- Email: [email protected]
Dye coupling in Purkinje cells of organotypic slice cultures
Developmental Brain Research 160 (2005) 101 – 105 www.elsevier.com/locate/devbrainres
Short Communication
Dye coupling in Purkinje cells of organotypic slice cultures Karl Meller, Kerstin Krah, Carsten Theiss* Medizinische Fakulta¨t, Institut fu¨r Anatomie, Abteilung fu¨r Cytologie, Ruhr-Universita¨t Bochum, Universita¨tsstraße 150, D-44780 Bochum, Germany Accepted 4 August 2005 Available online 16 September 2005
Abstract Cerebellar slice cultures of newborn rats showed poorly developed dendritic arborization of Purkinje cells, whereas cultures of 10-day-old rats revealed prominent dendritic branching. Gap junctional intercellular communication between Purkinje cells, investigated as dye transfer of microinjected neurobiotin, occurred through dendro-dendritic contacts, with decreased dye spreading in old cell cultures. These results indicate a possible correlation of gap junctional intercellular communication and the development of Purkinje cells. D 2005 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Formation and specificity of synapses Keywords: Cerebellar slice culture; Dye spreading; Gap junction; Microinjection; Purkinje cell
Gap junctional intercellular communication (GJIC) between neurons in various regions is a feature of the mammalian brain. Gap junctions are channels between adjacent neighboring cells formed by two opposing hexamers—the connexins (cx) together termed as one connexon. One aspect of the mammalian brain is the cell type specific but overlapping expression pattern of connexins. For instance, neurons are known to express predominantly cx36, cx32 and cx26, whereas cx31, cx43, cx37 and cx45 are expressed in non-neuronal cells, e.g., in glial cells [7,9]. Recently, in the developing mice brain, cx45 was detected in cerebellar basket and stellate cells [22], whereas in the chick cerebellum, cx39 was detected in neurons of the granular layer [5,23]. The gap junctional channels contribute to the structural basis for the direct transfer of metabolites and are therefore considered as one potential type of cellular communication. Such a flux on information is described to carry various functions, e.g., the control of cellular proliferation and diffeAbbreviations: cx, connexin; div, days in vitro; GJIC, gap junctional intercellular communication * Corresponding author. Fax: +49 234 3214476. E-mail address: [email protected] (C. Theiss). 0165-3806/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2005.08.007
rentiation [8]. Gap junctions are also discussed to be involved in the migration of neuronal precursor cells as well as in the formation of chemical synapses during the juvenile development. In the neocortex, they occur predominantly during the postnatal development, whereas in the adult phase, their number is diminished [13]. Besides this, in the adult brain, GJIC between neurons seems to be essential to generate synchronized activity of neuronal networks [18, 21,34]. Recently, another class of molecules called pannexins has been identified, which also form electrical junctions between neuronal cells in the mammalian brain [4]. A technical approach to study intercellular communication is the microinjection of tracer molecules into a cell. Low weight tracer such as the fluorescent Lucifer Yellow [27,36] or neurobiotin [35] is spread through gap junctions into adjacent cells. Using neurobiotin, it was shown that GJIC in the mammalian retina is realized by interneurons of the inner layer [35] as well as among ganglion cells [26,37] and amacrine cells [37]. In the neocortex, Peinado et al. [24] confirmed an extensive dye coupling among the pyramidal neurons forming clusters of up to 80 cells, and in the cerebellum dye coupling between molecular layer inhibitory interneurons was demonstrated subsequent injection of biocytin [21]. Besides this, dendro-dendritic and somato-
102
K. Meller et al. / Developmental Brain Research 160 (2005) 101 – 105
dendritic gap junctions in the rat cerebellar cortex have been reported by Sotelo and Triller [29]. Among the various kinds of tissues and cells, scanty information about the formation of gap junctions or pannexins in developing cerebellar Purkinje cells has been reported. Adopting the rollertube cultures of cerebellar slices, we investigated GJIC in Purkinje cells of the cerebellar cortex of different age. Slice cultures of rat cerebellum were obtained from newborn rat pups (P0) and at postnatal day 10 (P10) and incubated according to the rollertube technique [11,17]. Tracing studies were performed using 8% neurobiotin. After 11 up to 15 days in vitro (div), microinjection of tracer substances into single Purkinje cells was carried out as described elsewhere [33]. Treated tissues were post-incubated for 1 h to allow a sufficient spread of the intracellular dye. Specimens were fixed with 4% paraformaldehyde supplemented with 0.2% picric acid at 4 -C, and neurobiotin was visualized with an avidin –biotin enzyme complex (Vector Laboratories, USA) and diaminobenzidine. To intensify the reaction product, incubation in 5% cobalt chloride was preceded. Used antibodies were mouse monoclonal IgG (mAb) to calbindin (C-8666, 1:100, Sigma, Germany), rabbit polyclonal IgG to Cx36 (36 –4600, 1:100, Zymed, USA) and secondary goat anti-mouse IgG (Alexa 546 conjugated, 1:250, 11018, Molecular Probes, Germany) or goat anti-rabbit IgG (TRITC conjugated, 1.200, T-5268, Sigma). To cover the whole extension of the Purkinje cell dendrites, optical sections were collected in 2-Am steps by confocal laser scanning microscopy (LSM 510, Zeiss Meta, Germany), covering the thickness of the cell prolongation, and reconstructions were obtained using the Zeiss image analysis software. Freeze fracture replica of cerebellar slice cultures were performed after fixation with 2.5% glutaraldehyde and freezing using a Balzer’s Cryojet apparatus (Balzers, Liechtenstein). The specimens were then fractured and etched in a Balzer’s freeze-etching device. Platinum was applied at an angle of 45- and carbon at an angle of 90-. During cultivation, cerebellar slice cultures flattened to ¨100 Am in thickness, leading to a nearly two-dimensional system. Anyhow the third dimension was lost during cultivation, several characteristic features of the cytoarchitecture of the cerebellum were still retained. Whereas P1 cultures mostly showed clusters of Purkinje cells instead of a band of cells in the Purkinje cell layer, especially in the P10 cultures, a highly organized three-layered cerebellar cortex was preserved (Fig. 1). Characteristic features for these cell cultures were the organization of dendritic arbors. In P1 cerebellar slice cultures, Purkinje cells showed slightly developed cell prolongations after 6 div (Fig. 1a), whereas in the subsequent 15 div, these cells usually developed prominent dendritic arborization with an averaged of 3 –4 dendrites (Fig. 1b). These dendrites arose from the cell body in several directions with multiple branches. In P10 cerebellar slice cultures dendritic arborization arose from a stem-dendrite at the apical pole of the cell body with extensive branching in the molecular layer (Figs. 1c, d).
