Brain Structure and Function

, Volume 223, Issue 3, pp 1121–1132 | Cite as

High thickness histological sections as alternative to study the three-dimensional microscopic human sub-cortical neuroanatomy

  • Eduardo Joaquim Lopes AlhoEmail author
  • Ana Tereza Di Lorenzo Alho
  • Lea Grinberg
  • Edson AmaroJr.
  • Gláucia Aparecida Bento dos Santos
  • Rafael Emídio da Silva
  • Ricardo Caires Neves
  • Maryana Alegro
  • Daniel Boari Coelho
  • Manoel Jacobsen Teixeira
  • Erich Talamoni Fonoff
  • Helmut Heinsen
Original Article


Stereotaxy is based on the precise image-guided spatial localization of targets within the human brain. Even with the recent advances in MRI technology, histological examination renders different (and complementary) information of the nervous tissue. Although several maps have been selected as a basis for correlating imaging results with the anatomical locations of sub-cortical structures, technical limitations interfere in a point-to-point correlation between imaging and anatomy due to the lack of precise correction for post-mortem tissue deformations caused by tissue fixation and processing. We present an alternative method to parcellate human brain cytoarchitectural regions, minimizing deformations caused by post-mortem and tissue-processing artifacts and enhancing segmentation by means of modified high thickness histological techniques and registration with MRI of the same specimen and into MNI space (ICBM152). A three-dimensional (3D) histological atlas of the human thalamus, basal ganglia, and basal forebrain cholinergic system is displayed. Structure’s segmentations were performed in high-resolution dark-field and light-field microscopy. Bidimensional non-linear registration of the histological slices was followed by 3D registration with in situ MRI of the same subject. Manual and automated registration procedures were adopted and compared. To evaluate the quality of the registration procedures, Dice similarity coefficient and normalized weighted spectral distance were calculated and the results indicate good overlap between registered volumes and a small shape difference between them in both manual and automated registration methods. High thickness high-resolution histological slices in combination with registration to in situ MRI of the same subject provide an effective alternative method to study nuclear boundaries in the human brain, enhancing segmentation and demanding less resources and time for tissue processing than traditional methods.


Cytoarchitecture Thalamus Sub-cortical atlas Magnetic resonance imaging 



The authors would like to thank the team participating on the São Paulo-Würzburg collaborative project. This includes all members of the Brain Bank of the Brazilian Aging Brain Research Group (BBBABSG) of the University of São Paulo Medical School, Mrs. E. Broschk and Mrs. A. Bahrke from the Morphological Brain Research Unit of the University of Würzburg, Germany.

Compliance with ethical standards

Funding source

This study was supported by resources from the University of Sao Paulo School of Medicine, Brazil and University of Würzburg, Germany. The author Eduardo Joaquim Lopes Alho was supported by a scholarship from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) agency, Brazil, for doctoral studies at the University of Würzburg, Germany. The authors do not have personal financial or institutional interest in any of the drugs, materials, or devices described in this article.

Conflict of interest

The authors disclose any actual or potential conflict of interest including any financial, personal, or other relationships with other people or organizations within 3 years of beginning the submitted work that could inappropriately influence, or be perceived to influence, their work.

Ethical standards

The work described has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki).

Supplementary material

Supplementary material 1 (WMV 26324 kb)

Supplementary material 2 (WMV 21876 kb)

Supplementary material 3 (WMV 188761 kb)


