Skip to main content
Log in

Cerebellar Purkinje Cells are Reduced in a Subpopulation of Autistic Brains: A Stereological Experiment Using Calbindin-D28k

  • Published:
The Cerebellum Aims and scope Submit manuscript

Abstract

Although a decreased number of cerebellar Purkinje cells (PCs) in the autistic brain has been widely reported with a variety of qualitative and quantitative methods, the more accurate method of cell counting with modern stereology has not yet been employed. An additional possible problem with prior reports is the use of Nissl staining to identify the PCs, as this can miss cells due to staining irregularities. In the present study, PCs were immunostained for calbindin-D28k (CB), as this has been shown to be a more reliable marker for PCs than the Nissl stain, with more than 99% of the PCs immunopositive (Whitney, Kemper, Rosene, Bauman, Blatt, J Neurosci Methods 168:42–47, 2008). Using stereology and CB immunostaining, the density of PCs was determined in serial sections from a consistently defined area of the cerebellar hemisphere in four control and six autistic brains, with the density of PCs then correlated with the clinical severity of autism. Overall, there was no significant difference in the density of PCs between the autistic and control groups. However, three of six autistic brains had PC numbers that fell within the control range, whereas the remaining three autistic brains revealed a reduction compared with the control brains. These data demonstrate that a reduction in cerebellar PCs was not a consistent feature of these autistic brains and that it occurred without discernible correlation between their density and the clinical features or severity of autism.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.

Similar content being viewed by others

References

  1. American Psychiatric Association (1994) Diagnostic and statistical manual of mental disorders, 4th edn. American Psychiatric Association, Washington, DC

  2. Williams RS, Hauser SL, Purpura DP, DeLong GR, Swisher CN (1980) Autism and mental retardation: neuropathologic studies performed in four retarded persons with autistic behavior. Arch Neurol 37:749–753

    PubMed  CAS  Google Scholar 

  3. Bauman ML, Kemper TL (1985) Histoanatomic observations of the brain in early infantile autism. Neurology 35:866–874

    PubMed  CAS  Google Scholar 

  4. Ritvo ER, Freeman BJ, Scheibel AB, Duong T, Robinson H, Guthrie D, Ritvo A (1986) Lower Purkinje cell counts in the cerebella of four autistic subjects: initial findings of the UCLA-NSAC autopsy research report. Am J Psychiatry 146:862–866

    Google Scholar 

  5. Kemper TL, Bauman ML (1993) The contribution of neuropathologic studies to the understanding of autism. Neurol Clin 11:175–187

    PubMed  CAS  Google Scholar 

  6. Guerin P, Lyon G, Barthelemy C, Sostak E, Chevrollier V, Garreau B, Lelord G (1996) Neuropathological study of a case of autistic syndrome with severe mental retardation. Dev Med Child Neurol 38:203–211

    PubMed  CAS  Google Scholar 

  7. Bailey A, Luthert P, Dean A, Harding B, Janota I, Montgomery M, Rutter M, Lantos PA (1998) Clinicopathological study of autism. Brain 121:889–905

    Article  PubMed  Google Scholar 

  8. Lee M, Martin-Ruiz C, Graham A, Court J, Jaros E, Perry R, Iversen P, Bauman M, Perry E (2002) Nicotinic receptor abnormalities in the cerebellar cortex in autism. Brain 15:1483–1495

    Article  Google Scholar 

  9. Fatemi SH, Halt AR, Realmuto G, Earle J, Kist DA, Thuras P, Merz A (2002) Purkinje cell size is reduced in cerebellum of patients with autism. Cell Mol Neurobiol 22:171–175

    Article  PubMed  Google Scholar 

  10. Vargas DL, Nascimbene C, Krishnan C, Zimmerman AW, Pardo CA (2005) Neuroglial activation in the brain of patients with autism. Ann Neurol 57:67–81

    Article  PubMed  CAS  Google Scholar 

  11. Palmen S, van Engeland H, Hof PR, Schmitz C (2004) Neuropathological findings in autism. Brain 127:2572–2583

    Article  PubMed  Google Scholar 

  12. Saper CB (1996) Any way you cut it: a new journal policy for the use of unbiased counting methods. J Comp Neurol 364:5