Fig. 1. Purkinje cells of cerebellar rollertube slices after calbindin staining. (a – d) Calbindin immunostaining on cerebellar rollertube slice cultures from newborn rats (P1) revealed Purkinje cells to be arranged in wide clusters, with poorly developed dendrites after 6 days in vitro (6 div) (a), and prominent dendritic arborization into multiple directions after prolonged cultivation for 15 div (b). In P10 rollertube slices cultured for 11 days, the Purkinje cell layer was obvious as a band of neurons with prominent dendrites, characterized by numerous branches in the molecular layer (c, d). Scale bars: 50 Am.
These dendritic trees showed spiny branches at their distal parts, and they were convoluted with dendritic cell prolongations of neighboring Purkinje cells. Axons arose from the basal pole of the Purkinje cell bodies. The feasibility of in vitro dye-coupling studies demands adjacent cells, which indeed has been reported previously for cultured Purkinje cells in organotypic slice cultures [17]. Purkinje cells in vitro were arranged either in a layer similar to the lamination of the in vivo cerebellar cortex or as clusters with various dimensions. After microinjection of neurobiotin into single Purkinje cells, the tracer was allowed to be transported within a subsequent post-incubation time of 1 h. Histochemical staining demonstrated that neurobiotin was transported from the soma along its processes, dendrites and axon, but dye transfer to neighboring cells always occurred along the dendrite, therefore indicating GJIC through dendro-dendritic contacts. This was only found using the tracer neurobiotin. Microinjected Lucifer Yellow was not transported to adjacent cells, even after prolonged post-incubation (not shown). GJIC of neurobiotin usually occurred between Purkinje cells (Figs. 2a, c, d), but in a few cases of P1 cultures also dye spreading to other neuronal cells was evident (Fig. 2b). In P10 cultures, dye spreading was restricted to almost one adjacent Purkinje cell; however, sometimes, no dye transfer of microinjected neurobiotin was evident. Immunohistochemistry and freeze fracture replica revealed gap junctional plaques in Purkinje cells of P1 and
K. Meller et al. / Developmental Brain Research 160 (2005) 101 – 105
103
throughout the granular layer, but not in Purkinje cells. Besides this, the expression of two pannexin genes was recently demonstrated in the cerebellum, with strong pannexin 1 expression in the white matter, whereas the pannexin 2 riboprobe strongly labeled cells in the Purkinje cell layer [4]. In the present study, functional GJIC was analyzed as dye spreading of microinjected neurobiotin in cerebellar slice cultures of P1 as well as P10, cultured for up to 15 days. An artificial coupling created by the penetrating electrode as described by Gutnick and co-workers [12] can be excluded, as microinjected Lucifer Yellow showed no dye coupling between Purkinje cells in the same experiment. The data of GJIC between adjacent cells in the developing cerebellum are in line with previously reported studies analyzing intercellular communication in various tissues. The existence of gap junctions in neuronal tissues has already been demonstrated by dye coupling of Lucifer Yellow in juvenile neurons of the cat neocortex [6]. In dye-coupling studies, the molecular size defines the permeability of a tracer. Neurobiotin is a small molecule which facilitates appropriate gap junction permeability [24,26,35]. For instance, retinal amacrine neurons of the cat and rabbit were coupled
Fig. 2. Dye transfer in Purkinje cells in P1 and P10 cerebellar rollertube slices. Demonstration of neurobiotin transfer from a microinjected Purkinje cell (black arrow) to adjacent cells (white arrowheads) in P1 (a, b) and P10 (c, d) slice cultures through dendro-dendritic contacts after 11 (a, c) and 14 div (b, d). In P1 slice cultures 1 h after microinjection, dye spreading was evident between several Purkinje cells (a), and occasionally to other neurons in P1 cultures (b). In P10 slice cultures, cell coupling of Purkinje cells was restricted to one adjacent Purkinje cell (c, d). Scale bars: 50 Am.