  1. Afshar F, Watkins ES, Yap JC (1978) Stereotaxic atlas of the human brainstem and cerebellar nuclei: a variability study. Raven Press, New YorkGoogle Scholar
  2. Alegro M, Loring B, Alho E et al (2016) Multimodal whole brain registration: MRI and high resolution histology. Accessed 9 Sep 2016
  3. Alho ATDL, Hamani C, Alho EJL et al (2017) Magnetic resonance diffusion tensor imaging for the pedunculopontine nucleus: proof of concept and histological correlation. Brain Struct Funct. Google Scholar
  4. Alic L, Haeck JH (2010) Multi-modal image registration: matching MRI with histology. SPIE Medical Imaging, San DiegoGoogle Scholar
  5. Amunts K, Lepage C, Borgeat L et al (2013) BigBrain: an ultrahigh-resolution 3D human brain model. Science 340:1472–1475. CrossRefPubMedGoogle Scholar
  6. Andrew J, Watkins ES (1969) A stereotaxic atlas of the human thalamus and adjacent structures: a variability study. Williams and Wilkins, BaltimoreGoogle Scholar
  7. Aoki S, Okada Y, Nishimura K et al (1989) Normal deposition of brain iron in childhood and adolescence: MR imaging at 1.5 T. Radiology 172:381–385. CrossRefPubMedGoogle Scholar
  8. Avants BB, Epstein CL, Grossman M, Gee JC (2008) Symmetric diffeomorphic image registration with cross-correlation: evaluating automated labeling of elderly and neurodegenerative brain. Med Image Anal 12:26–41. CrossRefPubMedGoogle Scholar
  9. Bauchot R (1967) Modifications of brain weight in the course of fixation. J Für Hirnforsch 9:253–283Google Scholar
  10. Butler T, Zaborszky L, Pirraglia E et al (2014) Comparison of human septal nuclei MRI measurements using automated segmentation and a new manual protocol based on histology. NeuroImage 97:245–251. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Carballo-Barreda M, RodríGuez-Rojas R, Torres-Montoya A, LóPez-Flores G (2007) Computerized atlas for image-guided stereotactic functional neurosurgery. Neurocir Astur Spain 18:478–484CrossRefGoogle Scholar
  12. Casanova MF, Kreczmanski P, Trippe J 2nd et al (2008) Neuronal distribution in the neocortex of schizophrenic patients. Psychiatry Res 158:267–277. CrossRefPubMedGoogle Scholar
  13. Chakravarty MM, Bertrand G, Hodge CP et al (2006) The creation of a brain atlas for image guided neurosurgery using serial histological data. NeuroImage 30:359–376. CrossRefPubMedGoogle Scholar
  14. Crum WR, Camara O, Hill DLG (2006) Generalized overlap measures for evaluation and validation in medical image analysis. IEEE Trans Med Imaging 25:1451–1461. CrossRefPubMedGoogle Scholar
  15. Dandy WE (1918) Ventriculography following the injection of air into the cerebral ventricles. Ann Surg 68:5–11CrossRefPubMedPubMedCentralGoogle Scholar
  16. Dice LR (1945) Measures of the amount of ecologic association between species. Ecology 26:297–302. CrossRefGoogle Scholar
  17. Drayer B, Burger P, Darwin R et al (1986) MRI of brain iron. AJR Am J Roentgenol 147:103–110. CrossRefPubMedGoogle Scholar
  18. Duyn JH (2012) The future of ultra-high field MRI and fMRI for study of the human brain. Neuroimage 62:1241–1248. CrossRefPubMedGoogle Scholar
  19. Emmers R, Tasker R (1975) The human somesthetic thalamus: with maps for physiological target localization during stereotactic neurosurgery. Raven Press, New YorkGoogle Scholar
  20. Ewers M, Frisoni GB, Teipel SJ et al (2011) Staging Alzheimer’s disease progression with multimodality neuroimaging. Prog Neurobiol 95:535–546. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Ewert S, Plettig P, Li N et al (2017) Toward defining deep brain stimulation targets in MNI space: a subcortical atlas based on multimodal MRI, histology and structural connectivity. NeuroImage. PubMedCentralGoogle Scholar
