Spatiotemporal transcription of connexin45 during brain development results in neuronal expression in adult mice

Neuroscience 119 (2003) 689 –700

SPATIOTEMPORAL TRANSCRIPTION OF CONNEXIN45 DURING BRAIN DEVELOPMENT RESULTS IN NEURONAL EXPRESSION IN ADULT MICE ¨ GER,a K. SCHILLING,b S. MAXEINER,a O. KRU O. TRAUB,a S. URSCHELa AND K. WILLECKEa*

gap junction channel is formed by docking of two hemichannels (connexons) in the plasma membranes of adjacent cells. Each connexon is composed of six connexin (Cx) protein subunits. Cxs are coded by a multigene family, of which nineteen members have been so far identified in the mouse genome (Willecke, et al., 2002). Connexin45 (Cx45; Gja7) has been isolated as cDNA from F9 mouse embryonic carcinoma cells (Hennemann et al., 1992). By immunocytochemical analyses, the Cx45 protein has been localized in glomeruli and distal tubuli of the mouse kidney (Butterweck et al., 1994a), in the basal layer of embryonic skin (Butterweck et al., 1994b), in granulosa cells of the rat ovary (Okuma et al., 1996), in the deep muscular plexus of rat small intestine (Nakamura et al., 1998), and in the conductive myocardium (Alcolea´ et al., 1999; Coppen et al., 1999a, b). Furthermore, positive immunofluorescent signals of Cx45 protein were found in some cells of the rat hippocampus, colocalized with Cx32 immunosignals that were interpreted to be localized in oligodendrocytes (Kunzelmann et al., 1997; Dermietzel et al., 1997). Recently, Cx45 expression has been found in ␣-motoneurons of the spinal cord (Chang et al., 1999). Additionally, in vitro analyses and RT-PCR results suggest that Cx45 is expressed in Cx43-deficient astrocytes (Dermietzel et al., 2000) and a hippocampal neuronal cell line (Rozental et al., 2000). Various Cxs have been found in neural tissue. By immunogold labeling of freeze-fracture replicas from rat brain, Rash et al. (2001) found Cx32 to be exclusively expressed in membranes of oligodendrocytes, and Cx43 only in astrocytic membranes, whereas neither of these Cxs was detected in neurons. Previously, in situ hybridization and immunocytochemical results indicated that some populations of neurons do express Cx32 or Cx43 (Micevych and Abelson, 1991; Nadarajah et al., 1996; Simbu¨rger et al., 1997; for review, see Dermietzel, 1998; Dermietzel and Spray, 1998). Besides Cx32, the more recently characterized Cx29 (So¨hl et al., 2001) is also expressed in oligodendrocytes (Altevogt et al., 2002). In contrast, Cx36 appears to be almost exclusively expressed in neurons (Parenti et al., 2000; Belluardo et al., 2000). Transcripts for Cx26, -33, -36, -40, -43, -45 have been reported to be present during differentiation of a hippocampal cell line (for review, see Rozental et al., 2000). Furthermore, Cx47 mRNA has been found in brain and spinal cord and was reported to be expressed in neurons based on in situ hybridization data (Teubner et al., 2001), although expression of Cx47 protein in neurons remains to be shown.

a

Institut fu¨r Genetik, Abteilung fur Molekulargenetik, Rheinische Friedrich-Wilhelms Universita¨t Bonn, Ro¨merstraße 164, D-53117 Bonn, Germany

b Anatomisches Institut, Anatomie und Zellbiologie, Rheinische Friedrich-Wilhelms Universita¨t Bonn, Bonn, Germany