P10 cerebellar slice cultures (Fig. 3). Labeling with specific antibodies showed prominent Cx36 expression in Purkinje cells (Figs. 3a – c), with clearly marked gap junctional plaques along their primary dendrites (Figs. 3d, e). Cerebellar slice cultures have already been proven to be organotypic [1,10,17,28,30,31]. Comparing P1 and P10 cerebellar slice cultures, organotypic morphology of Purkinje cells was more retained in P10 slice, as revealed by Purkinje cell specific anti-calbindin immunostaining [17], however, although in P1 cerebellar slice cultures development of dendrites could be observed during culturing in vitro. On the basis of immunohistochemistry, in situ hybridization and Northern blotting studies, it is well described that neurons in the cerebellum express different connexins. For instance, in adult mouse and rat nervous system, Cx36 was shown to be present in the primary dendrites of the cerebellar Purkinje cells [32]. However, using a different Cx36 antibody in the adult rat brain immunoreactive puncta were homogeneously distributed in the molecular layer and scattered in small clusters in the granule cell layer of the cerebellum [2]. In the same study, Cx36 mRNA expression was detected in scattered cells
Fig. 3. Immunohistochemistry and freeze fracture replica in P10 cerebellar slices. In P10 slice cultures, calbindin-positive Purkinje cells (a) were labeled with Cx36 antibodies (b) after 6 div. The merged image (c) demonstrates expression of Cx36 in two Purkinje cells. Scale bar: 20 Am. Freeze fracture replica displayed gap junctional plaques (arrows) in a primary dendrite of a Purkinje cell (d, e) cultured for 6 div. Scale bars: (d) 500 nm, (e) 100 nm.
104
K. Meller et al. / Developmental Brain Research 160 (2005) 101 – 105
with neurobiotin but not with Lucifer Yellow [35]. Moreover, using neurobiotin in pyramidal cells of the neocortex led to dye spreading into clusters of up to 80 neurons among dendro-dendritic contacts [3,25]. These dendro-dendritic gap junctions might be important for the establishment of synaptic contacts as they are supposed to be responsible for synchronization of electrical activities among adjacent cells [14,25]. As a result, these early established contacts seem to be responsible for the differentiation of the adult neuronal network [15]. A similar coupling via gap junctions is demonstrated in the herein presented study, evidenced by the microinjection of neurobiotin into single Purkinje cells, immunohistochemistry and freeze fracture replica. Similarly to neurons of the neocortex or the retina in the present study, Purkinje cells form gap junctional contacts along their dendrites. Probably, the formation of gap junctions has the same functions in different brain regions, one of which might be the synchronization of early electrical activities to create a basis for the establishment of synaptic contacts. This hypothesis is strengthened by findings of the visual cortex. Injection of the tracer into individual cortical neurons revealed a temporal correlation between gap junction formation and the postnatal development of the nervous tissue [16]. Dye coupling continuously increased within the first 2 weeks after birth and diminished afterwards. Spontaneous electrical activity, however, increased up to 30-fold of the level of the adult cortex. It has been postulated that early gap junctions might have an effect on the organization of columns in the developing neocortex [19,38]. Anyhow, in cerebellum also climbing fiber spikes function as synchronization signals for Purkinje cells [20]. In conclusion, this study shows that slice cultures are suitable for further investigations on the biological function of the cell contacts in the developing cerebellum. For instance, the use of blocking reagents, known to affect the formation of gap junctions, might give further insights into the coherence of Purkinje cell maturation and GJIC.
[4]
[5]
[6]
[7]
[8]
[9] [10]
[11]
[12]
[13] [14] [15]
[16]
[17]
[18]
[19] [20]
Acknowledgments The authors especially thank C. Grzelak and A. Lodwig for their excellent technical assistance and to A. Ambrosat for the secretarial work.
[21]
[22]
References [1] J.A. Armengol, C. Sotelo, Early dendritic development of Purkinje cells in the rat cerebellum. A light and electron microscopic study using axonal tracing in Fin vitro_ slices, Brain Res. Dev. Brain Res. 64 (1991) 95 – 114. [2] N. Belluardo, G. Mudo, A. Trovato-Salinaro, S. Le Gurun, A. Charollais, V. Serre-Beinier, G. Amato, J.A. Haefliger, P. Meda, D.F. Condorelli, Expression of connexin36 in the adult developing rat brain, Brain Res. 865 (2000) 121 – 138. [3] K. Bittman, D.L. Becker, F. Cicirata, J.G. Parnavelas, Connexin
[23]
[24]
[25]