  22. Fonoff ET, Campos WK, Mandel M et al (2012) Bilateral subthalamic nucleus stimulation for generalized dystonia after bilateral pallidotomy. Mov Disord Off J Mov Disord Soc 27:1559–1563. CrossRefGoogle Scholar
  23. Fonov V, Evans AC, Botteron K et al (2011) Unbiased average age-appropriate atlases for pediatric studies. NeuroImage 54:313–327. CrossRefPubMedGoogle Scholar
  24. François C, Yelnik J, Percheron G (1996) A stereotaxic atlas of the basal ganglia in macaques. Brain Res Bull 41:151–158CrossRefPubMedGoogle Scholar
  25. Franklin K, Paxinos G (2008) The mouse brain in stereotaxic coordinates. Academic Press, New YorkGoogle Scholar
  26. Ganser KA, Dickhaus H, Metzner R, Wirtz CR (2004) A deformable digital brain atlas system according to Talairach and Tournoux. Med Image Anal 8:3–22CrossRefPubMedGoogle Scholar
  27. Grinberg L, Heinsen H (2007) Computer-assisted 3D reconstruction of the human basal forebrain complex. Dement Neuropsychol 1:140–146CrossRefPubMedPubMedCentralGoogle Scholar
  28. Grinberg LT, Amaro E Jr, Teipel S et al (2008) Assessment of factors that confound MRI and neuropathological correlation of human postmortem brain tissue. Cell Tissue Bank 9:195–203. CrossRefPubMedGoogle Scholar
  29. Grinberg LT, Amaro Junior E, da Silva AV et al (2009) Improved detection of incipient vascular changes by a biotechnological platform combining post mortem MRI in situ with neuropathology. J Neurol Sci 283:2–8. CrossRefPubMedGoogle Scholar
  30. Haakma W, Pedersen M, Froeling M et al (2016) Diffusion tensor imaging of peripheral nerves in non-fixed post-mortem subjects. Forensic Sci Int 263:139–146. CrossRefPubMedGoogle Scholar
  31. Hallgren B, Sourander P (1958) The effect of age on the non-haemin iron in the human brain. J Neurochem 3:41–51CrossRefPubMedGoogle Scholar
  32. Hassler R, Schaltenbrand G, Walker E (1982) Architectonic organization of the thalamic nuclei. In: Schaltenbrand G, Walker E (eds) Stereotaxy of the human brain: anatomical, physiological and clinical applications, 2nd edn. George Thieme Verlag, StuttgartGoogle Scholar
  33. Heckers S, Heinsen H, Heinsen Y, Beckmann H (1991) Cortex, white matter, and basal ganglia in schizophrenia: a volumetric postmortem study. Biol Psychiatry 29:556–566CrossRefPubMedGoogle Scholar
  34. Heinsen H, Heinsen YL (1991) Serial thick, frozen, gallocyanin stained sections of human central nervous system. J Histotechnol 14:167–173CrossRefGoogle Scholar
  35. Heinsen H, Henn R, Eisenmenger W et al (1994a) Quantitative investigations on the human entorhinal area: left–right asymmetry and age-related changes. Anat Embryol (Berl) 190:181–194CrossRefGoogle Scholar
  36. Heinsen H, Strik M, Bauer M et al (1994b) Cortical and striatal neurone number in Huntington’s disease. Acta Neuropathol (Berl) 88:320–333CrossRefGoogle Scholar
  37. Heinsen H, Rüb U, Gangnus D et al (1996) Nerve cell loss in the thalamic centromedian–parafascicular complex in patients with Huntington’s disease. Acta Neuropathol (Berl) 91:161–168CrossRefGoogle Scholar
  38. Heinsen H, Arzberger T, Schmitz C (2000) Celloidin mounting (embedding without infiltration)—a new, simple and reliable method for producing serial sections of high thickness through complete human brains and its application to stereological and immunohistochemical investigations. J Chem Neuroanat 20:49–59CrossRefPubMedGoogle Scholar