Abstract—Characterization of the expression pattern of connexins in neural tissue is a necessary prerequisite for understanding the functional relevance of the corresponding gap junction channels in brain. Here we describe the cell typespecific expression of connexin45 in the CNS and the spatiotemporal expression pattern from embryonic day 19.5 to adult brain using a recently described connexin45 LacZ-reporter mouse. The connexin45 gene is highly expressed during embryogenesis and up to 2 weeks after birth in nearly all brain regions. Afterward its expression is restricted to the thalamus, the CA3 region of hippocampus and the cerebellum. In adult mouse brain, the pattern of LacZ-staining in combination with the analysis of different neuronal and glial marker proteins strongly suggests that connexin45 is expressed in neurons, but presumably not in astrocytes or mature oligodendrocytes. Expression of the LacZ/connexin45 reporter gene in subsets of neurons, such as cerebral cortical, hippocampal and thalamic neurons as well as basket and stellate cells of cerebellum should be corroborated by functional investigations of connexin45 protein in electrical synapses. Based on its expression pattern during development, we suggest that the connexin45-containing gap junction channels have a rather ubiquitous role during brain development and may contribute to functional specification in certain subsets of neurons in the adult brain. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: gap junction, Cx45, thalamus, hippocampus, cerebellum, neuron.

During development, individual cells become organized into tissues and establish specialized contact sites to neighboring cells that can serve for structural and functional coordination. Gap junctions form intercellular conduits that allow the diffusional exchange of ions and metabolites for communication between neighboring cells. A *Corresponding author. Tel: ⫹49-228-734-210; fax: ⫹49-228-734263. E-mail address: [email protected] (K. Willecke). Abbreviations: CA, cornu ammonis; Cx, connexin; Cx45, connexin45; ED, day of embryonic development; ␤-Gal, ␤-galactosidase; GFAP, glial fibrillary acidic protein; NeuN, neural nuclei; OPC, oligodendrocyte precursor cell; PBS, phosphate-buffered saline; PFA, paraformaldehyde; pp, postpartum; X-Gal, 5-bromo-4-chloro3-indolyl-␤-galactoside.

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00077-0

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Recently, we (Kru¨ger et al., 2000) and others (Kumai et al., 2000) generated Cx45 reporter mice in which the coding region of Cx45 was replaced by the bacterial ␤-galactosidase (␤-Gal) (LacZ) gene via homologous recombination in embryonic stem cells. Taking advantage of the high sensitivity of the ␤-Gal assay, we demonstrated Cx45 expression in various tissues, where it had previously not been described, such as vascular and visceral smooth muscle cells and neural tissues. The present study aimed at establishing a cell type-specific profile of Cx45 transcription based on reporter-gene analyses in the developing and adult brain.

EXPERIMENTAL PROCEDURES Animals All animal handling was done in strict accordance to local governmental (European Communities Council Directive 86/609/EEC) and institutional animal care regulations. All efforts were made to reduce animal suffering and to plan the experiments such that the number of animals needed was minimized. Heterozygous Cx45deficient mice with a mixed background of C57BL/6 and Sv129/ Ola were used for all experiments, because general loss of Cx45 resulted in early embryonic lethality. These mice expressed the bacterial ␤-Gal protein under control of the endogenous Cx45 promoter (Kru¨ger et al., 2000). Animals were maintained using a 12-h light/dark cycle. Timed pregnancies were checked by vaginal plugs. The plug date was counted as embryonic day (ED) 0.5. Pregnant dams were killed at ED 18.5 and 19.5. Postnatal animals aged 1, 8, 14, 21 or 28 days or after 1 year were also analyzed.

Fixation and cryostat sectioning Animals were killed by cervical dislocation. Whole brains of embryonic and day 1 or dissected adult brains were rinsed briefly in phosphate-buffered saline (PBS). Vibratome sections (500 ␮m) were cut from unfixed brains/heads, and immediately fixed in 2% paraformaldehyde (PFA) in PBS over night at 4 °C. For cryostat sectioning, brains/heads were frozen on an aluminum sheet on dry ice and stored at ⫺70 °C until further use.

␤-Gal staining For ␤-Gal histochemistry fixed vibratome sections were incubated in a solution containing 5-bromo-4-chloro-3-indolyl-␤-galactoside (X-Gal). When ␤-Gal histochemical and immunohistochemical analyses were combined, 10 ␮m cryostat sections were used. Sections were cut at ⫺20 °C, and immediately fixed with 4% PFA for 10 min. They were stained for ␤-Gal activity as described (Schilling et al., 1991). Subsequently, they were postfixed in 2% PFA for 5 min and processed for immunohistochemical analysis or counterstained with eosine.

