pp 1–10 | Cite as

Dentate gyrus neurogenesis across different ages in male rats: an immunohistochemical approach

  • Ayman M. GhallabEmail author
  • Zeinab M. Alazouny
  • Mai A. Samak
  • Haidy G. Abdel Malek
Original Article


Dentate gyrus is a fundamental sub-region of the hippocampus which is directly engaged in higher memory and cognitive functions. This study was performed to describe the histological and immunohistochemical changes in dentate gyrus in experimental animals during postnatal development. Forty four male albino rats were classified into four equal groups: new born group aged one day, adult group aged 3–6 months, early senile group aged 18–20 months and late senile group aged 30–31 months. Specimens of hippocampus were processed and prepared for routine hematoxylin and eosin stains and immunohistochemical expressions of calretinin, glial fibrillary acidic protein and Ki67 (Kiel 67). Morphometric data were statistically analyzed. The present results showed significant reduction in thickness of granule cell layer of late senile group. Interestingly, the mean number of immature neurons was significantly increased in early senile group, while it was significantly reduced in late senile group. The number of mature granule cells showed marked reduction in both early and late senile groups. Furthermore, the number of astrocytes and optical density of glial fibrillary acidic protein revealed significant age-dependent increase. Measurable difference in number of calretinin positive interneurons was detected between adult and senile groups. However, mean number of Ki67 immune positive nuclei expressed significant age-dependent reduction. This study concluded that interneurons play a substantial role in modulating dentate gyrus neurogenesis which occurs throughout life and steadily decreases during aging. It is recommended to focus on the different stimuli and factors that potentiate neurogenesis to prevent or treat cognitive deficiencies associated with aging.


Dentate gyrus Calretinin Aging Neurogenesis, immunohistochemistry 



Kiel 67


dentate gyrus


glial fibrillary acidic protein


phosphate buffered saline


least significant difference


granule cell layer


subgranular zone


gamma aminobutyric acid


hematoxylin and eosin



The authors would like to thank Dr. Wael Galal in Community Medicine Department, Zagazig University for this help in the statistical work and Dr. Hanaa S. Mousa in Histology Department, Zagazig University; for her help in the morphometric study.

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.

Ethical approval

All procedures performed in the present study involving experimental animals were in accordance with the ethical standards of the Institutional Animal Care and Use Committee (IACUC), Faculty of Medicine, Zagazig University, Egypt.


