, 36:9737 | Cite as

Old-onset caloric restriction effects on neuropeptide Y- and somatostatin-containing neurons and on cholinergic varicosities in the rat hippocampal formation

  • Armando CardosoEmail author
  • Diogo Silva
  • Sara Magano
  • Pedro A. Pereira
  • José P. Andrade


Caloric restriction is able to delay age-related neurodegenerative diseases and cognitive impairment. In this study, we analyzed the effects of old-onset caloric restriction that started at 18 months of age, in the number of neuropeptide Y (NPY)- and somatostatin (SS)-containing neurons of the hippocampal formation. Knowing that these neuropeptidergic systems seem to be dependent of the cholinergic system, we also analyzed the number of cholinergic varicosities. Animals with 6 months of age (adult controls) and with 18 months of age were used. The animals aged 18 months were randomly assigned to controls or to caloric-restricted groups. Adult and old control rats were maintained in the ad libitum regimen during 6 months. Caloric-restricted rats were fed, during 6 months, with 60 % of the amount of food consumed by controls. We found that aging induced a reduction of the total number of NPY- and SS-positive neurons in the hippocampal formation accompanied by a decrease of the cholinergic varicosities. Conversely, the 24-month-old-onset caloric-restricted animals maintained the number of those peptidergic neurons and the density of the cholinergic varicosities similar to the 12-month control rats. These results suggest that the aging-associated reduction of these neuropeptide-expressing neurons is not due to neuronal loss and may be dependent of the cholinergic system. More importantly, caloric restriction has beneficial effects in the NPY- and SS-expressing neurons and in the cholinergic system, even when applied in old age.


Caloric restriction Hippocampus Neuropeptide Y Somatostatin Acetylcholine 



This work is supported by the National Funds through FCT, Fundação para a Ciência e a Tecnologia, within the scope of the Strategic Project Centro de Morfologia Experimental (CME/FM/UP), 2011–2012, and Project PEst-OE/SAU/UI0121/2011.

Conflict of interest

All authors state that there are no actual or potential conflicts of interest.


