Time-related changes in connexin mRNA abundance in the rat neocortex during postnatal development

Developmental Brain Research 119 Ž2000. 111–125 www.elsevier.comrlocaterbres

Research report

Time-related changes in connexin mRNA abundance in the rat neocortex during postnatal development Graham Prime, Gabi Horn, Bernd Sutor

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Institute of Physiology, UniÕersity of Munich, Pettenkoferstrasse 12, D-80336 Munich, Germany Accepted 24 August 1999

Abstract Gap junction coupling between neurons is important for the temporal and spatial co-ordination of neocortical development and can be visualised by dye-coupling. Neuronal dye-coupling in the rat neocortex is extensive during the first 2 postnatal weeks and diminishes rapidly thereafter. We used RT Žreverse transcriptase. –PCR to investigate the time-related changes in mRNA expression for the connexins ŽCx. Cx 26, Cx 30, Cx 32, Cx 36, Cx 37, Cx 40, Cx 43, Cx 45 and Cx 46 as well as for b-actin and GAPDH in rat neocortex during the first 6 postnatal weeks. The time courses for mRNA expression for GAPDH, Cx 30, Cx 36 and Cx 43 were also investigated by northern blotting. Cx 30 and Cx 45 mRNA abundance showed no time-dependent changes during the early postnatal period. The relative abundance of Cx 32, Cx 43 and Cx 46 mRNA increased significantly during the first 2–3 weeks and then remained relatively constant during weeks 3–6. The relative abundance of Cx 26, Cx 36, Cx 37 and Cx 40 mRNA also increased significantly during the first 10–15 postnatal days but then declined significantly from their peak values during weeks 3–6. b-actin mRNA expression showed no time-related changes but GAPDH mRNA expression increased significantly during the first postnatal week, then remained constant. The time-dependent changes in mRNA relative abundance for GAPDH, Cx 36 and Cx 43 determined by northern blotting corroborate the results from the RT–PCR study. None of the Cx exhibited time-dependent changes in mRNA expression in homogenates of rat neocortex which parallel the changes in neuronal dye-coupling during postnatal development. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Connexin; RT–PCR; Postnatal development; Neocortex; mRNA; Gap junction

1. Introduction Connexins ŽCx. are a family of plasma membrane spanning proteins which combine to form hexamers known as connexons. Normally two connexons, one from each of two closely apposed cells, align with each other to form a single continuous gap junction channel between the cells Žfor review, see Ref. w21x.. In recent years interest has focused on trying to elucidate the Cx composition of the various gap junctions expressed within and between populations of neurons and glia cells in the brain. Within the neocortex, neuronal dye-coupling, taken as an indication of gap junction coupling, is particularly extensive during embryonic and early postnatal development with groups of up to 80–90 coupled neurons w8,26,33,34,36x. The inci-

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dence of dye-coupling in rat neocortex was 100% during the period days 1 to 12 postnatal ŽP1–12. with the mean number of cells coupled to the injected neurons remaining stable at 40–45 until P13. Dye-coupling then declined dramatically from the end of the second postnatal week w36x. Changes in gap junction permeability influence the electrotonic properties of neocortical neurons, resulting in an altered efficacy of chemical synaptic transmission w36x. Since the period of increased dye-coupling and the period of enhanced synaptogenesis in the neocortex correlate in time, it can be hypothesised that gap junction permeability influences the formation of neuronal circuits within the neocortex w33,34x. The permeability of gap junctions depends on their Cx composition Žfor review, see Ref. w21x., therefore it is important to identify the CxŽs. responsible for dye-coupling in the neocortex. Time courses for the expression of Cx in the brain during early postnatal development have so far only been published for Cx 26, Cx 32 and Cx 43 w1,11,30x and Cx 36

0165-3806r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 9 . 0 0 1 3 2 - 7

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w41x. It seems unlikely, from the time courses reported so far, that changes in neuronal dye-coupling in the neocortex correlate with changes in expression of any one of these Cx alone. Since the first experiments of Dermietzel et al. w11x, the expression of several more Cx has been demonstrated in the brain ŽCx 37, w44x; Cx 40, w25x; Cx 30, w9x; Cx 45, w4,18x. but time-related changes in their expression during the postnatal period have not been reported. The expression of some of these Cx has been localised to specific cell populations, for example Cx 43 in astrocytes and some neurons w11,15,40x, Cx 32 in oligodendrocytes and some neurons w27,29x and Cx 36 in some neurons w7x. The expression of Cx 37 and Cx 40 has been localised to blood vessels in the brain w25,35x and Cx 45 is expressed in oligodendrocytes w23x. Cx 46 is expressed in Schwann cells in the peripheral nervous system w5x, but its expression in the brain has not been reported. Since there are no detailed reports of the time courses for the expression of the more recently characterised Cx identified in postnatal brain, we used RT Žreverse transcriptase. –PCR to investigate time-related changes in the expression of Cx 26, Cx 30, Cx 32, Cx 36, Cx 37, Cx 40, Cx 43, Cx 45 and Cx 46 mRNA in rat neocortex during the first 4 postnatal weeks. To enable a comparison of our data from the RT–PCR study with previously published data, we also determined the time courses for mRNA expression of Cx 30, Cx 36, Cx 43 and Glyceraldehyde-3phosphate dehydrogenase ŽGAPDH. by northern blotting, using total RNA samples from the same animals.

2. Materials and methods 2.1. Animals A total of 112 Wistar rats from 32 different litters were sacrificed on different days within the first 53 postnatal Table 1 X X DNA sequences Ž5 –3 . of the forward and reverse PCR primers

days. The animals were anaesthetised by inhalation of isoflurane ŽForene w , Abbott, Wiesbaden, Germany. and then decapitated. Neocortices were removed by dissection and either processed immediately to isolate the total RNA or frozen in liquid nitrogen and stored at y808C. 2.2. Total RNA and genomic DNA isolation Total RNA was isolated from the neocortex using TriReagent w ŽBiozol, Eching, Germany. according to the manufacturer’s instructions. The method is based on the single-step method for RNA isolation by Chomczynski and Sacchi w6x, using guanidinium thiocyanate–phenol–chloroform extraction. In all cases 75 mg tissue was used per 1 ml Tri-Reagent w and the RNA pellet was dissolved in 30 ml HPLC grade water ŽSigma–Aldrich, Steinheim, Germany.. The concentration and purity were determined photometrically ŽO.D. at l s 260 nm and l s 280 nm.. Total RNA yield was 0.8–0.9 mgrmg neocortex with O.D. 260r280 ratios of 1.85–1.95. Samples were stored at y808C. 2.3. mRNA isolation using oligo-dT magnetic bead separation Oligo-dT25 Dynalbeads ŽDeutsche Dynal, Hamburg, Germany. were used to isolate polyŽA.q mRNA from the total RNA samples. Fifteen microliter stock bead suspension was used for each sample. The beads were separated using the magnetic separator ŽMPG E-1; Deutsche Dynal., washed once in an equal volume of 2 = binding buffer Ž20 mM Tris–HCl, 1 M LiCl, 2 mM EDTA; pH 7.5., then separated and resuspended in a total volume of 2 = binding buffer equivalent to 8 mlrsample and RT-negative control. Eight microliter aliquots of the bead suspension were mixed with 6 mg total RNA in 8 ml HPLC grade water Ž0.75 mgrml.. Thus, the results relate to a standard-

G. Prime et al.r DeÕelopmental Brain Research 119 (2000) 111–125 Table 2 Length of the PCR products, number of PCR cycles and annealing temperatures ŽTa . for the amplification of the specific connexin, GAPDH and b-actin cDNA sequences Target cDNA

Product length Žbp.