    Article  PubMed  CAS  Google Scholar 

  13. Whitney ER, Kemper TL, Rosene DL, Bauman ML, Blatt GJ (2008) Calbindin-D28k is a more reliable marker of human Purkinje cells than standard Nissl stains: a stereological experiment. J Neurosci Methods 168:42–47

    Article  PubMed  CAS  Google Scholar 

  14. Schmahmann JD, Sherman JC (1998) The cerebellar cognitive affective syndrome. Brain 121:561–579

    Article  PubMed  Google Scholar 

  15. Levisohn L, Cronin-Golomb A, Schmahmann JD (2000) Neuropsychological consequences of cerebellar tumour resection in children: cerebellar cognitive affective syndrome in a paediatric population. Brain 123:1041–1050

    Article  PubMed  Google Scholar 

  16. Riva D, Giorgi C (2000) The cerebellum contributes to higher functions during development; evidence from a series of children surgically treated for posterior fossa tumours. Brain 123:1051–1061

    Article  PubMed  Google Scholar 

  17. Schmahmann JD (2004) Disorders of the cerebellum: ataxia, dysmetria of thought, and the cerebellar cognitive affective syndrome. J Neuropsychiatry Clin Neurosci 16:367–378

    PubMed  Google Scholar 

  18. Parvizi J, Joseph J, Press DZ, Schmahmann JD (2007) Pathological laughter and crying in patients with multiple system atrophy-cerebellar type. Mov Disord 22:798–803

    Article  PubMed  Google Scholar 

  19. Schmahmann JD, Weilburg JB, Sherman JC (2007) The neuropsychiatry of the cerebellum—insights from the clinic. Cerebellum 6:254–267

    Article  PubMed  Google Scholar 

  20. Grafman J, Litvan I, Massaquoi S, Stewart M, Sirigu A, Hallett M (1992) Cognitive planning deficit in patients with cerebellar atrophy. Neurology 43:1493–1496

    Google Scholar 

  21. Schmahmann JD, Doyon J, Toga AW, Petrides M, Evans AC (2000) MRI atlas of the human cerebellum. Academic, San Diego, CA, pp 3–20

    Google Scholar 

  22. Rosene DL, Roy NJ, Davis BJ (1986) A cryoprotection method that facilitates cutting frozen sections of whole monkey brains for histological and histochemical processing without freezing artifact. J Histochem Cytochem 34:1301–1315

    PubMed  CAS  Google Scholar 

  23. Crane AM, Goldman PS (1979) An improved method for embedding brain tissue in albumin-gelatin. Stain Technol 54:71–75

    PubMed  CAS  Google Scholar 

  24. Gundersen HJ (1986) Stereology of arbitrary particles. J Microsc 143:3–45

    PubMed  CAS  Google Scholar 

  25. West MJ, Slomianka L, Gundersen HJ (1991) Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Anat Rec 231:482–497

    Article  PubMed  CAS  Google Scholar 

  26. Hedreen JC (1998) Lost caps in histological counting methods. Anat Rec 250:366–372

    Article  PubMed  CAS  Google Scholar 

  27. Hall TC, Miller AKH, Corsellis JAN (1975) Variations in the human Purkinje cell population according to age and sex. Neuropathol Appl Neurobiol 1:267–292

    Article  Google Scholar 

  28. Spielmeyer W (1922) Histopathologie des Nervensystems. Julius Springer, Berlin, pp 56–79

    Google Scholar 

  29. Lawrence RD, Meyer A, Nevin S (1942) The pathological changes in the brain in fatal hypoglycaemia. Quart J Med 35:181–201

    Google Scholar 

  30. Blackwood W, McMenemey WH, Meyer A, Norman RM, Russell DS (1963) Greenfield’s neuropathology. William and Wilkins, Baltimore, pp 29–34

    Google Scholar 

  31. Ghatak NR, Santoso RA, McKinney WM (1976) Cerebellar degeneration following long-term phenytoin therapy. Neurology 26:818–820