  1. Adams MM, Shi L, Linville MC, Forbes ME, Long AB, Bennett C, Newton IG, Carter CS, Sonntag WE, Riddle DR, Brunso-Bechtold JK (2008) Caloric restriction and age affect synaptic proteins in hippocampal CA3 and spatial learning ability. Exp Neurol 211:141–149PubMedCentralPubMedCrossRefGoogle Scholar
  2. Aggleton JP, Brown MW (2006) Interleaving brain systems for episodic and recognition memory. Trends Cogn Sci 10:455–463PubMedCrossRefGoogle Scholar
  3. Amaral DG, Witter MP (1995) The hippocampal formation. In: Paxinos G (ed) The rat nervous system, 2nd edn. Academic Press, San Diego, pp 443–493Google Scholar
  4. Andrade JP, Lukoyanov NV, Paula-Barbosa MM (2002) Chronic food restriction is associated with subtle dendritic alterations in granule cells of the rat hippocampal formation. Hippocampus 12:149–164PubMedCrossRefGoogle Scholar
  5. Andrade JP, Mesquita R, Assuncão M, Pereira PA (2006) Effects of food restriction on synthesis and expression of brain-derived neurotrophic factor and tyrosine kinase B in dentate gyrus granule cells of adult rats. Neurosci Lett 399:135–140PubMedCrossRefGoogle Scholar
  6. Anton S, Leeuwenburgh C (2013) Fasting or caloric restriction for healthy aging. Exp Gerontol 48:1003–1005PubMedCentralPubMedCrossRefGoogle Scholar
  7. Azarbar A, McIntyre DC, Gilby KL (2010) Caloric restriction alters seizure disposition and behavioral profiles in seizure-prone (fast) versus seizure-resistant (slow) rats. Behav Neurosci 124:106–114PubMedCrossRefGoogle Scholar
  8. Baraban SC (2004) Neuropeptide Y and epilepsy: recent progress, prospects and controversies. Neuropeptides 38:261–265PubMedCrossRefGoogle Scholar
  9. Bough KJ, Valiyil R, Han FT, Eagles DA (1999) Seizure resistance is dependent upon age and calorie restriction in rats fed a ketogenic diet. Epilepsy Res 35:21–28PubMedCrossRefGoogle Scholar
  10. Bruce-Keller AJ, Umberger G, McFall R, Mattson MP (1999) Food restriction reduces brain damage and improves behavioral outcome following excitotoxic and metabolic insults. Ann Neurol 45:8–15PubMedCrossRefGoogle Scholar
  11. Cadacio CL, Milner TA, Gallagher M, Pierce JP (2003) Hilar neuropeptide Y interneuron loss in the aged rat hippocampal formation. Exp Neurol 183:147–158PubMedCrossRefGoogle Scholar
  12. Cardoso A, Castro JP, Pereira PA, Andrade JP (2013) Prolonged protein deprivation, but not food restriction, affects parvalbumin-containing interneurons in the dentate gyrus of adult rats. Brain Res 1522:22–30PubMedCrossRefGoogle Scholar
  13. Cardoso A, Freitas-da-Costa P, Carvalho LS, Lukoyanov NV (2010) Seizure-induced changes in neuropeptide Y-containing cortical neurons: potential role for seizure threshold and epileptogenesis. Epilepsy Behav 19:559–567PubMedCrossRefGoogle Scholar
  14. Cardoso A, Lukoyanova EA, Madeira MD, Lukoyanov NV (2011) Seizure-induced structural and functional changes in the rat hippocampal formation: comparison between brief seizures and status epilepticus. Behav Brain Res 225:538–546PubMedCrossRefGoogle Scholar
  15. Cardoso A, Paula-Barbosa MM, Lukoyanov NV (2006) Reduced density of neuropeptide Y neurons in the somatosensory cortex of old male and female rats: relation to cholinergic depletion and recovery after nerve growth factor treatment. Neuroscience 137:937–948PubMedCrossRefGoogle Scholar
  16. Cava E, Fontana L (2013) Will calorie restriction work in humans? Aging (Albany NY) 5:507–514Google Scholar
  17. Cha CI, Lee YI, Park KH, Baik SH (1996) Age-related change of neuropeptide Y-immunoreactive neurons in the cerebral cortex of aged rats. Neurosci Lett 214:37–40PubMedCrossRefGoogle Scholar