Immunohistochemical analysis For immunohistochemical analyses, ␤-Gal-stained cryostat sections were incubated in 10% methanol: 0.1% H2O2: 90% PBS for

10 min to block endogenous peroxidase activity. Afterward, sections were incubated for 10 min in PBS containing 0.5% Triton X-100 to permeabilize the cells. Blocking of unspecific binding sites was performed by incubation in 2% normal serum from the species in which the secondary antibody had been generated. Primary and secondary antibodies were diluted in blocking solution. The following antibodies were used in this study (supplier and final dilution in parenthesis): Pax2 (1:200; Zymed, San Francisco, USA), NG2 (1:400; Chemicon, Temecula, USA), neural nuclei (NeuN) (1:100; Chemicon, Temecula, USA), glial fibrillary acidic protein (GFAP) (1:250; Sigma, St. Louis, USA), MOSP (1:800; Chemicon, Temecula, USA), parvalbumin (1:250; Sigma), secondary biotinylated anti-rabbit antibodies (diluted 1:400) were obtained from Dianova (Hamburg, Germany). For the detection of mouse monoclonal antibodies, the M.O.M.-kit (Vector, Vector Laboratories Inc., Burlingame, USA) was used according to the manufacturer’s recommendations. Bound biotinylated secondary antibodies were analyzed using the Vectorstain Elite ABC kit (Vector). Sections were counterstained with Hoechst dye 33258 (0.5 ␮g/ml) prior to mounting with Entellan (Merck, Darmstadt, Germany).

Immunoblot analysis Specific brain regions of defined developmental stages were dissected on ice, immediately frozen in liquid nitrogen, and stored at ⫺70 °C until use. After lyophilization, the dry weight of tissue specimens was determined, and the tissue was taken up in 1⫻ Complete (Roche, Mannheim, Germany) at a final concentration of 20 ␮g/␮l. Samples were sonicated for 20 s. The protein concentration was determined using the bicinchoninic acid protein assay (Sigma, St. Louis, USA) and aliquots were mixed (3:1) with four-fold concentrated sample-loading buffer. For electrophoresis, 50 ␮g of protein were loaded on 12% SDS-polyacrylamide gels for analysis of Cx45 or on 8% gels for analysis of ␤-Gal. As controls we used 5 ␮g of HeLa-Cx45 homogenates (Butterweck et al., 1994a) and HeLa-Cx47 homogenates (Teubner et al., 2001) as well as 100 ␮g of total tissues from 9.5-day-old wild type and Cx45-deficient embryos. Separated proteins were transferred to 0.45 ␮m polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA, USA) by electrophoresis in transfer buffer (25 mM Tris, 192 mM glycine, in 20% [v/v] methanol). Membranes were blocked for 2 h at room temperature in blocking solution (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20 [TBS-Tween] and 5% skim milk powder), and then incubated with primary antibodies for 12–16 h at 4 °C in blocking solution. For the detection of Cx45, we used rabbit anti-Cx45 (Butterweck et al., 1994) at a dilution of 1:500. Rabbit anti-␤-Gal (ICN Biomedicals, Eschwege, Germany) was used at 1:600 in blocking buffer. After incubation with primary antibodies, membranes were washed for 1 h in blocking solution and then incubated for 1 h at room temperature with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Dianova) diluted 1:10,000 in blocking solution. Blots were washed twice in TBS-Tween, once in TBS, and then incubated for 2 min with ECL chemiluminescence reagent (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom).

Abbreviations used in the figures Ah Cb Co DG EGL Gl H

alveus hippocampi cerebellum cerebral cortex dentate gyrus external granule layer granule cell layer hippocampus

I IGL MI Pjl St T II and VI

inner layer inner granule layer molecular layer Purkinje cell layer striatum thalamus layer II and VI of cerebral cortex

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Fig. 1. (Caption overleaf).

