Archives of Oral Biology 47 (2002) 417–421

Short communication

Postnatal changes in the nicotinic acetylcholine receptorsubunits in rat masseter muscle

T. Saitoa, Y. Ohnukib, Y. Saekib, Y. Nakagawaa, K. Ishibashia,K. Yanagisawab, A. Yamanec,∗

a Second Department of Oral and Maxillo-facial Surgery, Tsurumi University School of Dental Medicine,2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan

b Department of Physiology, Tsurumi University School of Dental Medicine, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japanc Department of Pharmacology, Tsurumi University School of Dental Medicine, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan

Accepted 6 December 2001

Abstract

No published study on synaptogenesis in masseter muscle has focused on the shift of nicotinic acetylcholine receptors(nAChRs) from the embryonic type (�2-, �-, �- and�-subunits) to the adult-type (�2-, �-, ε- and�-subunits) and the eliminationof nAChRs outside the neuromuscular junction. To identify the time course of the nAChR transitions in rat masseter musclebetween 1 and 63 days of age, the expression of�-, ε- and�-subunit mRNAs was analysed by competitive polymerase chainreaction in combination with reverse transcription. The expression of the�-subunit was high between 1 and 7 days of age, thendecreased by 95% (P < 0.0001) between 7 and 28 days, suggesting that the nAChR elimination occurs during this period.The quantity of theε-subunit increased by approximately 600% (P < 0.0001) between 1 and 21 days of age, whereas thequantity of the�-subunit decreased by 85% (P < 0.0001) during the same period. This result indicates that the nAChR typeshift is terminated at 21 days of age. The feeding behaviour of the rats inevitably changed from suckling to biting after 19 daysof age, because they were weaned at that age. As the nAChR type shift was terminated soon after weaning, the terminationcould be related to the change in feeding behaviour. However, it might also be the case that nAChR elimination is not directlyrelated to the change in feeding behaviour, as the elimination continued at the same rate for 9 days after weaning (from 19 to28 days of age). © 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Synaptogenesis; Nicotinic acetylcholine receptor;�-, ε- and�-subunits; Masseter muscle; Competitive RT-PCR; Rat

The expression, distribution, and subunit composition ofthe nicotinic acetylcholine receptor (nAChR) are known tochange during the development of skeletal muscle (Brehmand Henderson, 1988; Hall and Sanes, 1993). EmbryonicnAChRs, composed of�2-, �-, �- and�-subunits, are ex-pressed throughout muscle cells. As the development ofskeletal muscle progresses, the�-subunit is replaced byan ε-subunit to become the adult-type receptor (�2-, �-, ε-

Abbreviations: RT-PCR, reverse transcriptase-polymerase chainreaction

∗ Corresponding author. Tel.:+81-45-581-1001;fax: +81-45-573-9599.

E-mail address: gah03667@nifty.ne.jp (A. Yamane).

and�-). The adult and embryonic types outside the neuro-muscular junction are eliminated, and only the adult-typecontinues to be expressed at that junction.

The feeding behaviour of the rat apparently changesfrom suckling to biting between 17 and 25 days after birth(Maeda et al., 1981a). During this postnatal period, markedchanges in the masseter muscle also reportedly occur, de-scribed as follows. The masseter myofibres grow rapidlyand markedly (Maeda et al., 1981b; Miyata et al., 1996); alarge amount of the neonatal myosin heavy chain disappears(Miyata et al., 1996); the diameter of the motoneuronesinnervating the masseter increases, and the electromyo-graphic pattern changes (Kubota et al., 1988; Miyata et al.,1996). As synaptogenesis is known to be closely related to

0003-9969/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.PII: S0003-9969(02)00010-9

418 T. Saito et al. / Archives of Oral Biology 47 (2002) 417–421

myogenesis (Buonanno et al., 1998), these findings implythat significant changes in synaptogenesis occur during thepostnatal period, including elimination of, and subunit shiftin the nAChR.

There are to date, to the best of our knowledge, nopublished data on synaptogenesis during the postnatal de-velopment of the murine masseter muscle. We have nowanalysed the expression of�-subunit mRNA, which occursrestrictedly in skeletal muscles throughout development(Lindstrom, 2000), to determine the time course of nAChRsubunit elimination. In addition, we have analysed theexpression ofε- and �-subunit mRNAs to identify thetime course of the subunit shift in the rat masseter musclebetween 1 and 63 days of age.

Pregnant female rats were purchased (Nippon Clea,Tokyo, Japan) and kept in separate cages under alternating12 h periods of light and dark. The pups were removed fromtheir mothers at 19 days of age (weaning) and fed a pelletdiet (CE-2; Nippon Clea, Tokyo, Japan) with tap water adlibitum thereafter. Groups of six rats were killed by exsan-guination under anaesthesia with pentobarbital sodium ata fatal overdose of 50 mg/kg at 1, 7, 14, 21, 28 and 63days of age. The middle portion of the superficial massetermuscle was dissected out, immediately frozen, and stored at−80◦C until subsequent analysis. Experimental protocolsfor animal handling were reviewed and approved by the In-stitutional Animal Care Committee of Tsurumi UniversitySchool of Dental Medicine.