  18. Cintra L, Díaz-Cintra S, Galván A, Kemper T, Morgane PJ (1990) Effects of protein undernutrition on the dentate gyrus in rats of three age groups. Brain Res 532:271–277PubMedCrossRefGoogle Scholar
  19. Colman RJ, Anderson RM, Johnson SC, Kastman EK, Kosmatka KJ, Beasley TM, Allison DB, Cruzen C, Simmons HA, Kemnitz JW, Weindruch R (2009) Caloric restriction delays disease onset and mortality in rhesus monkeys. Science 325:201–204PubMedCentralPubMedCrossRefGoogle Scholar
  20. Contestabile A, Ciani E, Contestabile A (2004) Dietary restriction differentially protects from neurodegeneration in animal models of excitotoxicity. Brain Res 1002:162–166PubMedCrossRefGoogle Scholar
  21. Cuello AC (2012) Gangliosides, NGF, brain aging and disease: a mini-review with personal reflections. Neurochem Res 37:1256–1260PubMedCrossRefGoogle Scholar
  22. Cuello AC, Maysinger D, Garofalo L (1992) Trophic factor effects on cholinergic innervation in the cerebral cortex of the adult rat brain. Mol Neurobiol 6:451–461PubMedCrossRefGoogle Scholar
  23. Del Arco A, Segovia G, de Blas M, Garrido P, Acuña-Castroviejo D, Pamplona R, Mora F (2011) Prefrontal cortex, caloric restriction and stress during aging: studies on dopamine and acetylcholine release, BDNF and working memory. Behav Brain Res 216:136–145PubMedCrossRefGoogle Scholar
  24. Drexel M, Kirchmair E, Wieselthaler-Hölzl A, Preidt AP, Sperk G (2012) Somatostatin and neuropeptide Y neurons undergo different plasticity in parahippocampal regions in kainic acid-induced epilepsy. J Neuropathol Exp Neurol 71:312–329PubMedCentralPubMedCrossRefGoogle Scholar
  25. Duan W, Lee J, Guo Z, Mattson MP (2001) Dietary restriction stimulates BDNF production in the brain and thereby protects neurons against excitotoxic injury. J Mol Neurosci 16:1–12PubMedCrossRefGoogle Scholar
  26. Duan W, Mattson MP (1999) Dietary restriction and 2-deoxyglucose administration improve behavioral outcome and reduce degeneration of dopaminergic neurons in models of Parkinson's disease. J Neurosci Res 57:195–206PubMedCrossRefGoogle Scholar
  27. Eichenbaum H (1999) The hippocampus and mechanisms of declarative memory. Behav Brain Res 103:123–133PubMedCrossRefGoogle Scholar
  28. Fontana L, Partridge L, Longo VD (2010) Extending healthy life span-from yeast to humans. Science 328:321–326PubMedCentralPubMedCrossRefGoogle Scholar
  29. Gavilán MP, Revilla E, Pintado C, Castaño A, Vizuete ML, Moreno-González I, Baglietto-Vargas D, Sánchez-Varo R, Vitorica J, Gutiérrez A, Ruano D (2007) Molecular and cellular characterization of the age-related neuroinflammatory processes occurring in normal rat hippocampus: potential relation with the loss of somatostatin GABAergic neurons. J Neurochem 103:984–996PubMedCrossRefGoogle Scholar
  30. Gillette-Guyonnet S, Vellas B (2008) Caloric restriction and brain function. Curr Opin Clin Nutr Metab Care 11:686–692PubMedCrossRefGoogle Scholar
  31. Gundersen HJ, Jensen EB (1987) The efficiency of systematic sampling in stereology and its prediction. J Microsc 147:229–263PubMedCrossRefGoogle Scholar
  32. Gundersen HJ, Jensen EB, Kiêu K, Nielsen J (1999) The efficiency of systematic sampling in stereology–reconsidered. J Microsc 193:199–211PubMedCrossRefGoogle Scholar
  33. Hartman AL, Stafstrom CE (2013) Harnessing the power of metabolism for seizure prevention: focus on dietary treatments. Epilepsy Behav 26:266–272PubMedCentralPubMedCrossRefGoogle Scholar
