Journal of Molecular Neuroscience

, Volume 59, Issue 2, pp 300–308 | Cite as

Structural and Morphometric Comparison of Lower Incisors in PACAP-Deficient and Wild-Type Mice

  • B. Sandor
  • K. Fintor
  • D. Reglodi
  • D. B. Fulop
  • Z. Helyes
  • I. Szanto
  • P. Nagy
  • H. Hashimoto
  • A. Tamas


Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with widespread distribution. PACAP plays an important role in the development of the nervous system, it has a trophic and protective effect, and it is also implicated in the regulation of various physiological functions. Teeth are originated from the mesenchyme of the neural crest and the ectoderm of the first branchial arch, suggesting similarities with the development of the nervous system. Earlier PACAP-immunoreactive fibers have been found in the odontoblastic and subodontoblastic layers of the dental pulp. Our previous examinations have shown that PACAP deficiency causes alterations in the morphology and structure of the developing molars of 7-day-old mice. In our present study, morphometric and structural comparison was performed on the incisors of 1-year-old wild-type and PACAP-deficient mice. Hard tissue density measurements and morphometric comparison were carried out on the mandibles and the lower incisors with micro-CT. For structural examination, Raman microscopy was applied on frontal thin sections of the mandible. With micro-CT morphometrical measurements, the size of the incisors and the relative volume of the pulp to dentin were significantly smaller in the PACAP-deficient group compared to the wild-type animals. The density of calcium hydroxyapatite in the dentin was reduced in the PACAP-deficient mice. No structural differences could be observed in the enamel with Raman microscopy. Significant differences were found in the dentin of PACAP-deficient mice with Raman microscopy, where increased carbonate/phosphate ratio indicates higher intracrystalline disordering. The evaluation of amide III bands in the dentin revealed higher structural diversity in wild-type mice. Based upon our present and previous results, it is obvious that PACAP plays an important role in tooth development with the regulation of morphogenesis, dentin, and enamel mineralization. Further studies are required to clarify the molecular background of the effects of PACAP on tooth development.


PACAP Raman Tooth Development Micro-CT 



The study was supported by: OTKA (K104984), TAMOP 4.2.4.A/2-11-1-2012-0001„National Excellence Program”, Arimura Foundation, Bolyai Scholarship, “Lendulet” Program of the Hungarian Academy of Sciences. The authors thank Tamas Kiss for his assistance in micro-CT examinations.


  1. Arimura A, Somogyvari-Vigh A, Weill C, et al. (1994) PACAP functions as a neurotrophic factor. Ann N Y Acad Sci 739:228–243Google Scholar
  2. Bartlett JD (2013) Dental enamel development: proteinases and their enamel matrix substrates. ISRN Dent 2013:684607 ReviewPubMedPubMedCentralGoogle Scholar
  3. Bei M (2009) Molecular genetics of tooth development. Curr Opin Genet Dev 19:504–510CrossRefPubMedPubMedCentralGoogle Scholar
  4. Blechman J, Levkowitz G (2013) Alternative splicing of the pituitary adenylate cyclase-activating polypeptide receptor PAC1: mechanisms of fine tuning of brain activity. Front Endocrinol (Lausanne) 4:55Google Scholar
  5. Boskey AL, Robey PG (2013) The regulatory role of matrix proteins in mineralization of bone in osteoporosis. Vol. 1. Marcus R, Feldman D, Dempster DW, Luckey M, Cauley JA; 4th ed. Elsevier Academic Press, Waltham, MA, pp. 235–255.Google Scholar
  6. Butler WT, Ritchie H (1995) The nature and functional significance of dentin extracellular matrix proteins. Int J Dev Biol 39:169–179PubMedGoogle Scholar