  1. Ables JL, Decarolis NA, Johnson MA, Rivera PD, Gao Z, Cooper DC, Radtke F, Hsieh J, Eisch AJ (2010) Notch 1 is required for maintenance of the reservoir of adult hippocampal stem cells. J Neurosci 30:10484–10492. CrossRefGoogle Scholar
  2. Amaral DG, Scharfman HE, Lavenex P (2007) The dentate gyrus: fundamental neuroanatomical organization (dentate gyrus for dummies). Prog Brain Res 163:3–22. CrossRefGoogle Scholar
  3. Ambrogini P, Lattanzi D, Ciuffoli S, Agostini D, Bertini L, Stocchi V, Santi S, Cuppini R (2004) Morpho-functional characterization of neuronal cells at different stages of maturation in granule cell layer of adult rat dentate gyrus. Brain Res 1017:21–31. CrossRefGoogle Scholar
  4. Andersen P, Soleng AF, Raastad M (2000) The hippocampal lamella hypothesis revisited1. Brain Res 886:165–171. CrossRefGoogle Scholar
  5. Andersen P., Morris R., Amaral D., Bliss T. & O’Keefe J. 2007. The hippocampus book. Oxford University Press, New York & Oxford. ISBN: 9780195100273Google Scholar
  6. Andrews-Zwilling Y, Bien-Ly N, Xu Q, Li G, Bernardo A, Yoon SY, Zwilling D, Yan TX, Chen L, Huang Y (2010) Apolipoprotein E4 causes age- and tau-dependent impairment of GABAergic interneurons, leading to learning and memory deficits in mice. J Neurosci 30:13707–13717. CrossRefGoogle Scholar
  7. Bancroft J.D. & Gamble M. 2008. Theory and practice of histological techniques. Churchill Livingstone, London. ISBN 9780443102790Google Scholar
  8. Ben-Ari Y (2002) Excitatory actions of gaba during development: the nature of the nurture. Nat Rev Neurosci 3:728–739. CrossRefGoogle Scholar
  9. Bertoldi K, Cechinel LR, Schallenberger B, Meireles L, Basso C, Lovatel GA, Bernardi L, Lamers ML, Siqueira IR (2017) Aging process alters hippocampal and cortical secretase activities of Wistar rats. Behav Brain Res 317:374–381. CrossRefGoogle Scholar
  10. Boldrini M, Fulmore CA, Tartt AN, Simeon LR, Pavlova I, Poposka V, Hen R (2018) Human hippocampal neurogenesis persists throughout aging. Cell Stem Cell 22(4):589–599. CrossRefGoogle Scholar
  11. Borlongan CV, Yamamoto M, Takei N, Kumazaki M, Ungsuparkorn C, Hida H, Sanberg PR, Nishino H (2000) Glial cell survival is enhanced during melatonin-induced neuroprotection against cerebral ischemia. FASEB J 14(10):1307–1317. CrossRefGoogle Scholar
  12. Brandt MD, Jessberger S, Steiner B, Kronenberg G, Reuter K, Bick-Sander A, Von Der Behrens W, Kempermann G (2003) Transient calretinin expression defines early postmitotic step of neuronal differentiation in adult hippocampal neurogenesis of mice. Mol Cell Neurosci 24:603–613. CrossRefGoogle Scholar
  13. Buckland M.D., Hall L., Mowlem A. & Whatley B.F. 2013. A guide to laboratory animal technology, William Heinemann Medical Books LTD, London, kindle ed. ISBN 0433045906Google Scholar
  14. Capilla-Gonzalez V, Herranz-Pérez V, García-Verdugo JM (2015) The aged brain: genesis and fate of residual progenitor cells in the subventricular zone. Front Cell Neurosci 9(365):1–11. Google Scholar
  15. Chohan MO, Li B, Blanchard J, Tung YC, Heaney AT, Rabe A, Iqbal K, Grundke-Iqbal I (2011) Enhancement of dentate gyrus neurogenesis, dendritic and synaptic plasticity and memory by a neurotrophic peptide. Neurobiol Aging 32:1420–1434. CrossRefGoogle Scholar
  16. Cummings BJ, Uchida N, Tamaki SJ, Salazar DL, Hooshmand M, Summers R, Gage FH, Anderson AJ (2005) Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc Natl Acad Sci 102:14069–14074. CrossRefGoogle Scholar
  17. de Guevara-Miranda DL, Millón C, Rosell-Valle C, Pérez-Fernández M, Missiroli M, Serrano A, Pavón F, Rodríguez de Fonseca F, Martínez-Losa M, Álvarez-Dolado M, Santín L, Castilla-Ortega E (2017) Long-lasting memory deficits in mice withdrawn from cocaine are concomitant to neuroadaptations in hippocampal basal activity, GABAergic interneurons and adult neurogenesis. Dis Models Mech 10(3):323–336. CrossRefGoogle Scholar
  18. Deisseroth K, Singla S, Toda H, Monje M, Palmer TD, Malenka RC (2004) Excitation-neurogenesis coupling in adult neural stem/progenitor cells. Neuron 42:535–552. CrossRefGoogle Scholar