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RESULTS Expression of Cx45 and ␤-Gal protein during perinatal development in comparison to adult mouse brain Cx45 expression was analyzed by visualizing ␤-Gal activity coded by the LacZ reporter gene in the brains of Cx45(⫹/⫺) heterozygous mice. Initially, we compared postnatal (1 week old) and adult animals to map Cx45 expression in the brain (Fig. 1). At day 8 postpartum (pp) (Fig. 1A), intense ␤-Gal activity was observed in nearly all regions of the thalamus, all sections of the hippocampus, the striatum and the cerebral cortex as well as throughout the cerebellum. In contrast, in the brains of adult Cx45(⫹/⫺) heterozygous mice only a residual and more restricted pattern of ␤-Gal expression was found (Fig. 1B). The age-related quantitative differences in Cx45 expression, suggested by the histochemical analysis of ␤-Gal expression, were confirmed by immunoblot analysis (Fig. 2) using affinity-purified polyclonal antibodies directed toward the C-terminal part of Cx45 (Butterweck et al., 1994). In HeLa-Cx45 transfectants and in brain lysates, these antibodies labeled two protein bands as previously described (Polontchouk et al., 2002). High levels of Cx45 protein were found in ED 18.5 and early postnatal (day 1 pp and day 8 pp) wild type brain, but declined during the second postnatal week to a lower level that could still be detected in adult brain tissue (Fig. 2A). The temporal decrease was also observed in Cx45 heterozygous animals but the amount of Cx45 which was clearly reduced (data not shown). Analysis of ␤-Gal by immunoblot confirmed the early postnatal decrease of Cx45 protein which led to the conclusion that the amount of ␤-Gal activity was proportional to the amount of Cx45 protein (Fig. 2B). In an additional set of experiments we tested for crossreactivity of the Cx45 antibodies with Cx47 protein, using a protein lysate from HeLa-Cx47 transfectants (Teubner et al., 2001). After Western blot analysis we found that the Cx45 antibodies recognized the Cx47 protein band at another position than the Cx45 protein (Fig. 2C, left side). The specificity of the antibody was confirmed by comparison of Cx45 wild type and Cx45-deficient tissue from day 9.5 embryos (Fig. 2C, right side). Identification of LacZ/Cx45-expressing cells in the developing and adult mouse cerebrum In order to identify cell type-specific expression of LacZ in adult brain, we combined staining for ␤-Gal activity with immunohistological analysis of cell type-specific markers on 10 ␮m frozen sections. The neuronal protein NeuN (Mullen et al., 1992), was utilized as a general marker for postmitotic

neurons. In our analysis of adult mouse brain, we found most ␤-Gal signals associated with NeuN-positive cell nuclei in the stratum pyramidale of the CA3 region of hippocampus (Fig. 3A, B). Consistently, there was no colocalization of ␤-Gal and the astrocytic marker GFAP (Fig. 3C, D). However, there remained a small number of ␤-Gal-positive cells which did not stain with neuronal markers. Similarly, in the cortex where ␤-Gal expression was particularly prominent in layers II and VI of the (parieto-) occipital cortex and reaching into the entorhinal cortex, it was consistently associated with NeuNpositive nuclei (Fig. 4A, A1, A2) but not with GFAP-positive cells (Fig. 4C). In general, the ␤-Gal signals that we observed in our histochemical analysis were always located close to the nucleus (confirmed by Hoechst 33258 dye staining, not shown) which facilitated further analyses on the colocalization with cell-specific marker proteins. Immunostaining for MOSP, a marker for adult oligodendrocytes (Dyer et al., 1991), showed that ␤-Gal did not localize to these cells. This cellular distribution was also confirmed in the thalamus, where numerous and intense ␤-Gal signals were found on NeuN-positive neurons (Fig. 5A, B), whereas areas of MOSP-positive oligodendrocytes appeared to be ␤-Gal-negative (Fig. 5C, D). The neuronal localization could also be confirmed by combining ␤-Gal and Nissl staining (Fig. 5E, F). ␤-Gal signals were only very rarely seen in the white matter, such as the alveus hippocampi (Fig. 4B) and the corpus callosum (not shown), which, however, intensely stained for MOSP and GFAP (not shown). To further characterize the Cx45-positive neurons, we stained for parvalbumin (Fig. 3G, H) known to be expressed in the cytosol of GABA-ergic interneurons (cf. DeFelipe, 1997; Parent et al., 1996). Although comparison of our sections to published data of parvalbumin-positive cells suggests that not all interneurons may have been labeled by our staining protocol, we never observed ␤-Gal signals in parvalbumin-positive cells in forebrain tissue samples. Since ␤-Gal was expressed neither in mature astrocytes nor in oligodendrocytes, we investigated its expression during oligodendrocyte development. For this purpose, we immunostained for NG2, a proteoglycan expressed by early precursor cells (Nishiyama et al., 1996; cf. Levine et al., 2001), that eventually give rise to type II astrocytes and oligodendrocytes (O2A cells), as well as immediate oligodendrocyte precursor cells (OPCs). Similar to MOSP-positive mature oligodendrocytes, NG2-positive OPCs that could be identified based on their highly branched processes (Figs. 3E, F and 4E), did not stain for ␤-Gal. In contrast, simple, spindle-shaped NG2-positive cells, resembling O2A cells (cf. Levine et al., 2001) that we have found at low frequencies, for instance, in the cerebral cortex, were found to be ␤-Gal positive (Fig. 4D).