  7. Cobourne MT, Hardcastle Z, Sharpe PT (2001) Sonic hedgehog regulates epithelial proliferation and cell survival in the developing tooth germ. J Dent Res 80:1974–1979CrossRefPubMedGoogle Scholar
  8. Dassule HR, Lewis P, Bei M, Maas R, McMahon AP (2000) Sonic hedgehog regulates growth and morphogenesis of the tooth. Development 127:4775–4785PubMedGoogle Scholar
  9. de Mul FF, Hottenhuis MH, Bouter P, Greve J, Arends J, ten Bosch JJ (1986) Micro-Raman line broadening in synthetic carbonated hydroxyapatite. J Dent Res 65:437–440CrossRefPubMedGoogle Scholar
  10. Dollish FR, Fateley WG, Bentley FF (1974) Characteristic Raman frequencies of organic compounds. Wiley, New YorkGoogle Scholar
  11. Faibish D, Ott SM, Boskey AL (2006) Mineral changes in osteoporosis: a review. Clin Orthop Relat Res 443:28–38 ReviewCrossRefPubMedPubMedCentralGoogle Scholar
  12. Featherstone JD, Lussi A (2006) Understanding the chemistry of dental erosion. Monogr Oral Sci 20:66–76CrossRefPubMedGoogle Scholar
  13. Fukae M, Shimizu M (1974) Studies on the proteins of developing bovine enamel. Arch Oral Biol 19:381–386CrossRefPubMedGoogle Scholar
  14. Gericke A, Qin C, Sun Y, et al. (2010) Different forms of DMP1 play distinct roles in mineralization. J Dent Res 89:355–359CrossRefPubMedPubMedCentralGoogle Scholar
  15. Goldberg M, Kellermann O, Dimitrova-Nakov S, Harichane Y, Baudry A (2014) Comparative studies between mice molars and incisors are required to draw an overview of enamel structural complexity. Front Physiol 5:359CrossRefPubMedPubMedCentralGoogle Scholar
  16. Gritli-Linde A, Bei M, Maas R, Zhang XM, Linde A, McMahon AP (2002) Shh signaling within the dental epithelium is necessary for cell proliferation, growth and polarization. Development 129:5323–5337CrossRefPubMedGoogle Scholar
  17. Hardcastle Z, Mo R, Hui CC, Sharpe PT (1998) The Shh signaling pathway in tooth development: defects in Gli2 and Gli3 mutants. Development 125:2803–2811PubMedGoogle Scholar
  18. Hargreaves KM, Berman LH (2016) Vital pulp therapy in Cohen’s pathways of the pulp expert consult, 11th edn. Elsevier, St. Louis, pp. 849–476Google Scholar
  19. Hart PS, Hart TC (2007) Disorders of human dentin. Cells Tissues Organs 186:70–77CrossRefPubMedPubMedCentralGoogle Scholar
  20. Hashimoto H, Shintani N, Tanaka K, et al. (2001) Altered psychomotor behaviors in mice lacking pituitary adenylate cyclase activating polypeptide (PACAP). Proc Natl Acad Sci U S A 98:13355–13360CrossRefPubMedPubMedCentralGoogle Scholar
  21. Hashimoto H, Hashimoto R, Shintani N, et al. (2009) Depression-like behavior in the forced swimming test in PACAP-deficient mice: amelioration by the atypical antipsychotic risperidone. J Neurochem 110:595–602CrossRefPubMedGoogle Scholar
  22. Hirose M, Niewiadomski P, Tse G, et al. (2011) Pituitary adenylyl cyclase-activating peptide counteracts hedgehog-dependent motor neuron production in mouse embryonic stem cell cultures. J Neurosci Res 89:1363–1374CrossRefPubMedPubMedCentralGoogle Scholar
  23. Ichikawa H, Sugimoto T (2003) Pituitary adenylate cyclase-activating polypeptide-immunoreactive nerve fibers in rat and human tooth pulps. Brain Res 980:288–292CrossRefPubMedGoogle Scholar
  24. Iwamoto T, Yamada A, Arakaki M, et al. (2011) Expressions and functions of neurotrophic factors in tooth development. J Oral Biosci 53:13–21CrossRefGoogle Scholar
  25. Juhasz T, Matta C, Katona E, et al. (2014) Pituitary adenylate cyclase-activating polypeptide (PACAP) signalling enhances osteogenesis in UMR-106 cell line. J Mol Neurosci 54:555–573CrossRefPubMedGoogle Scholar