  19. Dennis CV, Suh LS, Rodriguez ML, Kril JJ, Sutherland GT (2016) Human adult neurogenesis across the ages: an immunohistochemical study. Neuropathol Appl Neurobiol 42:621–638. CrossRefGoogle Scholar
  20. Dokter M, von Bohlen und Halbach O (2012) Neurogenesis within the adult hippocampus under physiological conditions and in depression. Neural Regen Res 7:552–559. Google Scholar
  21. Dringen R, Gutterer JM, Hirrlinger J (2000) Glutathione metabolism in brain. Eur J Biochem 267:4912–4916. CrossRefGoogle Scholar
  22. Espósito MS, Piatti VC, Laplagne DA, Morgenstern NA, Ferrari CC, Pitossi FJ, Schinder AF (2005) Neuronal differentiation in the adult hippocampus recapitulates embryonic development. J Neurosci 25:10074–10086. CrossRefGoogle Scholar
  23. Gulyás AI, Hájos N, Freund TF (1996) Interneurons containing calretinin are specialized to control other interneurons in the rat hippocampus. J Neurosci 16(10):3397–3411. CrossRefGoogle Scholar
  24. Herrup K (2010) Reimagining Alzheimer’s disease—an age-based hypothesis. J Neurosci 30:16755–16762. CrossRefGoogle Scholar
  25. Houser CR (2007) Interneurons of the dentate gyrus: an overview of cell types, terminal fields and neurochemical identity. Prog Brain Res 163:217–811. CrossRefGoogle Scholar
  26. Huusko N, Römer C, Ndode-Ekane XE, Lukasiuk K, Pitkänen A (2015) Loss of hippocampal interneurons and epileptogenesis: a comparison of two animal models of acquired epilepsy. Brain Struct Funct 220(1):153–191. CrossRefGoogle Scholar
  27. Kempermann G, Jessberger S, Steiner B, Kronenberg G (2004) Milestones of neuronal development in the adult hippocampus. Trends Neurosci 27(8):447–452. CrossRefGoogle Scholar
  28. Kiernan JA (2015) Histological and histochemical methods: theory and practice, 5th edn. Scion Publishing Ltd, Banbury, pp 103–118Google Scholar
  29. Kim JS, Jung J, Lee HJ, Kim JC, Wang H, Kim SH, Shin T, Moon C (2009) Differences in immunoreactivities of Ki-67 and doublecortin in the adult hippocampus in three strains of mice. Acta Histochem 111:150–156. CrossRefGoogle Scholar
  30. Larsson A, Wilhelmsson U, Pekna M, Pekny M (2004) Increased cell proliferation and neurogenesis in the hippocampal dentate gyrus of old GFAP-/-vim-/- mice. Neurochem Res 29(11):2069–2073. CrossRefGoogle Scholar
  31. Li B, Yamamori H, Tatebayashi Y, Shafit-Zagardo B, Tanimukai H, Chen S, Iqbal K, Grundke-Iqbal I (2008) Failure of neuronal maturation in Alzheimer disease dentate gyrus. J Neuropathol Exp Neurol 67:78–84. CrossRefGoogle Scholar
  32. Liu XS, Tilwalli S, Ye G, Lio PA, Pasternak JF, Trommer BL (2000) Morphologic and electrophysiologic maturation in developing dentate gyrus granule cells. Brain Res 856:202–212. CrossRefGoogle Scholar
  33. Llorens-Martín M, Rábano A, Ávila J (2016) The ever-changing morphology of hippocampal granule neurons in physiology and pathology. Front Neurosci 9(526):1–20. Google Scholar
  34. Maccaferri G, Lacaille JC (2003) Interneuron diversity series: hippocampal interneuron classifications – making things as simple as possible, not simpler. Trends Neurosci 26:564–571. CrossRefGoogle Scholar
  35. Masiulis I, Yun S, Eisch AJ (2011) The interesting interplay between interneurons and adult hippocampal neurogenesis. Mol Neurobiol 44:287–302. CrossRefGoogle Scholar
  36. Mercier F, Kitasako JT, Hatton GI (2002) Anatomy of the brain neurogenic zones revisited: fractones and the fibroblast/macrophage network. J Comp Neurol 451:170–188. CrossRefGoogle Scholar
  37. Paxinos G., Watson C., Petrides M., Rosa M. &Tokuno H. 2012. The marmoset brain in stereotaxic coordinates. Elsevier Academic Press. San Diego. ISBN 978-0124158184Google Scholar
  38. Pelkey KA, Chittajallu R, Craig MT, Tricoire L, Wester JC, McBain CJ (2017) Hippocampal GABAergic inhibitory interneurons. Physiol Rev 97(4):1619–1747. CrossRefGoogle Scholar
  39. Polak J.M.,Van Noorden S. 2003. Introduction to immunocytochemistry. 3rd edition, BIOS Scientific Publishers, Oxford, pp 141. ISBN 1859962084Google Scholar