Total RNA extraction was performed according to themanufacturer’s specifications (Fast RNA kit Green; Bio101, Vista, CA, USA). The treatment of deoxyribonucleaseI, reverse transcription, and competitive PCR were as de-scribed by Yamane et al. (2000a,b). In the conventional PCRtechnique, a small difference in the starting amount of targetDNA can result in a large change in the yield of the finalproduct because of the exponential nature of the PCR reac-tion. The plateau effect after many cycles can lead to an in-accurate estimation of the final product yield. Furthermore,because the PCR amplification depends on the reaction effi-ciency, a small change in the efficiency can lead to a majordifference in the final product yield. To overcome theseproblems, the competitor (internal standard), which has thesame primer sequences as those of the target DNA at the 3′

and 5′ ends, was amplified simultaneously with the target(Gilliland et al., 1990; Siebert and Larrick, 1992; Yamaneet al., 1998, 2000a). The competitors were constructedaccording to the manufacturer’s instructions for the PCRMIMIC Construction Kit (Clontech Laboratory Inc., PaloAlto, CA, USA) and were amplified with 50 ng of the totalcDNA in the presence of a primer pair specific to the targetgenes in a thermal cycler (TP3000; TaKaRa Biochemicals,Shiga, Japan). The sequences of primers specific for the�-and ε-subunits designed for the present study were as fol-lows: for the�-subunit, 5′-CAG CCG TCT ACA GTG GGATG-3′ and 5′-CTG CCA GTC GAA AGG GAA GTA-3′

(LaPolla et al., 1984); and for theε-subunit, 5′-ATT GAA

GAG CTT AGC CTG TA-3′ and 5′-TAC ACC TGC AAAATC GTC CT-3′ (Buonanno et al., 1989). The primer pair forthe�-subunit had been designed by Rohwedel et al. (1995).To determine the specificity of primers for theε-, �- and�-subunits of the nAChR in skeletal muscles, the sequencesof the resultant PCR products were analysed in an automatedsequencer and confirmed against the GenBank database.The primer pair for the house-keeping geneS16 (ribosomalprotein) for normalizing had been designed previously andits specificity was confirmed by Washabaugh et al. (1998).

Competitive PCR products were separated by elec-trophoresis on a 3% agarose gel containing ethidium bro-mide. An example of the electrophoretic gel pattern of the�-subunit and its competitor is shown in Fig. 1A. The sizesof the PCR products of the�-subunit and its competitorDNA fragment, which had identical primer-annealing sitesto those of the�-subunit, were 235 bp (the lower bands inFig. 1A) and 291 bp (the upper bands in Fig. 1A), respec-tively.

There was an observable inverse correlation in fluorescentintensity between the�-subunit and the competitor bands onthe gel. The fluorescent intensities of the bands of the tar-get genes and their respective competitors were measuredwith an image analyser (FLA-3000; Fujifilm, Tokyo, Japan).We then calculated the ratios of the fluorescent intensitiesof the target gene bands to those of their respective com-petitor bands. The cDNA quantity of each target gene wasestimated by the calculated ratio of the fluorescent inten-sity from a standard curve generated for each target gene,as described by Yamane et al. (2000a,b). Fig. 1B shows thestandard curve calculated by using the image-analysis dataof electrophoretic bands as shown in Fig. 1A. The slope ofthe regression line was 0.486 and the correlation coefficientwas 0.999, which was significantly different from zero (P <

0.001). The quantity of each target gene was divided by thequantity of S16 to normalise the variations in the yield oftotal RNA and the efficiency of reverse transcription. Theresulting ratio was expressed as a percentage relative to themean of each target gene at 1 day of age. Scheffe’s methodwas used to compare the means between any two groups.

Table 1 shows changes in body weight in groups of rats atvarious ages. The body weight increased by approximately377% (P < 0.001) between 1 and 14 days of age, then did

Table 1Weight of the rats at each time point

Age (days) Weight (g) n

1 5.3± 0.3 67 9.7± 1.2 6

14 25.3± 1.4 621 23.3± 2.6 628 66.9± 8.3 663 291.9± 12.2 6

Mean± 1 S.D. is shown in each groupn, the number of rats.

T. Saito et al. / Archives of Oral Biology 47 (2002) 417–421 419

Fig. 1. (A) Electrophoretic gel pattern of the nAChR�-subunit and its competitor after competitive PCR to examine the relation between theamount of PCR products and the concentration of the�-subunit cDNA. (B) The regression line for the�-subunit generated from the resultof image-analysis of the electrophoretic bands in (A). The formula of the regression line isy = 0.486x + 0.875, wherey is the logarithmicvalue of the ratio of the fluorescent intensity in the�-subunit band to that in its competitor band, andx is the logarithmic value of theconcentration of the�-subunit cDNA. MW, molecular weight markers; bp, base pairs.

not change significantly between 14 and 21 days of age. Thisretardation of growth might be related to the weaning at 19days of age. After that, the body weight began to increaseagain and showed a greater than 12-fold increase between21 and 63 days of age (P < 0.0001).

