedler, A. and Sievers, J., Light and electron microscopical studies on specific cells of the marginal zone in the developing rat cerebral cortex, Anat. Embryol., 149 (1976) 173-181. 13 Van der Loos, H., Anatomic and physiologic considerations. In R.E. Cood (Ed.), The Biologic Basis of Pediatric Practice, Sect. 16, The Regulating Systems, McGraw-Hill, New York, 1968, pp. 1177-1200. 14 Wolff, J.R. and Rickmann, M., Cytological characteristics of early stages of glial differentiation in the neocortex, Folia morph., 25 (1977) 235-237.