  34. Hattiangady B, Kuruba R, Shetty AK (2011) Acute seizures in old age leads to a greater loss of CA1 pyramidal neurons, an increased propensity for developing chronic TLE and a severe cognitive dysfunction. Aging Dis 2:1–18PubMedCentralPubMedGoogle Scholar
  35. Hattiangady B, Rao MS, Shetty GA, Shetty AK (2005) Brain-derived neurotrophic factor, phosphorylated cyclic AMP response element binding protein and neuropeptide Y decline as early as middle age in the dentate gyrus and CA1 and CA3 subfields of the hippocampus. Exp Neurol 195:353–371PubMedCrossRefGoogle Scholar
  36. Hauser WA (1992) Seizure disorders: the changes with age. Epilepsia 33(Suppl 4):S6–S14PubMedCrossRefGoogle Scholar
  37. Hipólito-Reis J, Pereira PA, Andrade JP, Cardoso A (2013) Prolonged protein deprivation differentially affects calretinin- and parvalbumin-containing interneurons in the hippocampal dentate gyrus of adult rats. Neurosci Lett 555:154–158PubMedCrossRefGoogle Scholar
  38. Huh Y, Kim C, Lee W, Kim J, Ahn H (1997) Age-related change in the neuropeptide Y and NADPH-diaphorase-positive neurons in the cerebral cortex and striatum of aged rats. Neurosci Lett 223:157–160PubMedCrossRefGoogle Scholar
  39. Jolkkonen J, Kahkonen K, Pitkanen A (1997) Cholinergic deafferentation exacerbates seizure-induced loss of somatostatin-immunoreactive neurons in the rat hippocampus. Neuroscience 80:401–411PubMedCrossRefGoogle Scholar
  40. Kim DW, Choi JH (2000) Effects of age and dietary restriction on animal model SAMP8 mice with learning and memory impairments. J Nutr Health Aging 4:233–238PubMedGoogle Scholar
  41. Kowalski C, Micheau J, Corder R, Gaillard R, Conte-Devolx B (1992) Age-related changes in cortico-releasing factor, somatostatin, neuropeptide Y, methionine enkephalin and beta-endorphin in specific rat brain areas. Brain Res 582:38–46PubMedCrossRefGoogle Scholar
  42. Lamour Y, Epelbaum J (1988) Interactions between cholinergic and peptidergic systems in the cerebral cortex and hippocampus. Prog Neurobiol 31:109–148PubMedCrossRefGoogle Scholar
  43. Lee J, Seroogy KB, Mattson MP (2002) Dietary restriction enhances neurotrophin expression and neurogenesis in the hippocampus of adult mice. J Neurochem 80:539–547PubMedCrossRefGoogle Scholar
  44. Lukoyanov NV, Andrade JP, Dulce Madeira M, Paula-Barbosa MM (1999) Effects of age and sex on the water maze performance and hippocampal cholinergic fibers in rats. Neurosci Lett 269:141–144PubMedCrossRefGoogle Scholar
  45. Marchal J, Dal-Pan A, Epelbaum J, Blanc S, Mueller S, Wittig Kieffer M, Metzger F, Aujard F, Consortium R (2013) Calorie restriction and resveratrol supplementation prevent age-related DNA and RNA oxidative damage in a non-human primate. Exp Gerontol 48:992–1000PubMedCrossRefGoogle Scholar
  46. Martel G, Dutar P, Epelbaum J, Viollet C (2012) Somatostatinergic systems: an update on brain functions in normal and pathological aging. Front Endocrinol (Lausanne) 3:154Google Scholar
  47. Mattison JA, Roth GS, Beasley TM, Tilmont EM, Handy AM, Herbert RL, Longo DL, Allison DB, Young JE, Bryant M, Barnard D, Ward WF, Qi W, Ingram DK, de Cabo R (2012) Impact of caloric restriction on health and survival in rhesus monkeys from the NIA study. Nature 489:318–321PubMedCrossRefGoogle Scholar
  48. Merry BJ (2004) Oxidative stress and mitochondrial function with aging-the effects of calorie restriction. Aging Cell 3:7–12PubMedCrossRefGoogle Scholar
  49. Milner TA, Wiley RG, Kurucz OS, Prince SR, Pierce JP (1997) Selective changes in hippocampal neuropeptide Y neurons following removal of the cholinergic septal inputs. J Comp Neurol 386:46–59PubMedCrossRefGoogle Scholar