  40. Rodríguez-Arellano JJ, Parpura V, Zorec R, Verkhratsky A (2016) Astrocytes in physiological aging and Alzheimer’s disease. Neuroscience 323:170–182. CrossRefGoogle Scholar
  41. Schwaller B (2014) Calretinin: from a “simple” Ca2+ buffer to a multifunctional protein implicated in many biological processes. Front Neuroanat 8:1–7. CrossRefGoogle Scholar
  42. Seidler F, Roy TS, Slotkin TA (2002) Prenatal nicotine exposure evokes alterations of cell structure in hippocampus and somatosensory cortex. J Pharmacol Exp Ther 300:124–133. CrossRefGoogle Scholar
  43. Seri B, García-Verdugo JM, McEwen BS, Alvarez-Buylla A (2001) Astrocytes give rise to new neurons in the adult mammalian hippocampus. J Neurosci 21:7153–7160. CrossRefGoogle Scholar
  44. Skilling Q, Roach J, Althaus AL, Murphy GG, Sander L, Zochowski M (2017) Modifications in network structure and excitability may drive differential activity-dependent integration of granule cells into dentate gyrus circuits during Normal and pathological adult neurogenesis. In: The rewiring brain, pp 409–423. CrossRefGoogle Scholar
  45. Somogyi P, Klausberger T (2005) Defined types of cortical interneurone structure space and spike timing in the hippocampus. J Physiol 562:9–26. CrossRefGoogle Scholar
  46. Suckow M.A., Weisbroth S.H. & Franklin C.L. 2006. In The Laboratory Rat, second. ed. Elsevier Academic Press, San Diego, CA. Chapter 4 – Morphophysiology, pp. 93–125. ISBN 9780120749034Google Scholar
  47. Takahashi H, Brasnjevic I, Rutten BPF, Van Der Kolk N, Perl DP, Bouras C, Steinbusch HWM, Schmitz C, Hof PR, Dickstein DL (2010) Hippocampal interneuron loss in an APP/PS1 double mutant mouse and in Alzheimer’s disease. Brain Struct Funct 214:145–160. CrossRefGoogle Scholar
  48. Varela-Nallar L, Aranguiz FC, Abbott AC, Slater PG, Inestrosa NC (2010) Adult hippocampal neurogenesis in aging and Alzheimer’s disease. Birth Defects Res Part C Embryo Today Rev 90:284–296. CrossRefGoogle Scholar
  49. Vinters H.V. & Kleinschmidt-DeMasters B.K. 2018. General pathology of the central nervous system. In Greenfield’s neuropathology, 9th edition. James Ironside, Arie Perry. CRC Press, Taylor& Francis group. Boca Raton.pp.25–82. ISBN9781498721288Google Scholar
  50. von Bohlen Und Halbach O (2007) Immunohistological markers for staging neurogenesis in adult hippocampus. Cell Tissue Res 329:409–420. CrossRefGoogle Scholar
  51. Wisse LEM, Biessels GJ, Heringa SM, Kuijf HJ, Koek DL, Luijten PR, Geerlings MI (2014) Hippocampal subfield volumes at 7T in early Alzheimer’s disease and normal aging. Neurobiol Aging 35:2039–2045. CrossRefGoogle Scholar
  52. Witter MP (2007) The perforant path: projections from the entorhinal cortex to the dentate gyrus. Prog Brain Res 163:43–61. CrossRefGoogle Scholar
  53. Wolf OT, Dyakin V, Vadasz C, De Leon MJ, McEwen BS, Bulloch K (2002) Volumetric measurement of the hippocampus, the anterior cingulate cortex, and the retrosplenial granular cortex of the rat using structural MRI. Brain Res Protocol 10(1):41–46. CrossRefGoogle Scholar
  54. Xiaoli W, Yun X, Fang W, Lihua T, Zhilong L, Honglian L, Shenghong L (2006) Aging-related changes of microglia and astrocytes in hypothalamus after intraperitoneal injection of hypertonic saline in rats. J Huazhong Univ Sci Technol Medical Sci 26:231–234. CrossRefGoogle Scholar
  55. Zhao C, Teng EM, Summers RG, Ming GL, Gage FH (2006) Distinct morphological stages of dentate granule neuron maturation in the adult mouse hippocampus. J Neurosci 26:3–11. CrossRefGoogle Scholar
  56. Zhao C, Deng W, Gage FH (2008) Mechanisms and functional implications of adult neurogenesis. Cell 132:645–660. CrossRefGoogle Scholar
  57. Zonta M, Angulo MC, Gobbo S, Rosengarten B, Hossmann KA, Pozzan T, Carmignoto G (2003) Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation. Nat Neurosci 6:43–50. CrossRefGoogle Scholar

Copyright information

© Institute of Molecular Biology, Slovak Academy of Sciences 2019

Authors and Affiliations

  1. 1.Department of Histology and Cell Biology, Faculty of MedicineZagazig UniversityZagazigEgypt
  2. 2.British University in Egypt (BUE)CairoEgypt

Personalised recommendations