Fig. 1. Comparison of ␤-Gal expression pattern in the brains of Cx45(⫹/⫺) mice at two developmental stages. Postnatal (day 8 pp) and adult brains (1 year) were cut into 500 ␮m vibratome sections horizontally (approximately horizontal section 77 according to Sidman et al., 1971) and stained with X-Gal. (A) Postnatal mouse brain was characterized by almost ubiquitous ␤-Gal expression, most prominently in cerebral cortex, striatum, CA fields and dentate gyrus of hippocampus, thalamus and cerebellum. The faint greenish appearance visible over some areas resulted from the overlap of yellowish LacZ-negative brain tissue and small LacZ-positive blood vessels, as determined by inspection at higher magnification. (B) Adult brain sections only revealed residual staining in some parts of mouse brain, such as CA3 region of hippocampus, various nuclei of the thalamus, some distinct layers of cerebral cortex and the molecular layer of cerebellum. Scale bar⫽2 mm.

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Fig. 2. Cx45 and ␤-Gal protein expression during brain development in wild type Cx45(⫹/⫹) and heterozygous Cx45(⫹/⫺) mice, respectively. (A) Immunoblot of brain homogenates probed with polyclonal Cx45 antibodies. Lysate of HeLa-Cx45-transfectants served as positive control. Affinity-purified Cx45 antibodies (Butterweck et al., 1994) detected two close bands in lysates of HeLa-Cx45 transfectants. Note the strong decrease in the intensity of signals between brains of 8-day pp and 14-day pp embryos. (B) Immunoblot of brain homogenates probed with the polyclonal ␤-Gal antibodies recognized a protein band migrating at 116 kDa. (C) The Cx45 antibodies recognized an additional band in HeLa-Cx47 transfectants (arrow) which migrated clearly separated from the Cx45 band. Comparison of Cx45 knockout and wild type embryonic tissue demonstrates the specificity of the Cx45 antibodies used for all immunoblot analyses.

LacZ/Cx45 expression in the developing and adult mouse cerebellum We have also studied LacZ/Cx45 expression in the cerebellum where interpretation is facilitated due to its simple