Number of cycles

Tannealing Ž8C.

Cx 26 Cx 30 Cx 32 Cx 36 Cx 37 Cx 40 Cx 43 Cx 45 Cx 46 GAPDH b-actin

408 494 633 619 438 659 1064 527 785 471 577 ŽcDNA. 1040 Žgenomic.

32 33 32 30 35 32 32 30 33 30 30

60 63 60 60 62 59 60 56 63 63 59

ised starting amount of 6 mg total RNA from each animal. After incubation for 5–10 min at room temperature ŽRT., the beads with attached mRNA were separated and the supernatant was discarded. The beads were washed once with 15 ml washing buffer containing SDS Ž10 mM Tris–

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HCl, 0.15 M LiCl, 1 mM EDTA, 0.1% SDS; pH 8.0., once with 15 ml washing buffer without SDS and then three times with 15 ml chilled RT buffer Ž10 mM Tris–HCl, 75 mM KCl; pH 8.3. by resuspending the beads in the appropriate buffer, then separating the beads and discarding the buffer. One 8-ml aliquot of magnetic beads in 2 = binding buffer was mixed with 8 ml HPLC grade water instead of a total RNA solution, but otherwise treated in exactly the same way as the other samples. This served as the RT-negative control. 2.4. First strand cDNA synthesis The beads with attached mRNA were resuspended in 20 ml reverse transcription mix containing 50 mM Tris–HCl ŽpH 8.3., 50 mM KCl, 4 mM MgCl 2 , 10 mM DTT, 1 mM each dNTP and 20 U Moloney murine leukemia virus ŽM-Mu-LV. reverse transcriptase ŽMBI Fermentas, Vilnius, Lithuania. and incubated at 378C for 60–75 min. The solid-phase first strand cDNA library was then separated, washed once in 50 ml 1 = PCR buffer Ž10 mM Tris–HCl ŽpH 8.3., 50 mM KCl, 1.5 mM MgCl 2 , 0.001% Žwrv. gelatin., then separated again, resuspended in 45 ml 1 = PCR buffer and stored at 48C.

Fig. 1. b-actin PCR product bands photographed under UV illumination after agarose gel electrophoresis and ethidium bromide staining showing; 1 kb DNA marker ladder Žlanes 1 and 8. and the products from P15 cDNA Žlane 2., P16 cDNA Žlane 3., a mixture of P15 cDNA and genomic DNA Žlane 4., genomic DNA only Žlane 5., the reverse transcription negative control Žlane 6. and the PCR negative control Žlane 7.. Note the longer b-actin PCR product from genomic DNA Ž1040 bp. than from cDNA Ž577 bp..

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Fig. 2. The percentage relative abundance of ŽA. GAPDH, ŽB. b-actin, ŽC. Cx 26, ŽD. Cx 37, ŽE. Cx 40 and ŽF. Cx 43 mRNA in five rats sacrificed on postnatal day 7. Each bar represents the mean " S.E.M. of duplicate samples prepared from each rat, relative to the maximum mean value in the group of five animals ŽP7a–e.. Inter-animal differences were analysed by Kruskal–Wallis non-parametric ANOVA and were only significant for Cx 26 Ž P s 0.005. and for Cx 40 Ž P s 0.01.. Differences between animals for Cx 32 and Cx 45 were not significant ŽNS, data not shown..

Fig. 3. PCR product bands for ŽA. b-actin and ŽB. GAPDH after agarose gel electrophoresis and ethidium bromide staining, photographed under UV illumination. The mean fluorescence gray value and area of each band was determined. For each gene, four to six such gels were analysed, each with 13–16 different animals spanning the P0–28 period.

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2.5. PCR analysis The same basic recipe was used for all the Cx, GAPDH and b-actin PCR reactions. The sequences of the forward and reverse primers are shown in Table 1 and the product lengths, annealing temperatures and cycle number for the specific reactions are shown in Table 2. The 50 ml PCR reaction mixture contained 10 mM Tris–HCl ŽpH 8.3., 50 mM KCl, 1.5 mM MgCl 2 , 0.001% Žwrv. gelatin, 15 mM each dNTP, 2% DMSO, 50 pmol each of a specific forward and reverse primer, 2 U Taq polymerase ŽAmplitaq Golde, Perkin-Elmer Applied Biosystems, Weiterstadt, Germany. and 5 ml first strand cDNArbead suspension. The cDNArbead suspension was replaced by 5 ml HPLC grade water for the PCR negative control. The reactions were overlaid with 60 ml liquid wax ŽChill-out 14e; MJ Research, Watertown, MA, USA.. The thermocycling programme was performed in a Perkin-Elmer DNA Thermocycler 480. The cycling times for all the PCR reactions were identical, but the annealing temperatures ŽTa . and the number of cycles was varied for the different Cx, GAPDH and b-actin Žsee Table 2.. The cycling times and other temperatures for all samples were as follows: 10 min at 958C Žto activate the Taq polymerase and an initial denaturation step., followed by 30–35 cycles of 50 s at 948C, 1 min 30 s at the specific annealing temperature ŽTa ., 1 min 30 s at 728C, then a final extension period of 10 min at 728C before cooling to a holding temperature of 48C.

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calculated and expressed as the percentage relative abundance, relative to the maximum fluorescence value during the P0–28 period on the gel. The mean relative abundance was calculated from the results from all the gels for the given Cx on a particular postnatal day, with the maximum mean relative abundance during the P0–28 period set at 100%. Statistical analysis was performed using the Prism2 computer programme ŽGraphpad Software, San Diego, USA.. The significance of the change in relative abundance with time was tested using Kruskal–Wallis nonparametric ANOVA. The mean relative abundance for the periods P0–4, P11–14 and P22–28 were compared by Dunn’s multiple comparisons test. 2.8. Northern blotting Digoxigenin ŽDIG.-labeled Cx 30, Cx 36, Cx 43 and GAPDH probes were synthesised by PCR using a PCR DIG-labeling mix ŽBoehringer Mannheim, Mannheim,

2.6. PCR product analysis PCR products were resolved by electrophoresis on a 1.5% agarose gel with a 1-kb DNA marker ŽMBI Fermentas, Vilnius, Lithuania.. Bands were visualised under UVlight after staining with ethidium bromide. Selected PCR products from each of the Cx, GAPDH and b-actin were sequenced on an ABI 377 DNA sequencer ŽMediGene, MartinsriedrMunich, Germany. to confirm the identity of the product. The PCR product sequences were compared with DNA sequences held in the Genbank Library at the National Center for Biotechnology Information ŽBethesda, USA.. 2.7. Data acquisition and analysis The ethidium bromide stained electrophoresis gels were photographed under UV-light illumination and a digitalised image was stored for computer analysis. The images were analysed using the Optimas 6.0 software programme ŽStemmer Imaging, Puchheim, Germany.. The RNA isolation, reverse transcription and specific PCR reactions were performed in three to five batches for each Cx, each time with 13–16 samples covering the time period P0–28. The band fluorescence value Žmean gray value = band area. for each animal for a given Cx was

Fig. 4. The time-related changes in the relative abundance of ŽA. b-actin mRNA and ŽB. GAPDH mRNA as a percentage of the maximum daily mean value observed for each gene during ŽP0–28.. For b-actin ŽA., each point represents the mean"S.E.M. calculated from two to four animals, except for P1, 27, 29, 31 and 40 Ž ns1.. For GAPDH ŽB., each point represents the mean"S.E.M. from two or three animals for each postnatal day, except for P8 and 19 Ž ns1..