  39. Hirai T, Jones EG (1989) A new parcellation of the human thalamus on the basis of histochemical staining. Brain Res Brain Res Rev 14:1–34CrossRefPubMedGoogle Scholar
  40. Horn A, Kühn AA (2015) Lead-DBS: a toolbox for deep brain stimulation electrode localizations and visualizations. NeuroImage 107:127–135. CrossRefPubMedGoogle Scholar
  41. Ilinsky IA, Kultas-Ilinsky K, Knosp B (2002) Stereotactic atlas of the Macaca mulatta thalamus and adjacent basal ganglia nuclei. Springer, BostonCrossRefGoogle Scholar
  42. Jenkinson M, Beckmann CF, Behrens TEJ et al (2012) FSL. NeuroImage 62:782–790. CrossRefPubMedGoogle Scholar
  43. Kilimann I, Grothe M, Heinsen H et al (2014) Subregional basal forebrain atrophy in Alzheimer’s disease: a multicenter study. J Alzheimers Dis JAD 40:687–700. PubMedGoogle Scholar
  44. König JFR, Klippel RA (1963) The rat brain: a stereotaxic atlas of the forebrain and lower parts of the brain stem. Williams and Wilkins, BaltimoreGoogle Scholar
  45. Konukoglu E, Glocker B, Ye DH et al (2012) Discriminative segmentation-based evaluation through shape dissimilarity. IEEE Trans Med Imaging 31:2278–2289CrossRefPubMedPubMedCentralGoogle Scholar
  46. Konukoglu E, Glocker B, Criminisi A, Pohl KM (2013) WESD—weighted spectral distance for measuring shape dissimilarity. IEEE Trans Pattern Anal Mach Intell 35:2284–2297CrossRefPubMedPubMedCentralGoogle Scholar
  47. Krauth A, Blanc R, Poveda A et al (2010) A mean three-dimensional atlas of the human thalamus: generation from multiple histological data. NeuroImage 49:2053–2062. CrossRefPubMedGoogle Scholar
  48. Kretschmann HJ, Tafesse U, Herrmann A (1982) Different volume changes of cerebral cortex and white matter during histological preparation. Microsc Acta 86:13–24PubMedGoogle Scholar
  49. Kroon JP, Riley AL (1986) A microcomputer-based system for stereotaxic coordinates in the rat brain. Physiol Behav 38:593–596CrossRefPubMedGoogle Scholar
  50. Kumazawa-Manita N, Katayama M, Hashikawa T, Iriki A (2013) Three-dimensional reconstruction of brain structures of the rodent Octodon degus: a brain atlas constructed by combining histological and magnetic resonance images. Exp Brain Res 231:65–74. CrossRefPubMedPubMedCentralGoogle Scholar
  51. Lanciego JL, Vázquez A (2012) The basal ganglia and thalamus of the long-tailed macaque in stereotaxic coordinates. A template atlas based on coronal, sagittal and horizontal brain sections. Brain Struct Funct 217:613–666. CrossRefPubMedGoogle Scholar
  52. Mai JK, Paxinos G, Voss T (2008) Atlas of the human brain. Academic Press, New YorkGoogle Scholar
  53. Martin RF, Bowden DM (1996) A stereotaxic template atlas of the macaque brain for digital imaging and quantitative neuroanatomy. NeuroImage 4:119–150. CrossRefPubMedGoogle Scholar
  54. Martinez RCR, Hamani C, de Carvalho MC et al (2013) Intraoperative dopamine release during globus pallidus internus stimulation in Parkinson’s disease. Mov Disord Off J Mov Disord Soc. Google Scholar
  55. Mesulam MM, Mufson EJ, Wainer BH, Levey AI (1983) Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Ch1–Ch6). Neuroscience 10:1185–1201CrossRefPubMedGoogle Scholar
  56. Morel A (2007) Stereotactic atlas of the human thalamus and basal ganglia. Informa Healthcare, New YorkCrossRefGoogle Scholar
  57. Morel A, Magnin M, Jeanmonod D (1997) Multiarchitectonic and stereotactic atlas of the human thalamus. J Comp Neurol 387:588–630CrossRefPubMedGoogle Scholar
  58. Niemann K, van Nieuwenhofen I (1999) One Atlas–Three Anatomies: relationships of the Schaltenbrand and Wahren Microscopic Data. Acta Neurochir (Wien) 141:1025–1038CrossRefGoogle Scholar