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three-layered structure. We used the same neuronal (NeuN, parvalbumin) and glia-specific markers (i.e. GFAP for astrocytes as well as MOSP and NG2 for oligodendrocyte development) as analyzed in the forebrain (cf. Figs. 3–5), in order to identify Cx45-expressing cell types. In the adult cerebellar cortex, anti-NeuN labeled exclusively granule cells (A. Weyer and K.S., unpublished observations), some of which were ectopically located in the molecular layer (Landis, 1973) (Fig. 6). NeuN-positive cells did not show ␤-Gal expression (Fig. 6A, B). Oligodendrocytes are predominantly located in the deep cerebellar matter, whereas GFAP-positive astrocytes are found in the granule cell layer and, as Bergmann glia, in the Purkinje layer. GFAP and MOSP immunosignals did not colocalize with ␤-Gal signals, which were almost completely absent from the granule cell layer and the white matter of the deep cerebellar mass (shown for GFAP in Fig. 6C). The molecular layer is known to harbor the perikarya of basket and stellate cells. These cells, like the Purkinje cells located between the granule cell layer and the molecular layer, express parvalbumin. By ␤-Gal staining and immunohistochemical analysis of parvalbumin (Fig. 6E) we found LacZ expression only in basket and stellate cells but not Purkinje cells. The latter cells can easily be distinguished from basket and stellate cells due to their position and their large cell body. Within the molecular layer of the cerebellum, we found NG2-positive, spindle-shaped cells positive for ␤-Gal activity (Fig. 6D) indicating LacZ expression in O2A precursor cells as we had already detected in forebrain (cf. Fig. 4D). We also analyzed Cx45 expression in the cerebellum during development and histogenesis. At day 8 pp, Pax2positive precursors in the deeper parts of the cerebellar anlage and the future white matter which develop into Golgi II, basket and stellate cells (Maricich and Herrup, 1999) were found to be ␤-Gal positive (Fig. 6F, F1). A conspicuous band of ␤-Gal-positive dots was found immediately above the Purkinje cell layer (Fig. 6F1), where early differentiating basket and stellate cells are known to be located (Maricich and Herrup, 1999). The other cell population, intensely stained for ␤-Gal, are cells of the external granule layer (EGL) (Fig. 6F1), and notably those in the outer part of the EGL as confirmed by nuclear staining with Hoechst dye 33258 (cf. Fig. 6F1, F2). These are proliferating granule cell precursors (Fujita, 1967, 1969).

DISCUSSION In this paper we have shown that Cx45 is strongly expressed in many regions of the developing brain at day 8 after birth but becomes restricted in adult brain mainly to the thalamus, the CA3/CA4 region of the hippocampus, layers II and VI in the parieto-occipital and entorhinal cerebral cortex and the molecular layer of the cerebellum. These findings are based on activation of the LacZ reporter gene product, ␤-Gal, which is expressed instead of Cx45 in heterozygous Cx45 (⫹/⫺) mice (Kru¨ger et al., 2000). In addition, we have studied the expression level of Cx45 protein and ␤-Gal by immunoblotting in whole brain during

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Fig. 3.

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Fig. 4. Cerebral cortex: Adult brain tissue showed only some residual X-Gal staining (A). NeuN-positive cells of layer II (A1), IV (not shown) and VI (A2) were characterized by blue signals indicating expression of the LacZ reporter gene. The marker protein of mature oligodendrocytes, MOSP, did not appear to colocalize with blue spots in the cerebral cortex (B). Furthermore, astrocytes expressing GFAP showed no expression of LacZ (C). Late stages of oligodendrocyte development characterized by NG2 expression did not show blue ␤-Gal signals (arrowhead in E), whereas the bipolar O2A precursor cells that were also positive for NG2 (arrowhead), expressed the reporter gene (D). Scale bar⫽250 ␮m (A, B, C); 50 ␮m (A1, A2,); 25 ␮m (D, E).

development. Both experimental approaches resulted in consistent findings, strongly suggesting that the expres-

sion of the reporter gene faithfully reflects cognate Cx45 expression, although there is a slight overexpression of

Fig. 3. LacZ expression in the adult hippocampus: (A, B) LacZ expression was primarily seen as perinuclear signal in NeuN-positive (brown) cells in the stratum pyramidale of the CA3/4 region. The cell type of the rare ␤-Gal-positive, NeuN-negative cells seen in the left half of the micrograph remains to be identified. GFAP-positive astrocytes (C, D) and late NG2-positive oligodendrocyte precursors (E, F arrowhead) did not show any colocalization with the reporter gene product after X-Gal treatment. GABA-ergic interneurons which express the cytosolic marker parvalbumin were found in the CA2 region and did not express the LacZ reporter gene product (G, H). Scale bar⫽250 ␮m (A, C, E, G); 50 ␮m (B, D, F, H).