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Fig. 5. PCR product bands for ŽA. Cx 45, ŽB. Cx 43 and ŽC. Cx 26 after agarose gel electrophoresis and ethidium bromide staining, photographed under UV illumination. The mean fluorescence gray value and area of each band was determined. For each gene, four to six such gels were analysed, each with 13–15 different animals spanning the P0–28 period.

Fig. 6. PCR product bands for ŽA. Cx 32, ŽB. Cx 37 and ŽC. Cx 40 after agarose gel electrophoresis and ethidium bromide staining, photographed under UV illumination. The mean fluorescence gray value and area of each band was determined. For each gene, four to six such gels were analysed, each with 13–15 different animals spanning the P0–28 period.

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Germany.. The primers and the thermocycler protocol were as described above. The PCR product mixture was cleaned using a PCR purification column ŽQiagen, Hilden, Germany. and an aliquot was resolved by agarose gel electrophoresis Žsee above. to estimate the PCR product concentration by comparison with standard quantities of DNA resolved and stained simultaneously. Aliquots of 15 mg total RNA were denatured by addition of 2 vol. of glyoxal-mix Ž1.8 M glyoxal, 0.75% Žvrv. DMSO, 15 mM Na 2 HPO4rNaH 2 PO4 ; pH 6.5. to 1 vol. of total RNA solution and incubation at 508C for 1 h. The total RNA was resolved by electrophoresis on a 1.2% agarose gel with 10 mM Na 2 HPO4rNaH 2 PO4 running buffer ŽpH 6.5. at 2 Vrcm. The RNA was transferred by capillary blotting in 20 = standard saline citrate buffer Ž20 = SSC; 3 M NaCl, 0.3 M sodium citrate. onto nylon membranes ŽNylon NX; Amersham, Braunschweig, Germany. and fixed by UV crosslinking Ž1200 Jrcm2 . ŽUV Stratalinker 1800; Stratagene, Heidelberg, Germany.. Membranes were prehybridised in hybridisation buffer containing 50% formamide, 5 = SSC, 0.5% SDS, and 0.5% blocking reagent ŽBoehringer Mannheim. for 2 h at 408C–428C and hybridised overnight in the same buffer with 100 ngrml of heat denatured DIG-labeled Cx or GAPDH probe at the same temperature. The membranes were washed three times, each time for 20 min, in 20 mM Na 2 HPO4rNaH 2 PO4 buffer containing 1 mM EDTA and

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1% SDS at the hybridisation temperature. The DIG detection procedure was based on the method of Engler-Blum et al. w14x using alkaline phosphatase conjugated anti-DIG FAB fragments ŽBoehringer Mannheim. and the alkaline phosphatase substrate disodium 3-Ž4-methoxyspiro-w1,2-dioxetane-3-2 X-Ž5X-chloro .tricyclo w3.3.1.13,7 xdecanex-4-yl. phenyl phosphate ŽCSPD; Boehringer Mannheim.. Briefly, the membranes were incubated for 5 min at RT in maleic acid wash buffer containing 0.1 M maleic acid ŽpH 8.0., 3 M NaCl and 0.3% Žvrv. Tween and then incubated for 45 min at RT in Blocking solution containing 0.1 M maleic acid, 0.3 M NaCl, 0.3% Žvrv. Tween and 0.5% blocking reagent ŽBoehringer Mannheim.. The blots were then transferred to fresh Blocking solution containing 50 mUrml alkaline phosphatase conjugated anti-DIG FAB fragments Ž1:15 000 dilution; Boehringer Mannheim. and incubated at RT for 30 min. After three washes, each 20 min, in maleic acid wash buffer at RT, the membranes were incubated for 5 min at RT in substrate buffer containing 10 mM Tris–HCl ŽpH 9.5. and 0.1 M NaCl and then incubated for about 5 min, in darkness, in fresh substrate buffer containing 0.25 mM CSPD. Excess buffer was allowed to drip off and the membranes were sealed in clear plastic bags and exposed to X-ray films ŽKodak X-OMAT AR, Eastman Kodak, New York, USA. for periods of 3–16 h. Digitalised images of the bands were analysed as described above for the PCR product bands.

Fig. 7. PCR product bands for ŽA. Cx 36, ŽB. Cx 30 and ŽC. Cx 46 after agarose gel electrophoresis and ethidium bromide staining, photographed under UV illumination. The mean fluorescence gray value and area of each band was determined. For each gene, four to six such gels were analysed, each with 13–15 different animals spanning the P0–28 period.

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3. Results 3.1. Exclusion of genomic DNA contamination To ensure the absence of genomic DNA in the first strand cDNA mixture, an aliquot of every solid-phase cDNA sample was used in an ‘‘intron-differential RT– PCR’’ w20x with a b-actin primer pair, selected so that the primers were located in different exons of the b-actin gene. Genomic DNA isolated from rat neocortex gave a longer PCR product Ž1040 bp. compared to the cDNA PCR product Ž577 bp. ŽFig. 1.. Every sample proved negative for genomic DNA.

70% to 90%, otherwise the abundance of GAPDH remained high and stable at 90%–100% for the rest of the period from P9 to 28 and in the adult ŽFig. 4B.. The difference in relative abundance was highly significant between P0–4 Ž78%. and P11–14 Ž95%. Ž P - 0.001. and between P0–4 Ž78%. and P22–28 Ž94%. Ž P - 0.01.. The difference between P11–14 and P22–28 was not significant Ž P ) 0.05..

3.2. Variation in Cx expression in indiÕidual animals sacrificed at the same age To test to what extent the variation in individual PCR band fluorescence, and hence amount of PCR product was attributable to variations between animals or due to the method, duplicate samples of solid-phase cDNA were prepared separately from total RNA isolated from the neocortices of five rats sacrificed on P7. Aliquots were used in PCR with primers for Cx 26, Cx 32, Cx 37, Cx 40, Cx 43, and Cx 45 and for GAPDH and b-actin. For each animal and PCR product the mean band fluorescence was calculated from the duplicate samples. The results for GAPDH, b-actin, Cx 26, Cx 37, Cx 40 and Cx 43 are presented in histograms as the percentage relative abundance of mRNA, compared with the maximum mean value within the group of five animals ŽFig. 2.. The inter-animal variation was not significant for b-actin, GAPDH, Cx 32, Cx 37, Cx 43 or Cx 45. The variation between animals ŽKruskal–Wallis non-parametric ANOVA. was significant for Cx 26 Ž P s 0.005. and Cx 40 Ž P s 0.010.. Considering the difference in the values within each duplicate, the lowest variation, as illustrated by the S.E.M. bars in Fig. 2, was observed for Cx 26 duplicates, followed in order of increasing variability by Cx 45, GAPDH, Cx 32, Cx 43, Cx 40, b-actin and Cx 37. 3.3. Postnatal expression of b-actin and GAPDH in rat neocortex Examples of the PCR product bands obtained for GAPDH and b-actin on various postnatal days are illustrated in Fig. 3. The results from all the gels, in terms of percentage relative abundance, are summarised in Fig. 4. There was no significant change in the relative abundance of b-actin with time during P0–28 Ž P s 0.093.. b-actin expression remained high and stable throughout the postnatal period and apparently into adulthood ŽFig. 4A.. The differences in the mean relative abundance between P0–4 Ž84%., P11–14 Ž92%. and P22–28 Ž90%. were not significant Ž P ) 0.05.. The change in GAPDH expression observed during P0–28 was highly significant Ž P s 0.0002.. The relative abundance increased during P0–8 from about