  26. Juhasz T, Helgadottir SL, Tamas A, Reglodi D, Zakany R (2015a) PACAP and VIP signaling in chondrogenesis and osteogenesis. Peptides 66:51–57CrossRefPubMedGoogle Scholar
  27. Juhasz T, Szentleleky E, Somogyi C, et al. (2015b) Pituitary adenylate cyclase activating polypeptide (PACAP) pathway is induced by mechanical load and reduces the activity of hedgehog signaling in chondrogenic micromass cell cultures. Int J Mol Sci 16:17344–17367Google Scholar
  28. Jussila M, Thesleff I (2012) Signaling networks regulating tooth organogenesis and regeneration, and the specification of dental mesenchymal and epithelial cell lineages. Cold Spring Harb Perspect Biol 4:a008425CrossRefPubMedPubMedCentralGoogle Scholar
  29. Kiss P, Farkas J, Horvath G, et al. (2010) Neurobehavioral development in PACAP (pituitary adenylate cyclase activating polypeptide) knockout mice. FENS Abstr., vol. 5, 042.7Google Scholar
  30. Kieffer S, Peterkova R, Vonesch JL, Ruch JV, Peterka M, Lesot H (1999) Morphogenesis of the lower incisor in the mouse from the bud to early bell stage. Int J Dev Biol 43:531–539PubMedGoogle Scholar
  31. Kyrylkova K, Kyryachenko S, Biehs B, Klein O, Kioussi C, Leid M (2012) BCL11B regulates epithelial proliferation and asymmetric development of the mouse mandibular incisor. PLoS One 7:e37670CrossRefPubMedPubMedCentralGoogle Scholar
  32. Lelievre V, Ghiani CA, Seksenyan A, Gressens P, deVellis J, Waschek JA (2006) Growth factor-dependent actions of PACAP on oligodendrocyte progenitor proliferation. Regul Pept 137:58–66CrossRefPubMedGoogle Scholar
  33. Luukko K, Moshnyakov M, Sainio K, Saarma M, Sariola H, Thesleff I (1996) Expression of neurotrophin receptors during rat tooth development is developmentally regulated, independent of innervation, and suggests functions in the regulation of morphogenesis and innervation. Dev Dyn 206:87–99CrossRefPubMedGoogle Scholar
  34. Luukko K, Arumae U, Karavanov A, et al. (1997) Neurotrophin mRNA expression in the developing tooth suggests multiple roles in innervation and organogenesis. Dev Dyn 210:117–129CrossRefPubMedGoogle Scholar
  35. Matthaus C, Bird B, Miljkovic M, Chernenko T, Romeo M, Diem M (2008) Infrared and Raman microscopy in cell biology (chapter 10). Methods Cell Biol 89:275–308CrossRefPubMedPubMedCentralGoogle Scholar
  36. McCabe PS, Dummer PM (2012) Pulp canal obliteration: an endodontic diagnosis and treatment challenge. Int Endod J 45:177–197 ReviewCrossRefPubMedGoogle Scholar
  37. Mitsiadis TA, Luukko K (1995) Neurotrophins in odontogenesis. Int J Dev Biol 39:195–202PubMedGoogle Scholar
  38. Nanci A (2008) Dentin-pulp complex in ten Cate’s oral histology: development, structure, and function, 7th edn. Mosby, St. Louis, pp. 191–238Google Scholar
  39. Nicot A, Lelievre V, Tam J, Waschek JA, DiCicco-Bloom E (2002) Pituitary adenylate cyclase activating polypeptide and sonic hedgehog interact to control cerebellar granule precursor cell proliferation. J Neurosci 22:9244–9254PubMedGoogle Scholar
  40. Niewiadomski P, Zhujiang A, Youssef M, Waschek JA (2013) Interaction of PACAP with sonic hedgehog reveals complex regulation of the hedgehog pathway by PKA. Cell Signal 25:2222–2230CrossRefPubMedPubMedCentralGoogle Scholar
  41. Nonaka S, Kitaura H, Kimura K, Ishida M, Takano-Yamamoto T (2013) Expression of pituitary adenylate cyclase-activating peptide (PACAP) and PAC1 in the periodontal ligament after tooth luxation. Cell Mol Neurobiol 33:885–892CrossRefPubMedGoogle Scholar
  42. Nosrat CA, Fried K, Ebendal T, Olson L (1998) NGF, BDNF, NT3, NT4 and GDNF in tooth development. Eur J Oral Sci 106:94–99CrossRefPubMedGoogle Scholar