expression in homotypic and heterotypic cell coupling in the developing cerebral cortex, J. Comp. Neurol. 443 (2002) 201 – 212. R. Bruzzone, S.G. Hormuzdi, M.T. Barbe, A. Herb, H. Monyer, Pannexins, a family of gap proteins expressed in brain, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 13644 – 13649. F. Cicirata, A. Nicotra, D. Cicero, R. Parenti, A. Zappala, Cloning and expression pattern of connexin39, a new member of the gap junction gene family isolated from the neural tube of chicken embryos, Gene 17 (2004) 121 – 126. B.W. Connors, L.S. Bernado, D.A. Prince, Coupling between neurons of the developing rat neocortex, J. Neurosci. 3 (1983) 773 – 782. R. Dermietzel, Diversification of gap junction proteins (connexins) in the central nervous system and the concept of functional compartments, Cell Biol. Int. 22 (1998) 719 – 730. R. Dermietzel, Gap junction wiring: a new principle in cell-to-cell communication in the nervous system? Brain Res. Brain Res. Rev. 26 (1998) 176 – 183. R. Dermietzel, D.C. Spray, Gap junctions in the brain: where, what type, how many and why? Trends Neurosci. 16 (1993) 186 – 192. I. Dusart, M.S. Airaksinen, C. Sotelo, Purkinje cell survival and axonal regeneration are age dependent: an in vitro study, J. Neurosci. 15 (1997) 3710 – 3726. B.H. Gaehwiler, Morphological differentiation of nerve cells in thin organotypic cultures derived from rat hippocampus and cerebellum, Proc. R. Soc. London, Ser. B 211 (1980) 287 – 290. M.J. Gutnick, R. Lobel-Yaakov, G. Rimon, Incidence of neuronal dyecoupling in neocortical slices depends on the plane of section, Neuroscience 15 (1985) 659 – 666. K. Kandler, Coordination of neuronal activity by gap junctions in the developing neocortex, Semin. Dev. Biol. 8 (1995) 43 – 51. K. Kandler, L.C. Katz, Neuronal coupling and uncoupling in the developing nervous system, Curr. Opin. Neurobiol. 5 (1995) 98 – 105. K. Kandler, L.C. Katz, Coordination of neuronal activity in developing visual cortex by gap junction-mediated biochemical communication, J. Neurosci. 18 (1998) 1419 – 1427. K. Kandler, L.C. Katz, Relationship between dye coupling and spontaneous activity in developing ferret visual cortex, Dev. Neurosci. 20 (1998) 59 – 64. K. Krah, K. Meller, Axonal and dendritic transport in Purkinje cells of cerebellar slice cultures studied by microinjection of horseradish peroxidase, Cell Tissue Res. 295 (1999) 55 – 64. F.E. LeBeau, R.D. Traub, H. Monyer, M.A. Whittington, E.H. Buhl, The role of electrical signaling via gap junctions in the generation of fast network oscillations, Brain Res. Bull. 15 (2003) 3 – 13. J.J. Lo Turco, A.R. Kriegstein, Clusters of coupled neuroblasts in embryonic neocortex, Science 252 (1991) 563 – 566. R. Llinas, K. Sasaki, The functional organization of the olivocerebellar system as examined by multiple Purkinje cell recordings, Eur. J. Neurosci. 1 (1989) 587 – 602. P. Mann-Metzer, Y. Yarom, Electrotonic coupling interacts with intrinsic properties to generate synchronized activity in cerebellar networks of inhibitory interneurons, J. Neurosci. 19 (1999) 3298 – 3306. S. Maxeiner, O. Kruger, K. Schilling, O. Traub, S. Urschel, K. Willecke, Spatiotemporal transcription of connexin45 during brain development results in neuronal expression in adult mice, Neuroscience 119 (2003) 689 – 700. A. Nicotra, F. Cicirata, S. Martinez, Analysis of cCx39 expression pattern during chick development, Brain Res. Dev. Brain Res. 150 (2004) 77. A. Peinado, R. Yuste, L.C. Katz, Extensive dye coupling between rat neocortical neurons during the period of circuit formation, Neuron 10 (1993) 103 – 114. A. Peinado, R. Yuste, L.C. Katz, Gap junctional communication and the development of local circuits in neocortex, Cereb. Cortex 3 (1993) 488 – 498.
K. Meller et al. / Developmental Brain Research 160 (2005) 101 – 105 [26] A.A. Penn, R.O.L. Wong, C.J. Shatz, Neuronal coupling in the developing mammalian retina, J. Neurosci. 14 (1994) 3805 – 3815. [27] J.L. Rae, C. Bartling, J. Rae, R.T. Mathias, Dye transfer between cells of the lens, J. Membr. Biol. 150 (1996) 89 – 103. [28] F.J. Seil, R.M. Herndon, K.L. Tiekotter, N.K. Blank, Reorganization of organotypic cultures of mouse cerebellum exposed to cytosine arabinoside: a timed ultrastructural study, J. Comp. Neurol. 313 (1991) 193 – 212. [29] C. Sotelo, A. Triller, in: L. Stjarne, P. Hedyvist, H. Lagercrantz, A. Wennhalm (Eds.), Chemical Neurotransmission: 75 Years, Academic Press, New York, 1981, pp. 13 – 28. [30] L. Stoppini, P.A. Buchs, D. Muller, A simple method for organotypic cultures of nervous tissue, J. Neurosci. Methods 37 (1991) 173 – 182. [31] U. Tauer, B. Volkand, B. Heimrich, Differentiation of Purkinje cells in cerebellar slice cultures: an immunocytochemical and golgi EM study, Neuropathol. Appl. Neurobiol. 22 (1996) 361 – 369. [32] B. Teubner, J. Degen, G. Sohl, M. Guldenagel, F.F. Bukauskas, E.B. Trexler, V.K. Verselis, C.I. DeZeeuw, C.G. Lee, C.A. Kozak, E. Petrasch-Parwez, R. Dermietzel, K. Willecke, Functional expression
[33]
[34]
[35]
[36] [37]
[38]
105
of the murine connexin 36 gene coding for a neuron-specific gap junctional protein, J. Membr. Biol. 176 (2000) 249 – 262. C. Theiss, K. Meller, Microinjected anti-actin antibodies decrease gap junctional intercellular communication in cultured astrocytes, Exp. Cell Res. 281 (2002) 197 – 204. R.D. Traub, I. Pais, A. Bibbig, F.E.N. LeBeau, E.H. Buhl, S.G. Hormuzdi, H. Monyer, M.A. Whittington, Contrasting roles of axonal (pyramidal cell) and dendritic (interneuron) electrical coupling in the generation of neuronal network oscillations, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 1370 – 1374. D.I. Vaney, Many diverse types of retinal neurons show tracer coupling when injected biocytin or neurobiotin, Neurosci. Lett. 125 (1991) 187 – 190. D.I. Vaney, Territorial organization of direction-selective ganglion cells in rabbit retina, J. Neurosci. 14 (1994) 6301 – 6316. D. Xin, S.A. Bloomfield, Tracer coupling pattern of amacrine and ganglion cells in the rabbit retina, J. Comp. Neurol. 383 (1997) 512 – 528. R. Yuste, A. Peinado, L.C. Katz, Neuronal domains in developing neocortex, Science 257 (1992) 665 – 669.