Fig. 8. The time-related changes in the relative abundance of ŽA. Cx 45, ŽB. Cx 43 and ŽC. Cx 26 mRNAs as a percentage of the maximum daily mean value observed for each gene during the first 28 postnatal days ŽP0–28.. For Cx 45 ŽA., each point represents the mean"S.E.M. calculated from two or three animals, except for P8, 17 and 27 Ž ns1.. For Cx 43 ŽB. and for Cx 26 ŽC., each point represents the mean"S.E.M. from two or three animals for each postnatal day, except for P8 and 19 Ž ns1..

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in Fig. 7. The changes in percentage relative abundance are summarised in the histograms in Figs. 8–10, respectively. Cx 45 did not show any significant changes in relative abundance during P0–28 Ž P s 0.143.. There was no significant difference in mean relative abundance between

Fig. 9. The time-related changes in the relative abundance of ŽA. Cx 32, ŽB. Cx 37 and ŽC. Cx 40 mRNAs as a percentage of the maximum daily mean value observed for each gene during the first 28 postnatal days ŽP0–28.. For Cx 32 ŽA., each point represents the mean"S.E.M. calculated from three to five animals, except for P1, 15, 17, 20, 23, 24, 26, 27, and 30 Ž ns 2. and P29, 31 and 40 Ž ns1.. For Cx 37 ŽB. each point represents the mean"S.E.M. calculated from two to four animals except for P17, 20, 27 29 and 40 Ž ns1. and for Cx 40 ŽC., each point represents the mean"S.E.M. from two to four animals for each postnatal day, except for P10, 20, 24, 26, 27, 29 and 40 Ž ns1..

3.4. Postnatal expression of Cx mRNA in rat neocortex Examples of the PCR product bands obtained for Cx 45, Cx 43 and Cx 26 are illustrated in Fig. 5, for Cx 32, Cx 37 and Cx 40 in Fig. 6 and for Cx 36, Cx 30 and Cx 46

Fig. 10. The time-related changes in the relative abundance of ŽA. Cx 30, ŽB. Cx 46 and ŽC. Cx 36 mRNAs as a percentage of the maximum daily mean value observed for each gene during the first 28 postnatal days ŽP0–28.. For Cx 30 ŽA., each point represents the mean"S.E.M. calculated from two animals, except for P8, 14, 22, 29, 31 and 40 Ž ns1.. For Cx 46 ŽB. each point represents the mean"S.E.M. calculated from two animals except for P10, 14, 24, 27, 29, 31 and 42 Ž ns1. and for Cx 36 ŽC., each point represents the mean"S.E.M. from two animals for each postnatal day, except for P14 and 40 Ž ns1..

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P0–4 Ž88%. and P11–14 Ž92%. or P22–28 Ž88%. ŽFig. 8A.. The changes in the relative abundance of Cx 43 during P0–28 were highly significant Ž P s 0.0004.. The time course for Cx 43 can be roughly divided into two phases. During the first 2 postnatal weeks the relative abundance increased from about 35% to about 80%. The differences in mean relative abundance were highly significant Ž P 0.01. between P0–4 Ž45%. and P11–14 Ž73%. and between P0–4 Ž45%. and P22–28 Ž75%. Ž P - 0.001.. During weeks 3 and 4 postnatal and in adult tissue levels remained stable Ž70%–90%. ŽFig. 8B.. The difference in mean relative abundance between P11–14 and P22–28 Ž73% vs. 75%. was not significant. A highly significant change in the mean relative abundance of Cx 26 was observed during P0–28 Ž P s 0.0015.. During the first 2–3 postnatal days the relative abundance of Cx 26 increased from around 45% to about 75%. The difference between P0–4 Ž61%. and P11–14 Ž90%. was highly significant Ž P - 0.01.. Expression levels then remained approximately constant at 80%–95% during the second and third weeks postnatal before starting to decline in the fourth week. The differences between P11–14 Ž90%. and P22–28 Ž83%. and between P0–4 Ž61%. and P22–28 Ž83%. were not significant. Cx 26 mRNA levels in the adult tissue were similar to the P0 level ŽFig. 8C.. The change in relative abundance of Cx 32 during P0–28 was highly significant Ž P - 0.0001.. During the first 3 postnatal weeks Cx 32 mRNA abundance increased steadily from about 20% on P0 to 100% on day 19. Mean relative abundance then remained about 80–95% during the fourth postnatal week, similar to the level in the adult samples ŽFig. 9A.. The difference in mean relative abundance between P0–4 Ž35%. and P11–14 Ž68%. was significant Ž P - 0.05.. Highly significant differences Ž P 0.001. were also observed between P0–4 Ž35%. and P22–

28 Ž94%. and between P11–14 Ž68%. and P22–28 Ž94%. Ž P - 0.01.. The change in relative abundance of Cx 37 was highly significant during P0–28 Ž P s 0.0006.. Mean relative abundance increased steadily from about 50% at birth to around 100% on P14 and then decreased to about 50% again around P28. Mean relative abundance during P11–14 Ž91%. was significantly higher Ž P - 0.001. than during P0–4 Ž57%. and was significantly higher Ž P - 0.01. than during P22–28 Ž55%.. The difference between P0–4 Ž57%. and P22–28 Ž55%. was not significant. Cx 37 mRNA relative abundance in the adult samples was similar to that observed in the early postnatal period ŽFig. 9B.. There was a significant change Ž P s 0.0001. in the relative abundance of Cx 40 during P0–28. The mean relative abundance during P11–14 Ž79%. was significantly higher Ž P - 0.001. than during P0–4 Ž29%. or during P22–28 Ž52%. Ž P - 0.05.. The difference between P0–4 Ž29%. and P22–28 Ž52%. was also significant Ž P - 0.05. ŽFig. 9C.. The relative abundance of Cx 30 mRNA showed no significant change during P0–28 Ž P s 0.052.. The mean relative abundance during P0–4 Ž40%., P11–14 Ž64%. and P22–28 Ž40%. did not differ significantly. A relatively high S.E.M. for the mean relative abundance was evident on most days during P0–28 ŽFig. 10A.. Cx 46 mRNA mean relative abundance was low Ž25%. during the first few postnatal days and increased steadily to maximum values Ž80%–100%. around P15 ŽFig. 10B.. Cx 46 mRNA abundance then remained relatively constant during postnatal weeks 3–6. The mean relative abundance during P11–14 Ž76%. and during P22–28 Ž64%. was significantly higher than during P0–4 Ž27%. Ž P - 0.001 and P - 0.01, respectively. but the difference in Cx 46 mRNA mean relative abundance during P11–14 Ž76%. and P22–28 Ž64%. was not significant.

