Age-Related and Function-Dependent Regional Alterations of Free L- and D-Aspartate in Postembryonic Chick Brain


D-aspartate (D-Asp) modulates adult neural plasticity and embryonic brain development by promoting cell proliferation, survival and differentiation. Here, developmental changes of the excitatory amino acids (EAAs) L-Glu, L-Asp and D-Asp were determined during the frst postembryonic days, a time window for early learning, in selected brain regions of domestic chickens after chiral separation and capillary electrophoresis. Extracellular concentration (ECC) of EAAs was measured in microdialysis samples from freely moving chicks. ECC of D-Asp (but not L-EAAs) decreased during the frst week of age, with no considerable regional or learning-related variation. ECC of L-Asp and L-Glu (but not of D-Asp) were elevated in the mSt/Ac in response to a rewarding stimulus, suggesting importance of Asp-Glu co-release in synaptic plasticity of basal ganglia. Potassium-evoked release of D-Asp, with a protracted transient, was also demonstrated. D-Asp constitutes greater percentage of total aspartate in the extracellular space than in whole tissue extracts, thus the bulk of D-Asp detected in tissue appears in the extracellular space. Conversely, only a fraction of tissue L-EAAs can be detected in extracellular space. The lack of changes in tissue D-Asp following avoidance learning indicates a tonic, rather than phasic, mechanism in the neuromodulatory action of this amino acid.


  1. 1.

    Ádám, A. S., Csillag, A. (2006) Differential distribution of L-aspartate- and L-glutamate-immunoreactive structures in the arcopallium and medial striatum of the domestic chick (Gallus domesticus). J. Comp. Neurol. 498, 266–276.

    PubMed  Google Scholar 

  2. 2.

    Balázs, D., Csillag, A., Gerber, G. (2012) L-aspartate effects on single neurons and interactions with glutamate in striatal slice preparation from chicken brain. Brain Res. 1474, 1–7.

    PubMed  Google Scholar 

  3. 3.

    Bálint, E., Csillag, A. (2007) Nucleus accumbens subregions: hodological and immunohistochemical study in the domestic chick (Gallus domesticus). Cell Tissue Res. 327, 221–230.

    PubMed  Google Scholar 

  4. 4.

    Bateson, P. (1966) The characteristics and context of imprinting. Biol. Rev. 41, 177–220.

    CAS  PubMed  Google Scholar 

  5. 5.

    Csillag, A. (1999) Striato-telencephalic and striato-tegmental circuits: relevance to learning in domestic chicks. Behav. Brain Res. 98, 227–236.

    CAS  PubMed  Google Scholar 

  6. 6.

    Daisley, J. N., Gruss, M., Rose, S. P. R., Braun, K. (1998) Passive avoidance training and recall are associated with increased glutamate levels in the intermediate medial hyperstriatum centrale of the day-old chick. Neural Plast. 6, 53–61.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    D’Aniello, A. (2007) D-Aspartic acid: an endogenous amino acid with an important neuroendocrine role. Brain Res. Rev. 53, 215–234.

    PubMed  Google Scholar 

  8. 8.

    D’Aniello, A., Guiditta, A. (1977) Identifcation of D-aspartic acid in the brain of Octopus vulgaris Lam. J. Neurochem. 29, 1053–1057.

    PubMed  Google Scholar 

  9. 9.

    D’Aniello, S., Somorjai, I., Garcia-Fernàndez, J., Topo, E., D’Aniello, A. (2011) D-Aspartic acid is a novel endogenous neurotransmitter. FASEB J. 25, 1014–1027.

    PubMed  Google Scholar 

  10. 10.

    Dermon, C. R., Zikopoulos, B., Panagis, L., Harrison, E., Lancashire, C. L., Mileusnic, R., Stewart, M. G. (2002) Passive avoidance training enhances cell proliferation in 1-day-old chicks. Eur. J. Neurosci. 16, 1267–1274.

    CAS  Google Scholar 

  11. 11.

    Dunlop, D. S., Neidle, A., McHale, D., Dunlop, D. M., Lajtha, A. (1986) The presence of free D-aspartic acid in rodents and man. Biochem. Biophys. Res. Commun. 141, 27–32.

    CAS  Google Scholar 

  12. 12.

    Errico, F., Rossi, S., Napolitano, F., Catuogno, V., Topo, E., Fisone, G., D’Aniello, A., Centonze, D., Usiello, A. (2008) D-aspartate prevents corticostriatal long-term depression and attenuates schizophrenia-like symptoms induced by amphetamine and MK-801. J. Neurosci. 28, 10404–10414.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Errico, F. et al. (2014) Free D-aspartate regulates neuronal dendritic morphology, synaptic plasticity, gray matter volume and brain activity in mammals. Transl. Psychiatry 4, e417.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Fagg, G. E., Matus, A. (1984) Selective association of N-methyl aspartate and quisqualate types of L-glutamate receptor with brain postsynaptic densities. Proc. Natl. Acad. Sci. USA 81, 6876–6880.