Short Communication
Dye coupling in Purkinje cells of organotypic slice cultures Karl Meller, Kerstin Krah, Carsten Theiss* Medizinische Fakulta¨t, Institut fu¨r Anatomie, Abteilung fu¨r Cytologie, Ruhr-Universita¨t Bochum, Universita¨tsstraße 150, D-44780 Bochum, Germany Accepted 4 August 2005 Available online 16 September 2005
Abstract Cerebellar slice cultures of newborn rats showed poorly developed dendritic arborization of Purkinje cells, whereas cultures of 10-day-old rats revealed prominent dendritic branching. Gap junctional intercellular communication between Purkinje cells, investigated as dye transfer of microinjected neurobiotin, occurred through dendro-dendritic contacts, with decreased dye spreading in old cell cultures. These results indicate a possible correlation of gap junctional intercellular communication and the development of Purkinje cells. D 2005 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Formation and specificity of synapses Keywords: Cerebellar slice culture; Dye spreading; Gap junction; Microinjection; Purkinje cell
Gap junctional intercellular communication (GJIC) between neurons in various regions is a feature of the mammalian brain. Gap junctions are channels between adjacent neighboring cells formed by two opposing hexamers—the connexins (cx) together termed as one connexon. One aspect of the mammalian brain is the cell type specific but overlapping expression pattern of connexins. For instance, neurons are known to express predominantly cx36, cx32 and cx26, whereas cx31, cx43, cx37 and cx45 are expressed in non-neuronal cells, e.g., in glial cells [7,9]. Recently, in the developing mice brain, cx45 was detected in cerebellar basket and stellate cells [22], whereas in the chick cerebellum, cx39 was detected in neurons of the granular layer [5,23]. The gap junctional channels contribute to the structural basis for the direct transfer of metabolites and are therefore considered as one potential type of cellular communication. Such a flux on information is described to carry various functions, e.g., the control of cellular proliferation and diffeAbbreviations: cx, connexin; div, days in vitro; GJIC, gap junctional intercellular communication * Corresponding author. Fax: +49 234 3214476. E-mail address: [email protected] (C. Theiss). 0165-3806/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2005.08.007
rentiation [8]. Gap junctions are also discussed to be involved in the migration of neuronal precursor cells as well as in the formation of chemical synapses during the juvenile development. In the neocortex, they occur predominantly during the postnatal development, whereas in the adult phase, their number is diminished [13]. Besides this, in the adult brain, GJIC between neurons seems to be essential to generate synchronized activity of neuronal networks [18, 21,34]. Recently, another class of molecules called pannexins has been identified, which also form electrical junctions between neuronal cells in the mammalian brain [4]. A technical approach to study intercellular communication is the microinjection of tracer molecules into a cell. Low weight tracer such as the fluorescent Lucifer Yellow [27,36] or neurobiotin [35] is spread through gap junctions into adjacent cells. Using neurobiotin, it was shown that GJIC in the mammalian retina is realized by interneurons of the inner layer [35] as well as among ganglion cells [26,37] and amacrine cells [37]. In the neocortex, Peinado et al. [24] confirmed an extensive dye coupling among the pyramidal neurons forming clusters of up to 80 cells, and in the cerebellum dye coupling between molecular layer inhibitory interneurons was demonstrated subsequent injection of biocytin [21]. Besides this, dendro-dendritic and somato-
102
K. Meller et al. / Developmental Brain Research 160 (2005) 101 – 105
dendritic gap junctions in the rat cerebellar cortex have been reported by Sotelo and Triller [29]. Among the various kinds of tissues and cells, scanty information about the formation of gap junctions or pannexins in developing cerebellar Purkinje cells has been reported. Adopting the rollertube cultures of cerebellar slices, we investigated GJIC in Purkinje cells of the cerebellar cortex of different age. Slice cultures of rat cerebellum were obtained from newborn rat pups (P0) and at postnatal day 10 (P10) and incubated according to the rollertube technique [11,17]. Tracing studies were performed using 8% neurobiotin. After 11 up to 15 days in vitro (div), microinjection of tracer substances into single Purkinje cells was carried out as described elsewhere [33]. Treated tissues were post-incubated for 1 h to allow a sufficient spread of the intracellular dye. Specimens were fixed with 4% paraformaldehyde supplemented with 0.2% picric acid at 4 -C, and neurobiotin was visualized with an avidin –biotin enzyme complex (Vector Laboratories, USA) and diaminobenzidine. To intensify the reaction product, incubation in 5% cobalt chloride was preceded. Used antibodies were mouse monoclonal IgG (mAb) to calbindin (C-8666, 1:100, Sigma, Germany), rabbit polyclonal IgG to Cx36 (36 –4600, 1:100, Zymed, USA) and secondary goat anti-mouse IgG (Alexa 546 conjugated, 1:250, 11018, Molecular Probes, Germany) or goat anti-rabbit IgG (TRITC conjugated, 1.200, T-5268, Sigma). To cover the whole extension of the Purkinje cell dendrites, optical sections were collected in 2-Am steps by confocal laser scanning microscopy (LSM 510, Zeiss Meta, Germany), covering the thickness of the cell prolongation, and reconstructions were obtained using the Zeiss image analysis software. Freeze fracture replica of cerebellar slice cultures were performed after fixation with 2.5% glutaraldehyde and freezing using a Balzer’s Cryojet apparatus (Balzers, Liechtenstein). The specimens were then fractured and etched in a Balzer’s freeze-etching device. Platinum was applied at an angle of 45- and carbon at an angle of 90-. During cultivation, cerebellar slice cultures flattened to ¨100 Am in thickness, leading to a nearly two-dimensional system. Anyhow the third dimension was lost during cultivation, several characteristic features of the cytoarchitecture of the cerebellum were still retained. Whereas P1 cultures mostly showed clusters of Purkinje cells instead of a band of cells in the Purkinje cell layer, especially in the P10 cultures, a highly organized three-layered cerebellar cortex was preserved (Fig. 1). Characteristic features for these cell cultures were the organization of dendritic arbors. In P1 cerebellar slice cultures, Purkinje cells showed slightly developed cell prolongations after 6 div (Fig. 1a), whereas in the subsequent 15 div, these cells usually developed prominent dendritic arborization with an averaged of 3 –4 dendrites (Fig. 1b). These dendrites arose from the cell body in several directions with multiple branches. In P10 cerebellar slice cultures dendritic arborization arose from a stem-dendrite at the apical pole of the cell body with extensive branching in the molecular layer (Figs. 1c, d).