Fig. 11. Northern blotting signals from DIG-labeled GAPDH, Cx 36 and Cx 43 oligonucleotides hybridised to 15 mg total RNA per lane, from individual animals sacrificed on various postnatal days ŽP1–40.. The signal intensity and band area on four blots were determined for each animal and each gene to calculate a mean " S.E.M. for each postnatal day. No hybridisation signals were detected using DIG-labeled Cx 30 probes.

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Cx 36 mRNA relative abundance increased steadily from about 50% on P0 to reach maximum levels around P12–15 Ž90%–100%. before declining steadily during the third and fourth weeks to values similar to those observed at birth ŽFig. 10C.. The overall change in Cx 36 mRNA

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mean relative abundance during P0–28 was highly significant Ž P s 0.0002.. Relative abundance during P11–14 Ž97%. was significantly higher Ž P - 0.001. than during P0–4 Ž62%. and during P22–28 Ž68%. Ž P - 0.01.. The difference in mean relative abundance between P0–4 Ž62%. and P22–28 Ž68%. was not significant. 3.5. GAPDH, Cx 30, Cx 43 and Cx 36 expression time courses determined by Northern blotting

Fig. 12. The time-related changes in the relative abundance of ŽA. GAPDH, ŽB. Cx 43 and ŽC. Cx 36 mRNAs as a percentage of the maximum daily mean value observed for each gene during the first 28 postnatal days ŽP0–28. after Northern blotting. For GAPDH ŽA., each point represents the mean"S.E.M. calculated from two animals, except for P30 and 40 Ž ns 4. and P22, 24, 26, 45 and 53 Ž ns1.. For Cx 46 ŽB. each point represents the mean"S.E.M. calculated from two animals except for P42 Ž ns 4. and P12, 14, 16 and 18 Ž ns1. and for Cx 36 ŽC., each point represents the mean"S.E.M. from two animals for each postnatal day.

We were unable to detect Cx 30 mRNA in any of the samples by Northern blotting. Representative Northern blots for GAPDH and the Cx 36 and 43 are illustrated in Fig. 11 and the results from all the animals and time points are summarised in Fig. 12. Cx mRNA relative abundance was not normalised to GAPDH abundance. The relative abundance of GAPDH mRNA increased during the first postnatal week, from about 65% at birth rising to around 95% by P8, then remained unchanged between 70% and 90% from the end of the second week into adulthood. The time-related changes in GAPDH mRNA relative abundance were significant Ž P s 0.0045.. The mean relative abundance during P0–4 Ž61%. was significantly lower Ž P - 0.01. than during P11–14 Ž86%. but not significantly different to the mean abundance during P23–28 Ž68%.. The difference between P11–14 Ž86%. and P22–28 Ž68%. was not significant ŽFig. 12A.. The relative abundance of Cx 43 mRNA, determined by Northern blotting, was relatively low Ž25%–30%. in the first few postpartum days, but steadily increased during the first 2 postnatal weeks. Expression then remained at a high, constant level into adulthood Ž85%–100%. ŽFig. 12B.. The changes in relative abundance during P0–28 were highly significant Ž P - 0.0002.. The mean relative abundance during P0–4 Ž26%. was significantly lower Ž P - 0.05. than during P11–14 Ž72%. and significantly lower Ž P - 0.001. than during P22–28 Ž81%.. The difference in mean relative abundance between P11–14 Ž72%. and P22–28 Ž81%. was not significant. Cx 36 mRNA expression determined by Northern blotting also showed highly significant time-related changes in mean relative abundance Ž P s 0.0001. ŽFig. 12C.. Levels increased steadily from 40%–50% at birth, to peak at 90%–100% around postnatal day 10, before declining during weeks 3 and 4 to relatively constant levels Ž10%– 15%. which were lower than those observed at birth. Mean relative abundance during P8–11 Ž92%. was significantly higher Ž P - 0.05. than during P0–4 Ž53%. and also significantly higher Ž P - 0.001. than during P22–28 Ž24%.. Mean relative abundance during P22–28 Ž24%. was significantly lower Ž P - 0.01. than during P0–4 Ž53%.. 4. Discussion The nine Cx selected for investigation in this study were chosen because they have all been shown to be

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expressed at the mRNA andror protein level in rodent nervous tissue and further, the expression of all but Cx 46 has been demonstrated in one or more cellular compartments in the brain. Cx 46 mRNA and protein expression has been localised in Schwann cells in the peripheral nervous system w5x. In this study we used an RT–PCR based method to determine the time-related changes in mRNA expression for the nine Cx in rat neocortex. Furthermore, we used a daily sampling interval during the first 3 postnatal weeks to obtain a high time resolution during the transient period of extensive dye-coupling between neocortical neurons w36x. The time courses for mRNA expression determined by this RT–PCR method show good agreement with previously published time courses for Cx 26, Cx 32, and Cx 43 mRNA abundance in the visual cortex w30x and for Cx 36 in brain w41x, determined by Northern blotting. Time-related changes in mRNA expression for Cx 30, Cx 36, Cx 37, Cx 40, Cx 45 and Cx 46 mRNA in the neocortex have not been previously reported. To compare directly the RT–PCR and Northern blotting methods, we also examined the postnatal changes in GAPDH, Cx 30, Cx 36 and Cx 43 mRNA abundance by Northern blotting. When considering Cx gene expression it is important to acknowledge that differences may exist between the pattern of expression at a functional level, at the protein level and at the mRNA level. There are reports indicating considerable differences between mRNA and protein levels for some Cx within certain tissues w22,43x suggesting that the functional expression of at least some Cx may be strongly regulated by post-transcriptional and post-translational processes. For example, Cx 37 and Cx 40 are highly expressed in the lung at the mRNA level although their protein levels in lung are low w44x. Similarly, Kren et al. w22x observed a transcriptional up-regulation of Cx 43 in regenerating hepatocytes, although Cx 43 protein, as in normal hepatocytes, was undetectable and Nadarajah et al. w30x also showed differences in the time course for expression at the mRNA level and at the protein level for Cx 26, Cx 32 and Cx 43 in the rat visual cortex. Dermietzel w10x suggested that the existence of cytoplasmic ‘‘pools’’ of Cx mRNA and protein may enable a rapid recruitment of gap junction coupling were appropriate.

The fluorescence of the PCR bands Žmean gray value = band area. was taken as a measure of the amount of product. To ensure that differences in the amount of amplified product between samples reflected only differences in the starting amount of a particular Cx mRNA in the 6-mg total RNA samples, the cDNA synthesis, PCR amplification and band visualisation protocols were standardised for each Cx to control experimenter determined variables. Factors such as RT efficiency, PCR primer hybridisation efficiency and the efficiency with which the Taq polymerase is able to amplify a particular cDNA sequence are constant for each particular Cx sequence and each primer pair, but they may vary for different Cx sequences. Thus, product yields can be compared within a time course for a given Cx PCR product but not compared between different Cx. We therefore make no statement about the relative abundance of mRNA between different Cx. Our observations relate specifically to the changes in abundance of a given Cx mRNA with time. The results for the Cx were not normalised with respect to GAPDH or b-actin values, since GAPDH exhibited its own time-dependent expression profile, with a significant increase in relative abundance during the first postnatal week. Normalisation of the individual values using b-actin was not performed, since it showed a constant expression profile and would have had no effect on the relative abundance profiles of the Cx. With the exception of Cx 26 and Cx 40, the inter-animal variation in the relative abundance of each Cx and ‘‘housekeeping’’ gene tested was not significant, indicating that observed differences reflected time-related changes. Cx 26 exhibited a significant inter-animal variation, although the differences in mean relative abundance between animals was similar to those observed for the other Cx, where no statistically significant difference was found. The statistical significance of the variation for Cx 26 was probably a consequence of the comparatively very small standard error of means, calculated from the duplicate samples. In contrast, Cx 40 showed large variations in the mean relative abundance between animals, suggesting that observed differences in mRNA expression levels may also be strongly influenced by factors independent of the time course.