  43. Nosrat I, Seiger A, Olson L, Nosrat CA (2002) Expression patterns of neurotrophic factor mRNAs in developing human teeth. Cell Tissue Res 310:177–187CrossRefPubMedGoogle Scholar
  44. Orsini G, Ruggeri A, Mazzoni A, et al. (2009) A review of the nature, role, and function of dentin non-collagenous proteins. Part 1: proteoglycans and glycoproteins. Endod Top 21:1–18CrossRefGoogle Scholar
  45. Otto C, Schutz G, Niehrs C, Glinka A (2000) Dissecting GHRH- and pituitary adenylate cyclase activating polypeptide-mediated signalling in Xenopus. Mech Dev 94:111–116CrossRefPubMedGoogle Scholar
  46. Pavelock KA, Girard BM, Schutz KC, Braas KM, May V (2007) Bone morphogeneticprotein down-regulation of neuronal pituitary adenylate cyclase-activating polypeptide and reciprocal effects on vasoactive intestinal peptide expression. J Neurochem 100:603–616CrossRefPubMedGoogle Scholar
  47. Penel G, Leroy G, Rey C, Sombret B, Huvenne JP, Bres E (1997) Infrared and Raman microspectrometry study of fluor-fluor-hydroxy and hydroxyl-apatite powders. J Mater Sci Mater Med 8:271–276CrossRefPubMedGoogle Scholar
  48. Penel G, Leroy G, Rey C, Bres E (1998) MicroRaman spectral study of the PO4 and CO3 vibrational modes in synthetic and biological apatites. Calcif Tissue Int 63:475–481CrossRefPubMedGoogle Scholar
  49. Plikus MV, Zeichner-David M, Mayer JA, et al. (2005) Morphoregulation of teeth: modulating the number, size, shape and differentiation by tuning Bmp activity. Evol Dev 7:440–457CrossRefPubMedPubMedCentralGoogle Scholar
  50. Qin C, Baba O, Butler WT (2004) Post-translational modifications of sibling proteins and their roles in osteogenesis and dentinogenesis. Crit Rev Oral Biol Med 15:126–136CrossRefPubMedGoogle Scholar
  51. Reglodi D, Kiss P, Lubics A, Tamas A (2011) Review on the protective effects of PACAP in models of neurodegenerative diseases in vitro and in vivo. Curr Pharm Des 17:962–972CrossRefPubMedGoogle Scholar
  52. Reglodi D, Kiss P, Szabadfi K, et al. (2012) PACAP is an endogenous protective factor-insights from PACAP-deficient mice. J Mol Neurosci 48:482–492CrossRefPubMedGoogle Scholar
  53. Shen S, Gehlert DR, Collier DA (2013) PACAP and PAC1 receptor in brain development and behavior. Neuropeptides 47:421–430CrossRefPubMedGoogle Scholar
  54. Sandor B, Fintor K, Felszeghy S, et al. (2014) Structural and morphometric comparison of the molar teeth in pre-eruptive developmental stage of PACAP-deficient and wild-type mice. J Mol Neurosci 54:331–341CrossRefPubMedGoogle Scholar
  55. Santos RV, Clayton RN (1995) The carbonate content in high temperature apatite; an analytical method applied to apatite from the Jacupiranga alkaline complex. Am Min 80:336–344CrossRefGoogle Scholar
  56. Smith CE, Nanci A (1989) A method for sampling the stages of amelogenesis on mandibular rat incisors using the molars as a reference for dissection. Anat Rec 225:257–266CrossRefPubMedGoogle Scholar
  57. Tajiri M, Hayata-Takano A, Seiriki K, et al. (2012) Serotonin 5-HT (7) receptor blockade reverses behavioral abnormalities in PACAP-deficient mice and receptor activation promotes neurite extension in primary embryonic hippocampal neurons: therapeutic implications for psychiatric disorders. J Mol Neurosci 48:473–481CrossRefPubMedGoogle Scholar
  58. Thesleff I, Nieminen P (1996) Tooth morphogenesis and cell differentiation. Curr Opin Cell Biol 8:844–850CrossRefPubMedGoogle Scholar
  59. Thesleff I (2006) The genetic basis of tooth development and dental defects. Am J Med Genet A 140:2530–2535CrossRefPubMedGoogle Scholar
  60. Thomas DB, Fordyce RE, Frew RD, Gordon KC (2007) A rapid, non-destructive method of detecting diagenetic alteration in fossil bone using Raman spectroscopy. J Raman Spectrosc 38:1533–1537CrossRefGoogle Scholar