Fig. 1. Purkinje cells of cerebellar rollertube slices after calbindin staining. (a – d) Calbindin immunostaining on cerebellar rollertube slice cultures from newborn rats (P1) revealed Purkinje cells to be arranged in wide clusters, with poorly developed dendrites after 6 days in vitro (6 div) (a), and prominent dendritic arborization into multiple directions after prolonged cultivation for 15 div (b). In P10 rollertube slices cultured for 11 days, the Purkinje cell layer was obvious as a band of neurons with prominent dendrites, characterized by numerous branches in the molecular layer (c, d). Scale bars: 50 Am.
These dendritic trees showed spiny branches at their distal parts, and they were convoluted with dendritic cell prolongations of neighboring Purkinje cells. Axons arose from the basal pole of the Purkinje cell bodies. The feasibility of in vitro dye-coupling studies demands adjacent cells, which indeed has been reported previously for cultured Purkinje cells in organotypic slice cultures [17]. Purkinje cells in vitro were arranged either in a layer similar to the lamination of the in vivo cerebellar cortex or as clusters with various dimensions. After microinjection of neurobiotin into single Purkinje cells, the tracer was allowed to be transported within a subsequent post-incubation time of 1 h. Histochemical staining demonstrated that neurobiotin was transported from the soma along its processes, dendrites and axon, but dye transfer to neighboring cells always occurred along the dendrite, therefore indicating GJIC through dendro-dendritic contacts. This was only found using the tracer neurobiotin. Microinjected Lucifer Yellow was not transported to adjacent cells, even after prolonged post-incubation (not shown). GJIC of neurobiotin usually occurred between Purkinje cells (Figs. 2a, c, d), but in a few cases of P1 cultures also dye spreading to other neuronal cells was evident (Fig. 2b). In P10 cultures, dye spreading was restricted to almost one adjacent Purkinje cell; however, sometimes, no dye transfer of microinjected neurobiotin was evident. Immunohistochemistry and freeze fracture replica revealed gap junctional plaques in Purkinje cells of P1 and
K. Meller et al. / Developmental Brain Research 160 (2005) 101 – 105
103
throughout the granular layer, but not in Purkinje cells. Besides this, the expression of two pannexin genes was recently demonstrated in the cerebellum, with strong pannexin 1 expression in the white matter, whereas the pannexin 2 riboprobe strongly labeled cells in the Purkinje cell layer [4]. In the present study, functional GJIC was analyzed as dye spreading of microinjected neurobiotin in cerebellar slice cultures of P1 as well as P10, cultured for up to 15 days. An artificial coupling created by the penetrating electrode as described by Gutnick and co-workers [12] can be excluded, as microinjected Lucifer Yellow showed no dye coupling between Purkinje cells in the same experiment. The data of GJIC between adjacent cells in the developing cerebellum are in line with previously reported studies analyzing intercellular communication in various tissues. The existence of gap junctions in neuronal tissues has already been demonstrated by dye coupling of Lucifer Yellow in juvenile neurons of the cat neocortex [6]. In dye-coupling studies, the molecular size defines the permeability of a tracer. Neurobiotin is a small molecule which facilitates appropriate gap junction permeability [24,26,35]. For instance, retinal amacrine neurons of the cat and rabbit were coupled
Fig. 2. Dye transfer in Purkinje cells in P1 and P10 cerebellar rollertube slices. Demonstration of neurobiotin transfer from a microinjected Purkinje cell (black arrow) to adjacent cells (white arrowheads) in P1 (a, b) and P10 (c, d) slice cultures through dendro-dendritic contacts after 11 (a, c) and 14 div (b, d). In P1 slice cultures 1 h after microinjection, dye spreading was evident between several Purkinje cells (a), and occasionally to other neurons in P1 cultures (b). In P10 slice cultures, cell coupling of Purkinje cells was restricted to one adjacent Purkinje cell (c, d). Scale bars: 50 Am.