4.1. Methodological considerations The use of Tri-Reagent w to isolate total RNA from neocortex homogenates and the subsequent isolation of polyŽA.q mRNA using magnetic bead separation proved to be a very effective way of excluding genomic DNA contamination. With the exceptions of Cx 35 and Cx 36, the Cx genes are uninterrupted by introns in their coding sequence w41x. b-Actin primers located in different exons of the gene were used for the ‘‘intron-differential’’ PCR test w20x to check for genomic DNA contamination. Every first strand cDNA sample proved negative for genomic DNA.

4.2. RT–PCR Õs. Northern blotting The time-related changes in mRNA expression for GAPDH, Cx 36 and Cx 43 determined by Northern blotting in this study are identical to those changes observed by RT–PCR. The increase in the relative abundance of Cx 43 mRNA during the first 2 weeks postpartum, followed by a relatively high and stable expression from the end of the second week, in both the RT–PCR study and after Northern blotting, is in agreement with the Northern blotting findings of Nadarajah et al. w30x and also reflects the

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Cx 43 protein expression time course in striatum as described by Dermietzel et al. w11x. However, it should be noted that the observations of Dermietzel et al. w11x describe the time course for the expression of Cx 26, Cx 32 and Cx 43 at the protein level in the rat striatum and that regional differences in Cx expression exist within the brain w7x. Striatal neurons exhibit a similar time course for dye-coupling compared with neocortical neurons, with stable coupling until about day 13, followed by a rapid decline, so that by P18 no striatal neurons are dye-coupled using neurobiotin w39x. However, striatal neurons show a lower incidence Ž50%–60%. and less extensive dye-coupling Žaverage five neurons coupled. during P1–10 w39x compared with neocortical neurons during the same period Žincidence of 100% and an average 45 neurons coupled. w36x. It is possible that the gap junctions coupling striatal neurons have different Cx compositions and different permeabilities to neurobiotin, therefore, the data from striatum may not necessarily accurately represent changes occurring in the neocortex. The time course for Cx 36 mRNA expression reported by Sohl ¨ et al. w41x in postnatal rat brain homogenates after Northern blotting also agrees very closely with our Northern blotting results from rat neocortex. Our RT–PCR results similarly show a significant increase followed by a significant decrease in Cx 36 mRNA expression during the first 4 postnatal weeks, although the post peak decline in mRNA levels after RT–PCR is apparently slower than that seen after the Northern blotting method. Interestingly, Condorelli et al. w7x failed to detect Cx 36 mRNA in rat striatum or cerebral cortex by Northern blotting. We observed weak PCR bands for Cx 30 throughout the first 4 postnatal weeks in neocortex but failed to detect a signal for Cx 30 from the same samples by Northern blotting. In comparison, Dahl et al. w9x also observed no Northern blotting hybridisation signal for Cx 30 on days P0 or P7 but a weak signal on P14 and abundant amounts of two Cx 30 transcripts on P31 in mouse brain. These observations indicate that the RT–PCR method is a suitable alternative to Northern blotting to follow changes in the relative abundance of an mRNA, with the advantage that RT–PCR is able to detect low abundance mRNA messages in cases where Northern blotting gives inconsistent or negative results. 4.3. Postnatal expression of Cx mRNA in rat neocortex The time-related changes in mRNA expression for the nine Cx can be broadly classified into three types. The first type is represented by Cx 30 and Cx 45 and shows no time-dependent changes in mRNA expression during the first 4 postnatal weeks. The second type represented by Cx 26, Cx 32, Cx 43 and Cx 46 shows a significant increase in mRNA expression during the first 2 postnatal weeks followed by a relatively high and stable expression or a gradual, non-significant decline in mRNA expression dur-

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ing the third and fourth postnatal weeks. The gradual decline in mRNA abundance may reach significance over a longer time span, as in the case of Cx 26 ŽFig. 8C.. The third type, represented by Cx 36, Cx 37 and Cx 40, shows significant increases in mRNA abundance during the first 2 postnatal weeks, followed by a significant decrease in abundance during the third and fourth postnatal weeks. None of the individual time courses for Cx mRNA relative abundance match closely the time course for neocortical neuronal dye-coupling w36x, particularly as all the Cx which did show time-related changes in mRNA expression in this study, showed a significant increase in mRNA abundance when dye-coupling is extensive and stable. One could speculate that the neuronal dye-coupling may be due to the expression of Cx not investigated here. Alternatively, the changes in neuronal dye-coupling could be due to a rearrangement of connexons to form different heterotypic gap junctions or a rearrangement of Cx to form different heteromeric combinations with altered neurobiotin permeability without requiring a net up- or downregulation of transcription of the Cx involved. Another possible explanation relates to the tissue used. The two published studies investigating time-related changes in postnatal expression of Cx mRNA have used brain or cortical homogenates as the tissue source w30,41x which as in the neocortex homogenates in this study, contain a mixture of cell types, including neurons, glial cells and vascular tissue. We cannot exclude the involvement of any of the Cx investigated in neuronal dye-coupling since it is possible that the Cx responsible for neuronal dye-coupling are also expressed in a much larger population of non-neuronal cells, in which case the changes in mRNA abundance in the neurons may be obscured by simultaneous changes in expression of the same mRNA in the non-neuronal cells. Cx 43 is one of the most abundant Cx expressed in the brain, where it has been localised in astrocytes, leptomeninges, endothelial cells, the ependymal layer and in various populations of neurons w11,12,27,29–32,40x. Similarly, Cx 32 is the main gap junction protein in oligodendrocytes w11,13,23,24,29,38x and has also been demonstrated in several different populations of neurons w11,27– 29x. Immunocytochemical studies of Cx 32 expression indicate that this Cx protein is not detectable in the prenatal brain and is first detectable in the rat cortex from about the end of the first week postnatal w11,30x. In contrast, Cx 32 mRNA was detectable at low levels in the neocortex at birth, in our RT–PCR study and by northern blotting in the study by Nadarajah et al. w30x, although Belliveau and Naus w1x first detected Cx 32 mRNA by in situ hybridisation in rat brain at P3 in the brainstem nuclei and not until P15 in the midbrain. The delayed first detection probably reflects differences in the sensitivities of the methods. Cx 32 immunoreactivity showed a large increase during the second and third weeks postnatal and then a small further increase in the fourth week w11,30x. This postnatal increase in Cx 32 protein expression mirrors the increase in Cx 32