  59. Niemann K, Naujokat C, Pohl G et al (1994) Verification of the Schaltenbrand and Wahren stereotactic atlas. Acta Neurochir (Wien) 129:72–81CrossRefGoogle Scholar
  60. Nowinski WL, Belov D (2003) The Cerefy Neuroradiology Atlas: a Talairach–Tournoux atlas-based tool for analysis of neuroimages available over the internet. NeuroImage 20:50–57CrossRefPubMedGoogle Scholar
  61. Nowinski WL, Fang A, Nguyen BT et al (1997) Multiple brain atlas database and atlas-based neuroimaging system. Comput Aided Surg Off J Int Soc Comput Aided Surg 2:42–66.<42:AID-IGS7>3.0.CO;2-N CrossRefGoogle Scholar
  62. Nowinski WL, Belov D, Thirunavuukarasuu A, Benabid AL (2005) A probabilistic functional atlas of the VIM nucleus constructed from pre-, intra- and postoperative electrophysiological and neuroimaging data acquired during the surgical treatment of Parkinson’s disease patients. Stereotact Funct Neurosurg 83:190–196. CrossRefPubMedGoogle Scholar
  63. Nowinski WL, Liu J, Thirunavuukarasuu A (2006) Quantification and visualization of the three-dimensional inconsistency of the subthalamic nucleus in the Schaltenbrand–Wahren brain atlas. Stereotact Funct Neurosurg 84:46–55. CrossRefPubMedGoogle Scholar
  64. Ono M, Kubik S, Abernathey CD (1990) Atlas of the cerebral sulci. G. Thieme Verlag/Thieme Medical Publishers, Stuttgart/New YorkGoogle Scholar
  65. Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates. Academic Press, New YorkGoogle Scholar
  66. Quester R, Schröder R (1997) The shrinkage of the human brain stem during formalin fixation and embedding in paraffin. J Neurosci Methods 75:81–89CrossRefPubMedGoogle Scholar
  67. Reese R, Pinsker MO, Herzog J et al (2012) The atypical subthalamic nucleus—an anatomical variant relevant for stereotactic targeting. Mov Disord Off J Mov Disord Soc 27:544–548. CrossRefGoogle Scholar
  68. Reuter M, Rosas HD, Fischl B (2010) Highly accurate inverse consistent registration: a robust approach. NeuroImage 53:1181–1196. CrossRefPubMedPubMedCentralGoogle Scholar
  69. Saleem KS, Logothetis NK (2012) A combined MRI and histology atlas of the rhesus monkey brain in stereotaxic coordinates. Academic Press, New YorkGoogle Scholar
  70. Schaltenbrand G, Bailey P (1959) Introduction to stereotaxis with an atlas of the human brain, vol I: Text, vol II: Plate 1–57, vol III: Plate 58–76. Georg Thieme, Grune & Stratton:Stuttgart, New YorkGoogle Scholar
  71. Schaltenbrand G, Hassler R, Wahren W (1977) Atlas for stereotaxy of the human brain: with an accompanying guide. Thieme, StuttgartGoogle Scholar
  72. Schulz G, Crooijmans HJA, Germann M et al (2011) Three-dimensional strain fields in human brain resulting from formalin fixation. J Neurosci Methods 202:17–27. CrossRefPubMedGoogle Scholar
  73. Schwarz AJ, Danckaert A, Reese T et al (2006) A stereotaxic MRI template set for the rat brain with tissue class distribution maps and co-registered anatomical atlas: application to pharmacological MRI. NeuroImage 32:538–550. CrossRefPubMedGoogle Scholar
  74. Shanta TR, Manocha SL, Bourne GH (1968) A stereotaxic atlas of the java monkey brain (Macaca irus). Basel, Karger, doi: 10.1159/000390681
  75. Simmons DM, Swanson LW (2009) Comparing histological data from different brains: sources of error and strategies for minimizing them. Brain Res Rev 60:349–367.  10.1016/j.brainresrev.2009.02.002 CrossRefPubMedPubMedCentralGoogle Scholar
  76. Small CS, Peterson DI (1982) The reliability of dimensions of formalin-fixed brains. Neurology 32:413–415CrossRefPubMedGoogle Scholar