P10 cerebellar slice cultures (Fig. 3). Labeling with specific antibodies showed prominent Cx36 expression in Purkinje cells (Figs. 3a – c), with clearly marked gap junctional plaques along their primary dendrites (Figs. 3d, e). Cerebellar slice cultures have already been proven to be organotypic [1,10,17,28,30,31]. Comparing P1 and P10 cerebellar slice cultures, organotypic morphology of Purkinje cells was more retained in P10 slice, as revealed by Purkinje cell specific anti-calbindin immunostaining [17], however, although in P1 cerebellar slice cultures development of dendrites could be observed during culturing in vitro. On the basis of immunohistochemistry, in situ hybridization and Northern blotting studies, it is well described that neurons in the cerebellum express different connexins. For instance, in adult mouse and rat nervous system, Cx36 was shown to be present in the primary dendrites of the cerebellar Purkinje cells [32]. However, using a different Cx36 antibody in the adult rat brain immunoreactive puncta were homogeneously distributed in the molecular layer and scattered in small clusters in the granule cell layer of the cerebellum [2]. In the same study, Cx36 mRNA expression was detected in scattered cells
Fig. 3. Immunohistochemistry and freeze fracture replica in P10 cerebellar slices. In P10 slice cultures, calbindin-positive Purkinje cells (a) were labeled with Cx36 antibodies (b) after 6 div. The merged image (c) demonstrates expression of Cx36 in two Purkinje cells. Scale bar: 20 Am. Freeze fracture replica displayed gap junctional plaques (arrows) in a primary dendrite of a Purkinje cell (d, e) cultured for 6 div. Scale bars: (d) 500 nm, (e) 100 nm.
104
K. Meller et al. / Developmental Brain Research 160 (2005) 101 – 105
with neurobiotin but not with Lucifer Yellow [35]. Moreover, using neurobiotin in pyramidal cells of the neocortex led to dye spreading into clusters of up to 80 neurons among dendro-dendritic contacts [3,25]. These dendro-dendritic gap junctions might be important for the establishment of synaptic contacts as they are supposed to be responsible for synchronization of electrical activities among adjacent cells [14,25]. As a result, these early established contacts seem to be responsible for the differentiation of the adult neuronal network [15]. A similar coupling via gap junctions is demonstrated in the herein presented study, evidenced by the microinjection of neurobiotin into single Purkinje cells, immunohistochemistry and freeze fracture replica. Similarly to neurons of the neocortex or the retina in the present study, Purkinje cells form gap junctional contacts along their dendrites. Probably, the formation of gap junctions has the same functions in different brain regions, one of which might be the synchronization of early electrical activities to create a basis for the establishment of synaptic contacts. This hypothesis is strengthened by findings of the visual cortex. Injection of the tracer into individual cortical neurons revealed a temporal correlation between gap junction formation and the postnatal development of the nervous tissue [16]. Dye coupling continuously increased within the first 2 weeks after birth and diminished afterwards. Spontaneous electrical activity, however, increased up to 30-fold of the level of the adult cortex. It has been postulated that early gap junctions might have an effect on the organization of columns in the developing neocortex [19,38]. Anyhow, in cerebellum also climbing fiber spikes function as synchronization signals for Purkinje cells [20]. In conclusion, this study shows that slice cultures are suitable for further investigations on the biological function of the cell contacts in the developing cerebellum. For instance, the use of blocking reagents, known to affect the formation of gap junctions, might give further insights into the coherence of Purkinje cell maturation and GJIC.
[4]
[5]
[6]
[7]
[8]
[9] [10]
[11]
[12]
[13] [14] [15]
[16]
[17]
[18]
[19] [20]
Acknowledgments The authors especially thank C. Grzelak and A. Lodwig for their excellent technical assistance and to A. Ambrosat for the secretarial work.
[21]
[22]
References [1] J.A. Armengol, C. Sotelo, Early dendritic development of Purkinje cells in the rat cerebellum. A light and electron microscopic study using axonal tracing in Fin vitro_ slices, Brain Res. Dev. Brain Res. 64 (1991) 95 – 114. [2] N. Belluardo, G. Mudo, A. Trovato-Salinaro, S. Le Gurun, A. Charollais, V. Serre-Beinier, G. Amato, J.A. Haefliger, P. Meda, D.F. Condorelli, Expression of connexin36 in the adult developing rat brain, Brain Res. 865 (2000) 121 – 138. [3] K. Bittman, D.L. Becker, F. Cicirata, J.G. Parnavelas, Connexin
[23]
[24]
[25]