    CAS  PubMed  Google Scholar 

  15. 15.

    Fujii, N. (2005) D-amino acid in elderly tissues. Biol. Pharm. Bull. 28, 1585–1589.

    CAS  PubMed  Google Scholar 

  16. 16.

    Gibbs, M., Johnston, A. N. B., Mileusnic, R., Crowe, S. F. (2008) A comparison of protocols for passive and discriminative avoidance learning tasks in the domestic chick. Brain Res. Bull. 76, 198–207.

    PubMed  Google Scholar 

  17. 17.

    Gundersen, V., Storm-Mathisen, J. (2000) Chapter II Aspartate-neurochemical evidence for a transmitter role. In: Ottersen, O. P., Storm-Mathisen, J. (eds), Handbook of Chemical Neuroanatomy, vol. 18. Amsterdam, Elsevier. pp. 45–62.

    CAS  Google Scholar 

  18. 18.

    Hanics, J., Bálint, E., Milanovich, D., Zachar, G., Adám, A., Csillag, A. (2012) Amygdalofugal axon terminals immunoreactive for L-aspartate or L-glutamate in the nucleus accumbens of rats and domestic chickens: a comparative electron microscopic immunocytochemical study combined with anterograde pathway tracing. Cell Tissue Res. 350, 409–423.

    CAS  PubMed  Google Scholar 

  19. 19.

    Hanics, J., Teleki, G., Alpár, A., Székely, A. D., Csillag, A. (2016) Multiple amygdaloid divisions of arcopallium send convergent projections to the nucleus accumbens and neighboring subpallial amygdala regions in the domestic chicken: a selective pathway tracing and reconstruction study. Brain Struct. Funct. 222, 301–315.

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Hashimoto, A., Kumashiro, S., Nishikawa, T., Oka, T., Takahashi, K., Mito, T., Takashima, S., Doi, N., Mizutani, Y., Yamazaki, T., Kaneko, T., Ootomo, E. (1993) Embryonic development and postnatal changes in free D-aspartate and D-serine in the human prefrontal cortex. J. Neurochem. 61, 348–351.

    CAS  PubMed  Google Scholar 

  21. 21.

    Hashimoto, A., Oka, T., Nishikawa, T. (1995) Anatomical distribution and postnatal changes in endogenous free D-Aspartate and D-Serine in rat-brain and periphery. Eur. J. Neurosci. 7, 1657–1663.

    CAS  PubMed  Google Scholar 

  22. 22.

    Horn, G. (2004) Pathways of the past: the imprint of memory. Nat. Rev. Neurosci. 5, 108–120.

    CAS  PubMed  Google Scholar 

  23. 23.

    Ito, T., Hayashida, M., Kobayashi, S., Muto, N., Hayashi, A., Yoshimura, T., Mori, H. (2016) Serine racemase is involved in D-aspartate biosynthesis. J. Biochem. 160, 345–353.

    CAS  PubMed  Google Scholar 

  24. 24.

    Matsushima, T., Izawa, E.-I., Yanagihara, S. (2001) D1-receptor dependent synaptic potentiation in the basal ganglia of quail chick. NeuroReport 12, 2831–2837.

    CAS  PubMed  Google Scholar 

  25. 25.

    McCabe, B. J. (2013) Imprinting. Interdiscip. Rev. Cogn. Sci. 4, 375–390.

    Google Scholar 

  26. 26.

    Mezey, S., Krivokuca, D., Balint, E., Adorjan, A., Zachar, G., Csillag, A. (2012) Postnatal changes in the distribution and density of neuronal nuclei and doublecortin antigens in domestic chicks (Gallus domesticus). J. Comp. Neurol. 520, 100–116.

    CAS  PubMed  Google Scholar 

  27. 27.

    Miller J. A. (1950) Do tumor proteins contain D-amino acids? A review of the controversy. Cancer Res. 10, 65–72.

    CAS  PubMed  Google Scholar 

  28. 28.

    Nadler, J. V., Vaca, K. W., White, W. F., Lynch, G. S., Cotman, C. W. (1976) Aspartate and glutamate as possible transmitters of excitatory hippocampal afferents. Nature 260, 538–540.

    CAS  PubMed  Google Scholar 

  29. 29.

    Nakamori, T., Maekawa, F., Sato, K., Tanaka, K., Ohki-Hamazaki, H. (2013) Neural basis of imprinting behavior in chicks. Dev. Growth Differ. 55, 198–206.

    CAS  PubMed  Google Scholar 

  30. 30.

    Neidle, A., Dunlop, D. S. (1990) Developmental changes in free D-aspartic acid in the chicken embryo and in the neonatal rat. Life Sci. 46, 1517–1522.

    CAS  Google Scholar 

  31. 31.

    Nuzzo, T. et al. (2017) Decreased free D-aspartate levels are linked to enhanced D-aspartate oxidase activity in the dorsolateral prefrontal cortex of schizophrenia patients. npj Schizophrenia 3, 16.