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mRNA abundance observed during the first 2 to 3 weeks postpartum, in both our RT–PCR experiments and the Northern blotting study of Nadarajah et al. w30x. With a 14-day sampling frequency, Nadarajah et al. w30x reported Cx 32 mRNA levels to be lower on P28 compared with P14 and to further decrease to P42, whereas our results suggest that Cx 32 mRNA abundance remains high and stable and similar to levels in the adult neocortex from the third week postnatal. Belliveau and Naus w1x also observed increasing Cx 32 mRNA expression during the period P3–30, with adult expression levels similar to those at P30. Cx 26 immunoreactivity in the adult brain is, according to most reports, confined to non-neuronal cells such as the leptomeninges, ependyma and pinealocytes w11,31,37,42x. Cx 26 antibodies, which positively stained Cx 26 in leptomeninges and liver did not label astrocytes, oligodendrocytes or neurons w11,13,31x. Dermietzel et al. w11x showed that in striatum Cx 26 immunoreactivity declined dramatically during P0–6 and then gradually decreased further during the second to fourth weeks postpartum. Cx 26 immunoreactivity was low but still detectable at P28 although not at P42. Nadarajah et al. w30x, on the other hand, reported a significant increase in immunoreactivity, in the rat visual cortex, during the period P0–7, with a further increase during the second week postnatal and then a dramatic decline in immunoreactivity during the third and fourth weeks, such that they observed no signal at P28. What is intriguing about the Cx 26 antibody used by Nadarajah et al. w30x is that it co-localised with a MAP-2 neuron specific marker and the signal was distributed throughout 35%–45% of the cortical cell population in the rat visual cortex, during P3–14. In the same study, the expression of Cx 26 mRNA, as determined by Northern blotting, indicated a different time course. At the end of the second postnatal week Cx 26 mRNA levels were about 15% higher than at P0 and by P28 had only fallen to about 80% of the P0 level. Moreover, Cx 26 mRNA was still detectable by Northern blotting at P42. The results from our RT–PCR study agree to a large extent with this mRNA profile and further, show that most of the increase in Cx 26 mRNA levels during the early postnatal period occurs during the first few postpartum days, followed by a smaller, gradual increase to stable levels during the second to fourth weeks. Cx 37 expression has been demonstrated in rat and mouse brain w17,19,44x but there are no reports describing a postnatal time course for Cx 37 mRNA expression. We observed a significant increase in Cx 37 expression during the first 2 postnatal weeks followed by a significant decrease, with levels at P28 similar to those during the very early postnatal period. Cx 37 mRNA in blood vessel endothelium in the brain w35x probably represents a significant source of the Cx 37 mRNA detected. Cx 40 mRNA expression was very variable between individuals, but significant differences in the relative abun-

dance did indicate time-related changes, similar to those for Cx 37, with an increase during the first 2 postnatal weeks being followed by a decline to adult levels by the end of the fourth week. Hennemann et al. w19x detected low levels of Cx 40 mRNA by Northern blotting in embryonic brain, but not in adult brain, even although Cx 40 has been demonstrated in blood vessels w2,3,16x and specifically in the microvessels from rat brain w25x. This discrepancy illustrates the difficulties in detecting low abundance mRNAs by Northern blotting. The similar time courses for Cx 37 and Cx 40 mRNA expression may reflect a time course related to angiogenesis. Cx 45 protein w4x and mRNA w18x expression has been demonstrated in brain and both Cx 45 mRNA and protein have been co-localised with Cx 32 mRNA and protein in oligodendrocytes w13,23x. There are no previous reports describing a time course for Cx 45 expression in brain. Our study demonstrates clearly that Cx 45 mRNA abundance in rat neocortex shows very little variation during the first 4 postnatal weeks. Overall, our results indicate clear differences in time-related changes in Cx mRNA expression in the rat neocortex during the postnatal period. The postnatal changes in mRNA expression presented here for Cx 26, Cx 32, Cx 36 and Cx 43 are in good agreement with, and complement, previously published results. The postnatal variations in mRNA expression for these four Cx and for those of Cx 30, Cx 37, Cx 40, Cx 45 and Cx 46, which have not been previously reported, do not demonstrate changes that can be obviously related to the time-related changes in postnatal neuronal dye-coupling.

Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft ŽSu 104r3-1. and by the Friedrich-BaurStiftung.

References w1x D.J. Belliveau, C.C. Naus, Cellular localization of gap junction mRNAs in developing rat brain, Dev. Neurosci. 17 Ž1995. 81–96. w2x E.C. Beyer, K.E. Reed, E.M. Westphale, H.L. Kanter, D.M. Larson, Molecular cloning and expression of rat connexin-40, a gap junction protein expressed in vascular smooth muscle, J. Membr. Biol. 127 Ž1992. 69–76. w3x R. Bruzzone, J.A. Haefliger, R.L. Gimlich, D.L. Paul, Connexin40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins, Mol. Biol. Cell 4 Ž1993. 7–20. w4x A. Butterweck, U. Gergs, C. Elfgang, K. Willecke, O. Traub, Immunochemical characterization of the gap junction protein connexin45 in mouse kidney and transfected human HeLa cells, J. Membr. Biol. 141 Ž1994. 247–256. w5x K.J. Chandross, J.A. Kessler, R.I. Cohen, E. Simburger, D.C. Spray, P. Bieri, R. Dermietzel, Altered connexin expression after peripheral nerve injury, Mol. Cell. Neurosci. 7 Ž1996. 501–518.