Because the�-subunit of the nAChR is present through-out the whole process of synaptogenesis, the amount of the�-subunit mRNA should reflect the amount of the receptor.In addition, its expression is restricted in skeletal muscles,which is different from the expression of the�- and�- sub-units (Lindstrom, 2000). Thus, to identify the time courseof the elimination of the nAChR during the development ofthe rat masseter, we analysed the expression of the�-subunitmRNA (Fig. 2A). That mRNA was expressed markedly inthe masseter at 1 and 7 days of age, and its expression de-creased almost linearly by 95% (P < 0.0001) between 7and 28 days of age. We interpreted these results as evidencethat the nAChR in the masseter was mainly eliminated dur-ing this period.

Elimination of the nAChR occurs between embryonic day15 and the newborn stage in mouse tongue muscle (Yamaneet al., 2001) and begins just before birth and ends at 18 daysof age after birth in mouse gastrocnemius muscle (Yamaneet al., 2001; Zoubine et al., 1996). Thus, its elimination

appears to occur later in murine masseter muscle than intongue and gastrocnemius muscles.

We determined the expression of theε-subunit (Fig. 2B)and�-subunit (Fig. 2C) mRNAs to identify the time courseof the subunit shift. Littleε-subunit mRNA was expressedat 1 day of age in the masseter muscle, indicating that thesubunit shift had already begun. After that, the subunit ex-pression showed an approximately seven-fold increase (P <

0.0001) by 21 days of age, suggesting that the amount ofadult-type nAChR had rapidly increased. After 21 days ofage, the amount of theε-subunit decreased by approximately60% (P < 0.0001) up to 28 days of age, then increasedagain by approximately 80% (P < 0.01) up to 63 days ofage. The decrease between 21 and 28 days of age mightreflect the elimination of the receptor and the increase be-tween 28 and 63 days of age might be due to an increasein the amount of adult-type receptor in the neuromuscularjunction (Steinbach, 1981).

The �-subunit mRNA was highly expressed at 1 day ofage, then showed a marked decrease (85%,P < 0.0001)in expression until 21 days of age (Fig. 2C). This decreaseindicates that the amount of embryonic type nAChR fellmarkedly. After that, its expression continued to decreasegradually until 63 days of age; 7% of the mean value at 1

420 T. Saito et al. / Archives of Oral Biology 47 (2002) 417–421

Fig. 2. Relative changes in the mRNA for the (A)�-, (B) ε- and(C) �- subunits in the rat masseter muscle at 1, 7, 14, 21, 28, and63 days of age assessed by competitive RT-PCR. Each point withits vertical bar represents the mean±1 S.D. of six samples, exceptat 63 days of age (where one sample was accidentally lost). Thevertical axis is expressed as a percentage of the mean value at 1day of age. The rats were weaned (arrow) at 19 days of age.

day remained at 63 days of age. Based on the expressionprofiles of theε- and�-subunit mRNAs, we suggest that thesubunit shift for the nAChR actively progresses after 1 dayof age and ends at 21 days of age.

We earlier reported that this subunit shift mainly occursbetween embryonic day 15 and the newborn stage in tonguemuscle (Yamane et al., 2001), so the present findings suggest

a later shift in masseter than in tongue muscle. The subunitshift occurs between 1 and 9 days of age in mouse tibialisanterior, extensor digitorum longus, sternomastoid, and di-aphragm muscles (Missias et al., 1996); the subunit shift inmasseter muscle, therefore, seems to begin at the same stage(perinatal) as for these muscles, but ends at a later stage.

Expression of the�-subunit persisted at 63 days of age.In mouse tibialis anterior, extensor digitorum longus, ster-nomastoid, diaphragm, and soleus muscles, the expressionof the �-subunit disappears by 14 days of age, whereas itpersists into adulthood in the extraocular muscles (Missiaset al., 1996). A study of Pax3/myf5 homozygous mutantmice suggested that craniofacial muscles, including themasseter and extraocular muscles, follow a unique develop-mental programme different from that of the trunk and limbmuscles (Tajbakhsh et al., 1997). The persistent expressionof the �-subunit mRNA at 63 days of age suggests thatthere may indeed be a unique programme for the postnataldevelopment of the masseter muscle.

We found that the subunit shift and elimination of thenAChR in the rat masseter are mostly completed at 21 and28 days of age, respectively. The feeding behaviour of ourrats inevitably changed from suckling to biting after 19 daysof age, because they were weaned at that age. As the re-ceptor subunit shift was terminated soon after weaning, thetermination might be related to the changes in feeding be-haviour. However, the elimination of the receptor continuedand the elimination rate did not appear to change by 9 daysafter weaning (from 19 to 28 days of age), so the elimi-nation might not be directly related to changes in feedingbehaviour.

Acknowledgements

We would like to thank Prof. M. Chiba, Tsurumi Univer-sity School of Dental Medicine, for his support and encour-agement throughout the present study. A part of the presentstudy was supported by Grants-in-Aid for funding scientificresearch (nos. 10671757 and 13671955), the Bio-venturesand High-Technology Research Center of the Ministry ofEducation, Culture, Sports, Science, and Technology ofJapan.

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