    PubMed  PubMed Central  Google Scholar 

  32. 32.

    Puelles, L. (2007) The chick brain in stereotaxic coordinates. Academic Press, San Diego.

    Google Scholar 

  33. 33.

    Punzo, D., Errico, F., Cristino, L., Sacchi, S., Keller, S., Belardo, C., Luongo, L., Nuzzo, T., Imperatore, R., Florio, E., De Novellis, V., Affnito, O., Migliarini, S., Maddaloni, G., Sisalli, M. J., Pasqualetti, M., Pollegioni, L., Maione, S., Chiariotti, L., Usiello, A. (2016) Age-related changes in D-Aspartate oxidase promoter methylation control extracellular D-Aspartate levels and prevent precocious cell death during brain aging. J. Neurosci. 36, 3064–3078.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Radzishevsky, I., Sason, H., Wolosker, H. (2013) D-serine: physiology and pathology. Curr. Opin. Clin. Nutr. Metab. Care. 16, 72–75.

    CAS  PubMed  Google Scholar 

  35. 35.

    Reiner, A. (2004) Revised nomenclature for avian telencephalon and some related brainstem nuclei. J. Comp. Neurol. 473, 377–414.

    PubMed  PubMed Central  Google Scholar 

  36. 36.

    Rose, S. P. (2000) God’s organism? The chick as a model system for memory studies. Learn. Mem. 7, 1–17.

    CAS  PubMed  Google Scholar 

  37. 37.

    Schell, M. J., Cooper, O. B., Snyder, S. H. (1997) D-aspartate localizations imply neuronal and neuroendocrine roles. Proc. Natl. Acad. Sci. USA 94, 2013–2018.

    CAS  PubMed  Google Scholar 

  38. 38.

    Stewart, M. G., Rusakov, D. A. (1995) Morphological changes associated with stages of memory formation in the chick following passive avoidance training. Behav. Brain Res. 66, 21–28.

    CAS  PubMed  Google Scholar 

  39. 39.

    Topo, E., Soricelli, A., Di Maio, A., D’Aniello, E., Di Fiore, M. M., D’Aniello, A. (2010) Evidence for the involvement of D-aspartic acid in learning and memory of rat. Amino acids 38, 1561–1569.

    CAS  PubMed  Google Scholar 

  40. 40.

    Venero, C., Sandi, C. (1997) Effects of NMDA and AMPA receptor antagonists on corticosterone facilitation of long-term memory in the chick. Eur. J. Neurosci. 9, 1923–1928.

    CAS  PubMed  Google Scholar 

  41. 41.

    Wagner, Z., Tabi, T., Zachar, G., Csillag, A., Szoko, E. (2011) Comparison of quantitative performance of three fuorescence labels in CE/LIF analysis of aspartate and glutamate in brain microdi-alysate. Electrophoresis 32, 2816–2822.

    CAS  PubMed  Google Scholar 

  42. 42.

    Wagner, Z., Tabi, T., Jako, T., Zachar, G., Csillag, A., Szoko, E. (2012) Chiral separation and determination of excitatory amino acids in brain samples by CE-LIF using dual cyclodextrin system. Anal. Bioanal. Chem. 404, 2363–2368.

    CAS  PubMed  Google Scholar 

  43. 43.

    Wolosker, H., D’Aniello, A., Snyder, S. H. (2000) D-aspartate disposition in neuronal and endocrine tissues: ontogeny, biosynthesis and release. Neuroscience 100, 183–189.

    CAS  PubMed  Google Scholar 

  44. 44.

    Yamanaka, M., Miyoshi, Y., Ohide, H., Hamase, K., Konno, R. (2012) D-Amino acids in the brain and mutant rodents lacking D-amino-acid oxidase activity. Amino Acids 43, 1811–1821.

    CAS  PubMed  Google Scholar 

  45. 45.

    Zachar, G., Wagner, Z., Tabi, T., Balint, E., Szoko, E., Csillag, A. (2012) Differential changes of extracellular aspartate and glutamate in the striatum of domestic chicken evoked by high potassium or distress: An in vivo microdialysis study. Neurochem. Res. 37, 1730–1737.

    CAS  PubMed  Google Scholar 

  46. 46.

    Zachar, G., Tóth, A. S., Balogh, M., Csillag, A. (2017) Effect of nucleus accumbens lesions on socially motivated behaviour of young domestic chicks. Eur. J. Neurosci. 45, 1606–1612.

    PubMed  Google Scholar 

Download references

Author information



Corresponding author

Correspondence to Gergely Zachar.

Rights and permissions

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zachar, G., Jakó, T., Vincze, I. et al. Age-Related and Function-Dependent Regional Alterations of Free L- and D-Aspartate in Postembryonic Chick Brain. BIOLOGIA FUTURA 69, 1–15 (2018).

Download citation


  • D-aspartic acid
  • avian brain
  • postembryonic development
  • arcopallium
  • amygdala