  61. Thomas DB, McGoverin CM, Fordyce RE, Frew RD, Gordon KC (2011) Raman spectroscopy of fossil bioapatite—a proxy for diagenetic alteration of the oxygen isotope composition. Palaeogeogr Palaeocl 310:62–70CrossRefGoogle Scholar
  62. Tummers M, Thesleff I (2009) The importance of signal pathway modulation in all aspects of tooth development. J Exp Zool B Mol Dev Evol 312B:309–319CrossRefPubMedGoogle Scholar
  63. Vaudry D, Falluel-Morel A, Bourgault S, et al. (2009) Pituitary adenylate cyclase-activating polypeptide and its receptors: 20 years after the discovery. Pharmacol Rev 61:283–357CrossRefPubMedGoogle Scholar
  64. Walker MP, Fricke BA (2006) Dentin-enamel junction of human teeth in: Akay M (ed) Wiley encyclopedia of biomedical engineering. Wiley, Hoboken, pp. 1061–1064Google Scholar
  65. Wang Y, Spencer P, Walker MP (2007a) Chemical profile of adhesive/caries-affected dentin interfaces using Raman microspectroscopy. J Biomed Mater Res A 81:279–286CrossRefPubMedPubMedCentralGoogle Scholar
  66. Wang XP, Suomalainen M, Felszeghy S, et al. (2007b) An integrated gene regulatory network controls stem cell proliferation in teeth. PLoS Biol 5:e159CrossRefPubMedPubMedCentralGoogle Scholar
  67. Watanabe J, Nakamachi T, Matsuno R, et al. (2007) Localization, characterization and function of pituitary adenylate cyclase-activating polypeptide during brain development. Peptides 28:1713–1719CrossRefPubMedGoogle Scholar
  68. Xu C, Yao X, Walker MP, Wang Y (2009) Chemical/molecular structure of the dentin-enamel junction is dependent on the intratooth location. Calcif Tissue Int 84:221–228CrossRefPubMedPubMedCentralGoogle Scholar
  69. Xu C, Wang Y (2012) Chemical composition and structure of peritubular and intertubular human dentine revisited. Arch Oral Biol 57:383–391CrossRefPubMedPubMedCentralGoogle Scholar
  70. Yang Z, Balic A, Michon F, Juuri E, Thesleff I (2015) Mesenchymal Wnt/β-catenin signaling controls epithelial stem cell homeostasis in teeth by inhibiting the antiapoptotic effect of Fgf10. Stem Cells 33:1670–1681CrossRefPubMedGoogle Scholar
  71. Yokohama-Tamaki T, Ohshima H, Fujiwara N, et al. (2006) Cessation of Fgf10 signaling, resulting in a defective dental epithelial stem cell compartment, leads to the transition from crown to root formation. Development 133:1359–1366CrossRefPubMedGoogle Scholar
  72. Yu R, Cui Z, Li M, Yang Y, Zhong J (2014) Dimer-dependent intrinsic/basal activity of the class B G protein-coupled receptor PAC1 promotes cellular anti-apoptotic activity through Wnt/β-catenin pathways that are associated with dimer endocytosis. PLoS One 9:e113913CrossRefPubMedPubMedCentralGoogle Scholar
  73. Zhang YD, Chen Z, Song YQ, Liu C, Chen YP (2005) Making tooth: growth factors, transcription factors and stem cells. Cell Res 15:301–316CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • B. Sandor
    • 1
    • 2
  • K. Fintor
    • 3
  • D. Reglodi
    • 2
  • D. B. Fulop
    • 2
  • Z. Helyes
    • 4
    • 5
  • I. Szanto
    • 1
  • P. Nagy
    • 4
  • H. Hashimoto
    • 6
  • A. Tamas
    • 2
  1. 1.Department of Dentistry, Oral and Maxillofacial SurgeryPecsHungary
  2. 2.Department of AnatomyMTA-PTE “Lendulet” PACAP Research TeamPecsHungary
  3. 3.Department of Mineralogy, Geochemistry and Petrology, Faculty of Science and InformaticsUniversity of SzegedSzegedHungary
  4. 4.Department of Pharmacology and Pharmacotherapy, Medical School, Szentagothai Research CenterUniversity of PecsPecsHungary
  5. 5.MTA-NAP B Chronic Pain Research GroupPecsHungary
  6. 6.School of Pharmaceutical SciencesOsaka UniversityOsakaJapan

Personalised recommendations