  50. Morris R (1984) Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 11:47–60PubMedCrossRefGoogle Scholar
  51. Niewiadomska G, Komorowski S, Baksalerska-Pazera M (2002) Amelioration of cholinergic neurons dysfunction in aged rats depends on the continuous supply of NGF. Neurobiol Aging 23:601–613PubMedCrossRefGoogle Scholar
  52. Niewiadomska G, Mietelska-Porowska A, Mazurkiewicz M (2011) The cholinergic system, nerve growth factor and the cytoskeleton. Behav Brain Res 221:515–526PubMedCrossRefGoogle Scholar
  53. Patrylo PR, Tyagi I, Willingham AL, Lee S, Williamson A (2007) Dentate filter function is altered in a proepileptic fashion during aging. Epilepsia 48:1964–1978PubMedCrossRefGoogle Scholar
  54. Paxinos G, Watson C (1998) The rat brain in stereotaxic coordinates, 4th edn. Academic Press, San DiegoGoogle Scholar
  55. Pereira PA, Santos D, Neves J, Madeira MD, Paula-Barbosa MM (2013) Nerve growth factor retrieves neuropeptide Y and cholinergic immunoreactivity in the nucleus accumbens of old rats. Neurobiol Aging 34:1988–1995PubMedCrossRefGoogle Scholar
  56. Perovic M, Tesic V, Mladenovic Djordjevic A, Smiljanic K, Loncarevic-Vasiljkovic N, Ruzdijic S, Kanazir S (2013) BDNF transcripts, proBDNF and proNGF, in the cortex and hippocampus throughout the life span of the rat. Age (Dordr) 35:2057–2070CrossRefGoogle Scholar
  57. Picca A, Fracasso F, Pesce V, Cantatore P, Joseph AM, Leeuwenburgh C, Gadaleta MN, Lezza AM (2013) Age- and calorie restriction-related changes in rat brain mitochondrial DNA and TFAM binding. Age (Dordr) 35:1607–1620CrossRefGoogle Scholar
  58. Potier B, Jouvenceau A, Epelbaum J, Dutar P (2006) Age-related alterations of GABAergic input to CA1 pyramidal neurons and its control by nicotinic acetylcholine receptors in rat hippocampus. Neuroscience 142:187–201PubMedCrossRefGoogle Scholar
  59. Rapp PR, Gallagher M (1996) Preserved neuron number in the hippocampus of aged rats with spatial learning deficits. Proc Natl Acad Sci U S A 93:9926–9930PubMedCentralPubMedCrossRefGoogle Scholar
  60. Rich NJ, Van Landingham JW, Figueiroa S, Seth R, Corniola RS, Levenson CW (2010) Chronic caloric restriction reduces tissue damage and improves spatial memory in a rat model of traumatic brain injury. J Neurosci Res 88:2933–2939PubMedGoogle Scholar
  61. Roth GS, Ingram DK, Lane MA (2001) Caloric restriction in primates and relevance to humans. Ann N Y Acad Sci 928:305–315PubMedCrossRefGoogle Scholar
  62. Roth LW, Polotsky AJ (2012) Can we live longer by eating less? A review of caloric restriction and longevity. Maturitas 71:315–319PubMedCrossRefGoogle Scholar
  63. Scheen AJ (2008) The future of obesity: new drugs versus lifestyle interventions. Expert Opin Investig Drugs 17:263–267PubMedCrossRefGoogle Scholar
  64. Schliebs R, Arendt T (2011) The cholinergic system in aging and neuronal degeneration. Behav Brain Res 221:555–563PubMedCrossRefGoogle Scholar
  65. Shetty PK, Galeffi F, Turner DA (2011) Age-induced alterations in hippocampal function and metabolism. Aging Dis 2:196–218PubMedCentralPubMedGoogle Scholar
  66. Singh R, Lakhanpal D, Kumar S, Sharma S, Kataria H, Kaur M, Kaur G (2012) Late-onset intermittent fasting dietary restriction as a potential intervention to retard age-associated brain function impairments in male rats. Age (Dordr) 34:917–933CrossRefGoogle Scholar
  67. Slomianka L, West MJ (1987) Asymmetry in the hippocampal region specific for one of two closely related species of wild mice. Brain Res 436:69–75PubMedCrossRefGoogle Scholar