    PubMed  CAS  Google Scholar 

  32. Rapport RL, Shaw CM (1977) Phenytoin-related cerebellar degeneration without seizures. Ann Neurol 2:437–439

    Article  PubMed  Google Scholar 

  33. McLain LW, Martin JT, Allen JH (1980) Cerebellar degeneration due to chronic phenytoin therapy. Ann Neurol 7:18–23

    Article  PubMed  Google Scholar 

  34. Schmahmann JD, Pandya DN (1989) Anatomical investigation of projections to the basis pontis from posterior parietal association cortices in rhesus monkey. J Comp Neurol 289:53–73

    Article  PubMed  CAS  Google Scholar 

  35. Schmahmann JD, Pandya DN (1991) Projections to the basis pontis from the superior temporal sulcus and superior temporal region in the rhesus monkey. J Comp Neurol 308:224–248

    Article  PubMed  CAS  Google Scholar 

  36. Schmahmann JD, Pandya DN (1993) Prelunate, occipitotemporal, and parahippocampal projections to the basis pontis in rhesus monkey. J Comp Neurol 337:94–112

    Article  PubMed  CAS  Google Scholar 

  37. Schmahmann JD, Pandya DN (1995) Prefrontal cortex projections to the basilar pons in rhesus monkey: implications for the cerebellar contribution to higher function. Neurosci Lett 199:175–178

    Article  PubMed  CAS  Google Scholar 

  38. Schmahmann JD, Pandya DN (1997) The cerebrocerebellar system. Int Rev Neurobiol 41:31–60

    Article  PubMed  CAS  Google Scholar 

  39. Schmahmann JD (2001) The cerebrocerebellar system: anatomic substrates of the cerebellar contribution to cognition and emotion. Int Rev Psychiatry 13:247–260

    Article  Google Scholar 

  40. Middleton FA, Strick PL (2001) Cerebellar projections to the prefrontal cortex in the primate. J Neurosci 21:700–712

    PubMed  CAS  Google Scholar 

  41. Zagrebelsky M, Strata P, Hawkes R, Rossi F (1997) Reestablishment of the olivocerebellar projection map by compensatory transcommissural reinnervation following unilateral transection of the inferior cerebellar peduncle in the inferior cerebellar peduncle in the newborn rat. J Comp Neurol 379:283–299

    Article  PubMed  CAS  Google Scholar 

  42. Sugihara I, Lohof AM, Letellier M, Mariani J, Sherrard RM (2003) Post-lesion transcommissural growth of olivary climbing fibres creates functional synaptic microzones. Eur J Neurosci 18:3027–3303

    Article  PubMed  Google Scholar 

  43. Yip J, Soghomonian JJ, Blatt GJ (2007) Decreased GAD67 mRNA levels in cerebellar Purkinje cells in autism: pathophysiological implications. Acta Neuropathol 113:559–568

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgment

We gratefully acknowledge the Harvard Brain Tissue Resource Center, Kathleen Price Bryan Brain Bank at Duke University Medical Center, and University of Maryland Brain Bank for providing brain tissue for this study. This work was supported by grants from the NIH-NICHD HD39459, the National Alliance for Autism Research (NAAR], the Nancy Laurie Marks Foundation, and the John and Lisa Hussman Foundation. We are grateful to Michael Bowley for his assistance with the stereology software instruction and maintenance of the system. We also thank several members of the laboratory for their assistance with tissue processing: Rita Marcon, Sandy Thevarkunnel, Melissa Martchek, Matthew Stoker, and Matthew Fields.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Elizabeth R. Whitney.

Appendix: Autistic Case Histories

Appendix: Autistic Case Histories

Autistic Case 4414

Twenty-six-year-old male born of an uncomplicated birth. He began showing signs of behavioral abnormalities during his toddler years and was diagnosed with autism at the age of five. He graduated from high school and enjoyed writing. He was able to communicate in sentences. At the age of 23, medical records reported that he was fully oriented and followed all simple commands. He did, however, have trouble with more complex commands. His motor exam showed normal strength and tone. Coordination was normal except for slightly slow and clumsy rapid alternating movements in his feet. His gait was normal and he was able to tandem walk. He began taking Prozac at the age of 23, which was reported to be “very effective” in managing behavioral problems. A seizure disorder became apparent at 10 years of age, with eight to ten seizures per year, for which he received Dilantin®. Medications at time of death included Dilantin® and Prozac®.