expression in homotypic and heterotypic cell coupling in the developing cerebral cortex, J. Comp. Neurol. 443 (2002) 201 – 212. R. Bruzzone, S.G. Hormuzdi, M.T. Barbe, A. Herb, H. Monyer, Pannexins, a family of gap proteins expressed in brain, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 13644 – 13649. F. Cicirata, A. Nicotra, D. Cicero, R. Parenti, A. Zappala, Cloning and expression pattern of connexin39, a new member of the gap junction gene family isolated from the neural tube of chicken embryos, Gene 17 (2004) 121 – 126. B.W. Connors, L.S. Bernado, D.A. Prince, Coupling between neurons of the developing rat neocortex, J. Neurosci. 3 (1983) 773 – 782. R. Dermietzel, Diversification of gap junction proteins (connexins) in the central nervous system and the concept of functional compartments, Cell Biol. Int. 22 (1998) 719 – 730. R. Dermietzel, Gap junction wiring: a new principle in cell-to-cell communication in the nervous system? Brain Res. Brain Res. Rev. 26 (1998) 176 – 183. R. Dermietzel, D.C. Spray, Gap junctions in the brain: where, what type, how many and why? Trends Neurosci. 16 (1993) 186 – 192. I. Dusart, M.S. Airaksinen, C. Sotelo, Purkinje cell survival and axonal regeneration are age dependent: an in vitro study, J. Neurosci. 15 (1997) 3710 – 3726. B.H. Gaehwiler, Morphological differentiation of nerve cells in thin organotypic cultures derived from rat hippocampus and cerebellum, Proc. R. Soc. London, Ser. B 211 (1980) 287 – 290. M.J. Gutnick, R. Lobel-Yaakov, G. Rimon, Incidence of neuronal dyecoupling in neocortical slices depends on the plane of section, Neuroscience 15 (1985) 659 – 666. K. Kandler, Coordination of neuronal activity by gap junctions in the developing neocortex, Semin. Dev. Biol. 8 (1995) 43 – 51. K. Kandler, L.C. Katz, Neuronal coupling and uncoupling in the developing nervous system, Curr. Opin. Neurobiol. 5 (1995) 98 – 105. K. Kandler, L.C. Katz, Coordination of neuronal activity in developing visual cortex by gap junction-mediated biochemical communication, J. Neurosci. 18 (1998) 1419 – 1427. K. Kandler, L.C. Katz, Relationship between dye coupling and spontaneous activity in developing ferret visual cortex, Dev. Neurosci. 20 (1998) 59 – 64. K. Krah, K. Meller, Axonal and dendritic transport in Purkinje cells of cerebellar slice cultures studied by microinjection of horseradish peroxidase, Cell Tissue Res. 295 (1999) 55 – 64. F.E. LeBeau, R.D. Traub, H. Monyer, M.A. Whittington, E.H. Buhl, The role of electrical signaling via gap junctions in the generation of fast network oscillations, Brain Res. Bull. 15 (2003) 3 – 13. J.J. Lo Turco, A.R. Kriegstein, Clusters of coupled neuroblasts in embryonic neocortex, Science 252 (1991) 563 – 566. R. Llinas, K. Sasaki, The functional organization of the olivocerebellar system as examined by multiple Purkinje cell recordings, Eur. J. Neurosci. 1 (1989) 587 – 602. P. Mann-Metzer, Y. Yarom, Electrotonic coupling interacts with intrinsic properties to generate synchronized activity in cerebellar networks of inhibitory interneurons, J. Neurosci. 19 (1999) 3298 – 3306. S. Maxeiner, O. Kruger, K. Schilling, O. Traub, S. Urschel, K. Willecke, Spatiotemporal transcription of connexin45 during brain development results in neuronal expression in adult mice, Neuroscience 119 (2003) 689 – 700. A. Nicotra, F. Cicirata, S. Martinez, Analysis of cCx39 expression pattern during chick development, Brain Res. Dev. Brain Res. 150 (2004) 77. A. Peinado, R. Yuste, L.C. Katz, Extensive dye coupling between rat neocortical neurons during the period of circuit formation, Neuron 10 (1993) 103 – 114. A. Peinado, R. Yuste, L.C. Katz, Gap junctional communication and the development of local circuits in neocortex, Cereb. Cortex 3 (1993) 488 – 498.
K. Meller et al. / Developmental Brain Research 160 (2005) 101 – 105 [26] A.A. Penn, R.O.L. Wong, C.J. Shatz, Neuronal coupling in the developing mammalian retina, J. Neurosci. 14 (1994) 3805 – 3815. [27] J.L. Rae, C. Bartling, J. Rae, R.T. Mathias, Dye transfer between cells of the lens, J. Membr. Biol. 150 (1996) 89 – 103. [28] F.J. Seil, R.M. Herndon, K.L. Tiekotter, N.K. Blank, Reorganization of organotypic cultures of mouse cerebellum exposed to cytosine arabinoside: a timed ultrastructural study, J. Comp. Neurol. 313 (1991) 193 – 212. [29] C. Sotelo, A. Triller, in: L. Stjarne, P. Hedyvist, H. Lagercrantz, A. Wennhalm (Eds.), Chemical Neurotransmission: 75 Years, Academic Press, New York, 1981, pp. 13 – 28. [30] L. Stoppini, P.A. Buchs, D. Muller, A simple method for organotypic cultures of nervous tissue, J. Neurosci. Methods 37 (1991) 173 – 182. [31] U. Tauer, B. Volkand, B. Heimrich, Differentiation of Purkinje cells in cerebellar slice cultures: an immunocytochemical and golgi EM study, Neuropathol. Appl. Neurobiol. 22 (1996) 361 – 369. [32] B. Teubner, J. Degen, G. Sohl, M. Guldenagel, F.F. Bukauskas, E.B. Trexler, V.K. Verselis, C.I. DeZeeuw, C.G. Lee, C.A. Kozak, E. Petrasch-Parwez, R. Dermietzel, K. Willecke, Functional expression
[33]
[34]
[35]
[36] [37]
[38]
105
of the murine connexin 36 gene coding for a neuron-specific gap junctional protein, J. Membr. Biol. 176 (2000) 249 – 262. C. Theiss, K. Meller, Microinjected anti-actin antibodies decrease gap junctional intercellular communication in cultured astrocytes, Exp. Cell Res. 281 (2002) 197 – 204. R.D. Traub, I. Pais, A. Bibbig, F.E.N. LeBeau, E.H. Buhl, S.G. Hormuzdi, H. Monyer, M.A. Whittington, Contrasting roles of axonal (pyramidal cell) and dendritic (interneuron) electrical coupling in the generation of neuronal network oscillations, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 1370 – 1374. D.I. Vaney, Many diverse types of retinal neurons show tracer coupling when injected biocytin or neurobiotin, Neurosci. Lett. 125 (1991) 187 – 190. D.I. Vaney, Territorial organization of direction-selective ganglion cells in rabbit retina, J. Neurosci. 14 (1994) 6301 – 6316. D. Xin, S.A. Bloomfield, Tracer coupling pattern of amacrine and ganglion cells in the rabbit retina, J. Comp. Neurol. 383 (1997) 512 – 528. R. Yuste, A. Peinado, L.C. Katz, Neuronal domains in developing neocortex, Science 257 (1992) 665 – 669.