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Fig. 5. Thalamic neurons highly expressed the LacZ/Cx45 reporter gene as shown by X-Gal treatment in combination with NeuN labeling by the brown DAB reaction product. About three to six blue dots per cell were closely associated with stained nuclei of these neurons (A, B). Long processes of mature oligodendrocytes were visible by staining of the myelin– oligodendrocyte surface protein, MOSP, intruding into the thalamus (C, D). Nissl staining confirmed the neuronal origin of the ␤-Gal signal (E, F). Neural cell bodies appeared purple and contained LacZ-positive spots. Scale bar⫽250 ␮m (A, C, E); 50 ␮m (B, D); 25 ␮m (F).

␤-Gal during postnatal day 14 –21. This could be due to the different half-life times of Cx45 transcript or protein, compared with ␤-Gal. We have used expression of the LacZ reporter gene in combination with various antibodies to identify the cell types in adult mouse brain in which the Cx45 gene is expressed. Our results exclude mature astrocytes and oligodendrocytes as sites of Cx45 expression, and clearly demonstrate expression of LacZ/Cx45 in certain

neuronal subpopulations. This is in conflict with the previously reported expression of Cx45 in adult/mature oligodendrocytes (Kunzelmann et al., 1997), based on immunostaining with anti-Cx45. The extensive sequence similarity between Cx45 protein and the recently discovered Cx47 protein (Teubner et al., 2001), however, raised the issue whether the previously used antiserum to Cx45 had been specific. Indeed, immunoblot analyses of lysates from Cx47 transfected HeLa cells

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Fig. 6. (Caption overleaf).

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suggest that this novel Cx protein is also recognized by the antibodies previously thought to be specific for Cx45 (Kunzelmann et al., 1997). In the present study, we have detected the expression of the LacZ/Cx45 reporter gene in NG2-positive O2A precursor cells. This group of glial precursor cells gives rise to oligodendrocytes as well as type II astrocytes (Trotter and Schachner, 1989) and can be found scattered in most parts of the brain, mainly after birth and at sites of brain lesion (Levine et al., 2001). Oligodendrocyte precursors in later developmental stages were negative for LacZ/Cx45 expression. The developing cerebellum is particularly well suited to correlate Cx45 expression to specific aspects of neuronal histogenesis. Strikingly, Cx45 expression was found in Pax2-positive cells of the cerebellar anlage, which represent migrating, and proliferating cerebellar inhibitory interneurons (i.e. basket, stellate and/or Golgi cells; Zhang and Goldman, 1996; Maricich and Herrup, 1999). At least in basket and stellate cells, Cx45 expression persists following their setting (after migration) in the molecular layer. An additional band of ␤-Gal signals above the Purkinje cell layer suggests Cx45 expression in Pax2-negative cells representing early differentiating basket and stellate cells (Altman and Bayer, 1997). Taken together, our results indicate expression of Cx45 in this special type of cerebellar interneurons (basket/stellate cells) during development, differentiation and mature stages. Previously Mann-Metzer and Yarom (1999) described dye coupling between cerebellar interneurons in sections of guinea-pig cerebellum suggesting functional gap junctions between these inhibitory interneurons. The expression of Cx45 in these obviously migrating cells, as well as in the OPCs, which lie scattered and isolated from each other, raises the question which cells might be coupled via Cx45 gap junction channels and what could be the functional relevance for migrating and proliferating cells. Besides this expression during the development of cerebrellar inhibitory interneurons, ␤-Gal signals were also found in mitotically active granule cell precursors which were visible as a small band of cells in the external granule cell layer at day 8 pp (Fujita, 1967, 1969). In contrast to cerebellar inhibitory neurons in the adult brain, Cx45 was not expressed in mature granule cells any more as indicated by the complete lack of ␤-Gal signals in adult granule cell layer. In addition to Cx45, other Cxs like Cx26, Cx32, Cx36, Cx43 and Cx47 have previously been suggested to be expressed in neurons of the CNS. Of these, only Cx36 has