Autistic Case 3845

Thirty-two-year-old male without complication during the prenatal or perinatal period. During her pregnancy, his mother had no history of infection and did not take any medications. She sensed that something was wrong in the neonatal period, as he was a very unresponsive baby and his general development was slower than that of his siblings. He was formally evaluated at 2.5 years. At that time, he seemed involved in his own world, had no interest in playing with other children, and never wanted to be held. He had not developed language skills; he only grunted. His parents suspected that he might be deaf, but formal education revealed intact hearing. Additionally, he frequently rocked and waved his arms and developed a fascination for spinning tops. As he got older, he was very difficult to manage. He was aggressive towards others, had daily tantrums, and frequently demonstrated self-abusive behaviors. He remained nonverbal throughout his life, but was able to dress and feed himself independently. Physical examination four months prior to his death revealed the absence of tremors and “unremarkable” gait. He began having seizures at the age of 8 years. Seizures were poorly controlled over the years despite receiving numerous medications including Dilantin®, Depakote®, Felbamate®, Gabapentin®, Tegretol®, and Primidone®.

Autistic Case 4099

Nineteen-year-old male born of an uncomplicated birth. His mother became concerned about him at 2 years of age when he began spinning plates. At this age, he also did not speak and was not yet walking. He began walking and speaking at 3 years of age. In addition to his diagnosis of autism, he was diagnosed with Duchenne’s muscular dystrophy when he was 8 years old. He attended a mainstream middle school with special classes for autistic children. At 10 years of age, his vocabulary consisted of 150 words. Medical records revealed that he was minimally cooperative during physician visits but was reported to recognize his mother and show affection toward her, able to answer yes/no questions, and converse in short simple phrases. He never experienced any seizures.

Autistic Case 2431

Fifty-four-year-old male who sustained trauma to the face, ear, and shoulder during a breech delivery. At 9 months of age, it was thought that he was deaf because he paid no attention to sound. Examination of his hearing revealed possible deafness, but his behavior was not in accordance with deafness alone. He attended a school for the deaf until the age of 12 when he was referred for further evaluation; at that time, his behavior was characterized as “withdrawn, mute, violent, destructive…strangely manneristic.” Examination also revealed normal hearing, and a diagnosis of infantile autism was made. He was first hospitalized at 12 years of age after he was found using an ax to destroy furniture in the family home. He remained hospitalized until his death. Reports consistently reference his aggressive behaviors without provocation. Temper tantrums frequently occurred if disruptions in his daily routine occurred. He remained non-verbal throughout his life, but was able to use some gestures to communicate and was able to follow simple three-step commands. Motor exam at the age of 5.7 years reported that he “has marvelous muscle coordination involving the smaller muscles; however, activities demanding use of large muscles seem to be very difficult for him.” He had no history of seizures. Medications at the time of his death included Ascriptin®, Thorazine®, Centrax®, Ativan®, and Serax®.

Autistic Case 4259

Thirteen-year-old female diagnosed with infantile autism as a toddler. Concerns were raised very early in the neonatal period as she did not cry or respond to noise. She had delayed motor milestones and remained non-verbal throughout her life. She displayed self-abusive hand biting but was rarely aggressive toward caregivers. She experienced her first seizure at 5 years of age for which Tegretol® was prescribed.

Autistic Case 3511

Twenty-seven-year-old male who was living in a residential home at the time of death. He was reportedly very difficult to manage behaviorally. He exhibited unpredictable episodes of aggressiveness with yelling, screaming, pacing, scratching his face, and attacking the staff. Routine was very important to him and he would perseverate on an activity until it was completed. He worked for a cleaning and landscaping service and was said to be an excellent worker. He would work for hours without stopping. He could follow very simple commands and was able to express simple needs in two- to three-word sentences. He had a history of two seizures during his lifetime. At the time of his death, he was taking Haloperidol® and Phenobarbital®.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Whitney, E.R., Kemper, T.L., Bauman, M.L. et al. Cerebellar Purkinje Cells are Reduced in a Subpopulation of Autistic Brains: A Stereological Experiment Using Calbindin-D28k. Cerebellum 7, 406–416 (2008). https://doi.org/10.1007/s12311-008-0043-y

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12311-008-0043-y

Keywords

Navigation