  77. Spiegel EA, Wycis HT, Freed H (1952) Stereoencephalotomy: thalamotomy and related procedures. J Am Med Assoc 148:446–451. CrossRefPubMedGoogle Scholar
  78. St-Jean P, Sadikot AF, Collins L et al (1998) Automated atlas integration and interactive three-dimensional visualization tools for planning and guidance in functional neurosurgery. IEEE Trans Med Imaging 17:672–680. CrossRefPubMedGoogle Scholar
  79. Talairach J (1957) Atlas d’anatomie stéréotaxique; repérage radiologique indirect des noyaux gris centraux des régions mésencéphalo-sous-optique et hypothalamique de l’homme. Masson, ParisGoogle Scholar
  80. Talairach J, Tournoux P (1988) Co-planar stereotaxic atlas of the human brain: 3-dimensional proportional system: an approach to medical cerebral imaging. Thieme, StuttgartGoogle Scholar
  81. Teipel SJ, Meindl T, Grinberg L et al (2008) Novel MRI techniques in the assessment of dementia. Eur J Nucl Med Mol Imaging 35(Suppl 1):S58–S69. CrossRefPubMedGoogle Scholar
  82. Teipel SJ, Meindl T, Grinberg L et al (2011) The cholinergic system in mild cognitive impairment and Alzheimer’s disease: an in vivo MRI and DTI study. Hum Brain Mapp 32:1349–1362. CrossRefPubMedGoogle Scholar
  83. Teipel SJ, Flatz W, Ackl N et al (2014) Brain atrophy in primary progressive aphasia involves the cholinergic basal forebrain and Ayala’s nucleus. Psychiatry Res 221:187–194. CrossRefPubMedGoogle Scholar
  84. Toga AW, Samaie M, Payne BA (1989) Digital rat brain: a computerized atlas. Brain Res Bull 22:323–333CrossRefPubMedGoogle Scholar
  85. Van Buren JM, Borke RC (1972) Variations and connections of the human thalamus. Springer, BerlinCrossRefGoogle Scholar
  86. Yelnik J, Bardinet E, Dormont D et al (2007) A three-dimensional, histological and deformable atlas of the human basal ganglia. I. Atlas construction based on immunohistochemical and MRI data. NeuroImage 34:618–638. CrossRefPubMedGoogle Scholar
  87. Yoshida M (1987) Creation of a three-dimensional atlas by interpolation from Schaltenbrand–Bailey’s atlas. Appl Neurophysiol 50:45–48PubMedGoogle Scholar
  88. Zaborszky L, Hoemke L, Mohlberg H et al (2008) Stereotaxic probabilistic maps of the magnocellular cell groups in human basal forebrain. NeuroImage 42:1127–1141. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Eduardo Joaquim Lopes Alho
    • 1
    • 2
    • 4
    • 7
    Email author
  • Ana Tereza Di Lorenzo Alho
    • 3
    • 4
  • Lea Grinberg
    • 3
    • 5
  • Edson AmaroJr.
    • 4
  • Gláucia Aparecida Bento dos Santos
    • 3
    • 4
  • Rafael Emídio da Silva
    • 4
  • Ricardo Caires Neves
    • 3
  • Maryana Alegro
    • 4
    • 5
  • Daniel Boari Coelho
    • 6
  • Manoel Jacobsen Teixeira
    • 2
  • Erich Talamoni Fonoff
    • 2
  • Helmut Heinsen
    • 1
    • 4
  1. 1.Morphological Brain Research Unit, Department of PsychiatryUniversity of WürzburgWürzburgGermany
  2. 2.Division of Functional Neurosurgery, Department of NeurologyUniversity of São Paulo Medical SchoolSão PauloBrazil
  3. 3.Department of PathologyUniversity of São Paulo Medical SchoolSão PauloBrazil
  4. 4.Department of RadiologyUniversity of São Paulo Medical SchoolSão PauloBrazil
  5. 5.Sandler Neurosciences Center, Memory and Aging Center, Department of NeurologyUniversity of California at San FranciscoSan FranciscoUSA
  6. 6.Human Motor Systems Laboratory, School of Physical Education and SportUniversity of São PauloSão PauloBrazil
  7. 7.São PauloBrazil

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