so far been unequivocally located in neurons in comparison to mice with ablated Cx36 protein (Gu¨ldenagel et al., 2001; Deans et al., 2001; Hormuzdi et al., 2001). In all other cases, the assignment of certain Cxs to brain neurons was based on results with Cx antibodies or in situ hybridization, which are prone to cross-reaction unless adequate Cx-deficient mice are used as controls. Our results on Cx45 expression are based on analyses of the LacZ reporter gene, and thus unlikely to be affected by crossreactivity. It is intriguing to note that the level of Cx26 and Cx45 mRNAs during brain development paralleled each other, i.e. they declined postnatally, whereas Cx32 and Cx47 mRNA were only synthesized at the beginning of the second week pp (So¨hl et al., 2001). The amount of the Cx43 mRNA did not vary significantly during embryogenesis or early juvenile stages. In contrast, Cx36 is expressed at the end of embryogenesis in brain tissue and rises within the first 2 postnatal weeks before it slightly declines to a lower level. The observation of coupled early postnatal cortical neurons (Peinado et al., 1993; Peinado, 2001; Kandler and Katz, 1998) as well as coupled neuroblasts (Lo Turco and Kriegstein, 1991) strikingly coincides with the expression of Cx26 and Cx45 and might indicate that either one or both Cxs could be involved in this phenomenon. The spatiotemporal profile of Cx45 expression could be associated with the specific functional properties of this gap junction protein. The unitary conductance of Cx45 channels is reduced to 50% at 13.4 mV voltage difference between contacting cells (Moreno et al., 1995), in contrast to 70 mV for Cx36 channels (Srinivas et al., 1999). Thus, the gating of neuronal Cx45 gap junction channels could be particularly sensitive to changes in membrane potential, caused by synaptic input and/or generation of action potentials. Electrophysiological analysis showed that mouse Cx45 gap junction channels expressed in Xenopus oocytes were most sensitive to changes in membrane potential and upon hyperpolarization (Barrio et al., 2000). It will be challenging to clarify the functional relevance of Cx45 gap junction channels expressed in neurons of adult thalamus, hippocampus, cortex and cerebellum. Acknowledgements—We thank Gerda Hertig and Shahrazad Schlu¨ter for expert technical help with the genotyping and histological analyses of Cx45 ⫾ mice. This work was supported by grants of the Deutsche Forschungsgemeinschaft through SFB 400, project E3 and the Fonds der Chemischen Industrie to K.W.

Fig. 6. Cerebellum: NeuN-positive granule cells of the granule cell layer as well as a few dispersed positive cells in the molecular layer of adult cerebellum were not marked by blue signals after X-Gal treatment (A, B). GFAP-positive astrocytes in the granule cell layer and white matter did not express ␤-Gal (C, D). The only blue signals in adult cerebellum were found in the molecular layer in basket and stellate cells which stained positive for parvalbumin (E), whereas Purkinje neurons at the border of granule cell and molecular layer did not express the reporter gene. Similar as in cerebral cortex (see Fig. 4D), NG2-positive bipolar oligodendrocyte precursors in the molecular layer were positive for ␤-Gal (D, arrows). The cerebellar anlage at day 8 pp showed additional ␤-Gal-positive cells in the external granule layer. In the nascent white matter, ␤-Gal signals were associated with Pax2-positive nuclei in cells that give rise to cerebellar inhibitory interneurons (stellate, basket, and Golgi cells) in adult brain. Furthermore, LacZ was expressed in Pax2-negative precursor cells of inhibitory interneurons, residing just above the Purkinje cell layer (red arrow: direction of cell migration). Nuclear staining with Hoechst 33258 dye showed that reporter-gene expression in the external granule layer occurred only in its outer part, where proliferating granule cell precursors were located (white arrow in F2, corresponding to black arrow in F1, indicates range of ␤-Gal-positive cells). Scale bar⫽250 ␮m (A, C, F); 50 ␮m (B, D, E, F1, F2).

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(Accepted 16 January 2003)