  68. Sohal RS, Agarwal S, Candas M, Forster MJ, Lal H (1994) Effect of age and caloric restriction on DNA oxidative damage in different tissues of C57BL/6 mice. Mech Ageing Dev 76:215–224PubMedCrossRefGoogle Scholar
  69. Sousa N, Almeida OF, Holsboer F, Paula-Barbosa MM, Madeira MD (1998) Maintenance of hippocampal cell numbers in young and aged rats submitted to chronic unpredictable stress. Comparison with the effects of corticosterone treatment. Stress 2:237–249PubMedCrossRefGoogle Scholar
  70. Spiegel AM, Koh MT, Vogt NM, Rapp PR, Gallagher M (2013) Hilar interneuron vulnerability distinguishes aged rats with memory impairment. J Comp Neurol 521:3508–3523PubMedCrossRefGoogle Scholar
  71. Stanley DP, Shetty AK (2004) Aging in the rat hippocampus is associated with widespread reductions in the number of glutamate decarboxylase-67 positive interneurons but not interneuron degeneration. J Neurochem 89:204–216PubMedCrossRefGoogle Scholar
  72. Stanley EM, Fadel JR, Mott DD (2012) Interneuron loss reduces dendritic inhibition and GABA release in hippocampus of aged rats. Neurobiol Aging 33(431):e431–413Google Scholar
  73. Thorsell A, Slawecki CJ, El Khoury A, Mathe AA, Ehlers CL (2006) The effects of social isolation on neuropeptide Y levels, exploratory and anxiety-related behaviors in rats. Pharmacol Biochem Behav 83:28–34PubMedCrossRefGoogle Scholar
  74. Vela J, Gutierrez A, Vitorica J, Ruano D (2003) Rat hippocampal GABAergic molecular markers are differentially affected by ageing. J Neurochem 85:368–377PubMedCrossRefGoogle Scholar
  75. Weindruch R (1996) Caloric restriction and aging. Sci Am 274:46–52PubMedCrossRefGoogle Scholar
  76. West MJ, Slomianka L, Gundersen HJ (1991) Unbiased stereological estimation of the total number of neurons in thesubdivisions of the rat hippocampus using the optical fractionator. Anat Rec 231:482–497PubMedCrossRefGoogle Scholar
  77. Wettstein JG, Earley B, Junien JL (1995) Central nervous system pharmacology of neuropeptide Y. Pharmacol Ther 65:397–414PubMedCrossRefGoogle Scholar
  78. Wong TP, Debeir T, Duff K, Cuello AC (1999) Reorganization of cholinergic terminals in the cerebral cortex and hippocampus in transgenic mice carrying mutated presenilin-1 and amyloid precursor protein transgenes. J Neurosci 19:2706–2716PubMedGoogle Scholar
  79. Ypsilanti AR, Girão da Cruz MT, Burgess A, Aubert I (2008) The length of hippocampal cholinergic fibers is reduced in the aging brain. Neurobiol Aging 29:1666–1679PubMedCrossRefGoogle Scholar
  80. Zhang ZJ, Lappi DA, Wrenn CC, Milner TA, Wiley RG (1998) Selective lesion of the cholinergic basal forebrain causes a loss of cortical neuropeptide Y and somatostatin neurons. Brain Res 800:198–206PubMedCrossRefGoogle Scholar
  81. Zhu XO, Waite PM (1998) Cholinergic depletion reduces plasticity of barrel field cortex. Cereb Cortex 8:63–72PubMedCrossRefGoogle Scholar

Copyright information

© American Aging Association 2014

Authors and Affiliations

  • Armando Cardoso
    • 1
    • 2
    • 3
    Email author
  • Diogo Silva
    • 1
    • 2
  • Sara Magano
    • 1
    • 2
  • Pedro A. Pereira
    • 1
    • 2
    • 3
  • José P. Andrade
    • 1
    • 2
    • 3
  1. 1.Department of Anatomy, Faculty of MedicineUniversity of PortoPortoPortugal
  2. 2.Center of Experimental Morphology (CME), Faculty of MedicineUniversity of PortoPortoPortugal
  3. 3.Center for Health Technology and Services Research (CINTESIS), Faculty of MedicineUniversity of PortoPortoPortugal

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