G. Prime et al.r DeÕelopmental Brain Research 119 (2000) 111–125 w6x P. Chomczynski, N. Sacchi, Single step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction, Anal. Biochem. 162 Ž1987. 156–159. w7x D.F. Condorelli, R. Parenti, F. Spinella, A.T. Salinaro, N. Belluardo, V. Cardile, F. Cicirata, Cloning of a new gap junction gene ŽCx36. highly expressed in mammalian brain neurons, Eur. J. Neurosci. 10 Ž1998. 1202–1208. w8x B.W. Connors, L.S. Bernado, D.A. Prince, Coupling between neurons of the developing rat neocortex, J. Neurosci. 3 Ž1983. 773–782. w9x E. Dahl, D. Manthey, Y. Chen, H.-J. Schwarz, Y.S. Chang, P.A. Lalley, B.J. Nicholson, K. Willecke, Molecular cloning and functional expression of mouse connexin-30, a gap junction gene highly expressed in adult brain and skin, J. Biol. Chem. 271 Ž1996. 17903–17910. w10x R. Dermietzel, Molecular diversity and plasticity of gap junctions in the nervous system, in: D.C. Spray, R. Dermietzel ŽEds.., Gap Junctions in the Nervous System, R.G. Landes, Austin, TX, USA, 1996, pp. 19 and 22. w11x R. Dermietzel, O. Traub, T.K. Hwang, E. Beyer, M.V.L. Bennett, D.C. Spray, K. Willecke, Differential expression of three gap junction proteins in developing and mature brain tissues, Proc. Natl. Acad. Sci. U. S. A. 86 Ž1989. 10148–10152. w12x R. Dermietzel, E.L. Hertberg, J.A. Kessler, D.C. Spray, Gap junctions between cultured astrocytes: immunocytochemical, molecular and electrophysiological analysis, J. Neurosci. 11 Ž1991. 1421–1432. w13x R. Dermietzel, M. Farooq, J.A. Kessler, H. Althaus, E.L. Hertzberg, D.C. Spray, Oligodendrocytes express gap junction proteins connexin32 and connexin45, Glia 20 Ž1997. 101–114. w14x G. Engler-Blum, M. Meier, J. Frank, G.A. Muller, Reduction of ¨ background problems in nonradioactive Northern and Southern blot analyses enables higher sensitivity than 32 P-based hybridizations, Anal. Biochem. 20 Ž1993. 235–244. w15x C. Giaume, C. Fromaget, A. el-Aoumari, J. Cordier, J. Glowinski, D. Gros, Gap junctions in cultured astrocytes: single-channel currents and characterization of channel-forming protein, Neuron 6 Ž1991. 133–143. w16x D. Gros, T. Jarry-Guichard, I. ten Velde, A. de Maziere, M.J. van Kempen, J. Davoust, J.P. Briand, A.F. Moorman, H.J. Jongsma, Restricted distribution of connexin40, a gap junctional protein in mammalian heart, Circ. Res. 74 Ž1994. 839–851. w17x J.-A. Haefliger, R. Bruzzone, N.A. Jenkins, D.J. Gilbert, N.G. Copeland, D.L. Paul, Four novel members of the connexin family of gap junction proteins: molecular cloning, expression and chromosome mapping, J. Biol. Chem. 267 Ž1992. 2057–2064. w18x H. Hennemann, H.-J. Schwarz, K. Willecke, Characterisation of gap junction genes expressed in F9 embryonic carcinoma cells: molecular cloning of mouse connexin31 and -45 cDNAs, Eur. J. Cell Biol. 57 Ž1992. 51–58. w19x H. Hennemann, T. Suchyna, H. Lichtenberg-Frate, ´ S. Jungbluth, E. Dahl, J. Schwarz, J. Nicholson, K. Willecke, Molecular cloning and functional expression of mouse connexin40, a second gap junction gene preferentially expressed in lung, J. Cell Biol. 117 Ž1992. 1299–1310. w20x H. Ji, Q. Zhang, B.S. Leung, Survey of oncogene and growth factorrreceptor gene expression in cancer cells by intron-differential RNArPCR, Biochem. Biophys. Res. Commun. 170 Ž1990. 569–575. w21x N.M. Kumar, N.B. Gilula, The gap junction communication channel, Cell 84 Ž1996. 381–388. w22x B.T. Kren, N.M. Kumar, S.Q. Wang, N.B. Gilula, C.J. Steer, Differential regulation of multiple gap junction transcripts and proteins during rat liver regeneration, J. Cell Biol. 123 Ž1993. 707–718. w23x P. Kunzelmann, I. Blumcke, O. Traub, R. Dermietzel, K. Willecke, Coexpression of connexin45 and -32 in oligodendrocytes of rat brain, J. Neurocytol. Ž1997. 17–22. w24x J. Li, E.L. Hertzberg, J.I. Nagy, Connexin32 in oligodendrocytes and association with myelinated fibers in mouse and rat brain, J. Comp. Neurol. 379 Ž1997. 571–591.

125

w25x T.L. Little, E.C. Beyer, B.R. Duling, Connexin 43 and connexin 40 gap junctional proteins are present in arteriolar smooth muscle and endothelium in vivo, Am. J. Physiol. 268 Ž1995. H729–H739. w26x J.J. Lo Turco, A.R. Kreigstein, Clusters of coupled neuroblasts in embryonic neocortex, Science 252 Ž1991. 563–566. w27x P.E. Micevych, L. Abelson, Distribution of mRNAs coding for liver and heart gap junction proteins in the rat central nervous system, J. Comp. Neurol. 305 Ž1991. 96–118. w28x P.E. Micevych, P. Popper, G.I. Hatton, Connexin 32 mRNA levels in the rat supraoptic nucleus: up-regulation prior to parturition and during lactation, Neuroendocrinology 63 Ž1996. 39–45. w29x B. Nadarajah, D. Thomaidou, W.H. Evans, J.G. Parnavelas, Gap junctions in the adult cerebral cortex: regional differences in their distribution and cellular expression of connexins, J. Comp. Neurol. 376 Ž1996. 326–342. w30x B. Nadarajah, A.M. Jones, W.H. Evans, J.G. Parnavelas, Differential expression of connexins during neocortical development and neuronal circuit formation, J. Neurosci. 17 Ž1997. 3096–3111. w31x J.I. Nagy, P.A. Ochalski, J. Li, E.L. Hertzberg, Evidence for the colocalization of another connexin with connexin-43 at astrocytic gap junctions in rat brain, Neuroscience 78 Ž1997. 533–548. w32x P.A. Ochalski, U.N. Frankenstein, E.L. Hertzberg, J.I. Nagy, Connexin-43 in rat spinal chord: localization in astrocytes and identification of heterotypic astro-oligodendrocytic gap junctions, Neuroscience 76 Ž1997. 931–945. w33x A. Peinado, R. Yuste, L.C. Katz, Gap junctional communication and the development of local circuits in neocortex, Cereb. Cortex Ž1993., 488–498. w34x A. Peinado, R. Yuste, L.C. Katz, Extensive dye coupling between rat neocortical neurons during the period of circuit formation, Neuron 10 Ž1993. 103–114. w35x K.E. Reed, E.M. Westphale, D.M. Larson, H.Z. Wang, R.D. Veenstra, E.C. Beyer, Molecular cloning and functional expression of human connexin37, an endothelial cell gap junction protein, J. Clin. Invest. 91 Ž1993. 997–1004. w36x B. Rorig, ¨ G. Klausa, B. Sutor, Intracellular acidification reduced gap junction coupling between immature rat neocortical pyramidal neurones, J. Physiol. 490 Ž1996. 31–49. w37x J.C. Saez, V.M. Berthoud, R. Kadle, O. Traub, B.J. Nicholson, M.V. Bennett, R. Dermietzel, Pinealocytes in rats: connexin identification and increase in coupling caused by norepinephrine, Brain Res. 568 Ž1991. 265–275. w38x S.S. Scherer, S.M. Deschenes, Y.T. Xu, J.B. Grinspan, K.H. Fischbeck, D.L. Paul, Connexin32 is a myelin-related protein in the PNS and CNS, J. Neurosci. 15 Ž1995. 8281–8294. w39x B. Schlosser, R. Lipowsky, G. ten Bruggencate, B. Sutor, Develop¨ mental changes in gap junction coupling between neostriatal neurons, Eur. J. Physiol. 433 Ž1997. R132, Suppl. 6. w40x E. Simburger, A. Stang, M. Kremer, R. Dermietzel, Expression of connexin43 mRNA in adult rodent brain, Histochem. Cell Biol. 107 Ž1997. 127–137. w41x G. Sohl, ¨ J. Degen, B. Teubner, K. Willecke, The murine gap junction gene connexin36 is highly expressed in mouse retina and regulated during brain development, FEBS Lett. 428 Ž1998. 27–31. w42x D.C. Spray, A.P. Moreno, J.A. Kessler, R. Dermietzel, Characterization of gap junctions between cultured leptomeningeal cells, Brain Res. 568 Ž1991. 1–14. w43x O. Traub, R. Eckert, H. Lichtenberg-Frate, ´ C. Elfgang, B. Bastide, K.H. Scheidtmann, D.F. Hulser, K. Willecke, Immunochemical and electrophysiological characterization of murine connexin40 and -43 in mouse tissues and transfected human cells, Eur. J. Cell Biol. 64 Ž1994. 101–112. w44x K. Willecke, R. Heynkes, E. Dahl, R. Stutenkemper, H. Hennemann, S. Jungbluth, T. Suchyna, B.J. Nicholson, Mouse connexin37: Cloning and functional expression of a gap junction gene highly expressed in lung, J. Cell Biol. 114 Ž1991. 1049–1057.