Reduced Purkinje Cell Size in the Cerebellar Vermis of Elderly Patients With Schizophrenia

The cerebellum has been viewed traditionally as a central nervous system (CNS) structure dedicated to motor control. However, recent evidence from neuro­behavioral and functional neuroimaging studies indicates additional roles for the cerebellum in such diverse cognitive processes as attention, sensory discrimination, verbal learning and memory, working memory, and complex problem solving (1). Furthermore, abnormalities in cerebellar structure and function have also been described (2). The purpose of the current neuropathological investigation was to determine linear density and cell size of the Purkinje cell layer in the cerebellum of elderly individuals with schizophrenia compared with nonneuropsychiatric subjects and to study possible relationships between these morphometric and clinical features.

METHOD

Subjects included 14 patients with chronic schizophrenia. Their mean age at death was 80.3 years (SD=8.0). Three were men and 11 were women. The mean postmortem interval was 11.0 hours (SD=3.6). The comparison group comprised 13 age-comparable, sex-comparable, and postmortem-interval-comparable normal subjects with no known history of neuropsychiatric disorder. Their mean age at death was 79.3 years (SD=11.8). Four were men and nine were women. The mean postmortem interval was 12.2 hours (SD=6.2). All subjects with schizophrenia had been participants in a prospective clinicopathological study at the University of Pennsylvania Mental Health Clinical Research Center (3). Diagnoses were established by consensus among clinicians using DSM-III-R criteria. All patients had been assessed with clinical rating scales within 1 year prior to death. All autopsies were conducted at our institution in a uniform manner. Minor neuropathological abnormalities (e.g., lacunar infarcts), none of which affected the cerebellum, were found in four patients with schizophrenia and three comparison subjects.

Parasagittal blocks of the superior cerebellar vermis (including the central lobule and culmen) were dissected at autopsy, immersed in alcohol fixative (70% ethanol, 150 mM NaCl) for 24 hours, and then paraffin-embedded. Ten-micron-thick sections were Nissl-stained with 1% cresyl violet, as previously described (4). All histologic data were obtained by a single operator (K.D.T.), who was blind to case information. Representative microscopic fields were randomly and systematically sampled in each section. Starting from a random point within the section, we identified successive clockwise fields at low magnification. At each point, magnification was raised and the field was video-captured for computer-assisted image analysis by using a Leitz DMRB research microscope, Sony CCD video camera, and Macintosh computer with NIH Image 1.59 software (National Institutes of Health, Rockville, Md.). The linear densities of Purkinje cells were determined by counting the number of Purkinje cells per millimeter line length in 15 fields magnified by 100 per case. Next, we obtained Purkinje cell cross-sectional areas in 50 Purkinje cells with clearly visible nucleoli per case from 9–29 fields magnified by 200. After digitization of the captured image, customized NIH Image software was used to automatically delineate Purkinje cell somata, as previously described (4). After further manual editing to exclude portions of proximal dendrites or other artifacts that were not automatically excluded, Purkinje cell cross-sectional areas were determined.

Preliminary scatterplots with correlation analyses were performed to assess possible effects of age, sex, and postmortem interval in each of the two groups. Between-group morphometric differences were analyzed by using Student’s t test (two-tailed) and the Mann-Whitney U test. We then studied possible correlations between Purkinje cell density and size and clinical variables, including age at onset, duration of illness, antipsychotic medication dose (converted to chlorpromazine equivalents), Mini-Mental State score, Brief Psychiatric Rating Scale (BPRS) score, Scale for the Assessment of Negative Symptoms (SANS) score, Scale for the Assessment of Positive Symptoms (SAPS) score, and Abnormal Involuntary Movement Scale (AIMS) score, as previously described (3).

RESULTS

Figure 1 shows representative Purkinje cells of a comparison subject and a schizophrenic patient magnified by 400. Preliminary analyses showed a significant correlation between age and Purkinje cell density in the comparison group (r=–0.58, df=11, p<0.04) but not the schizophrenia group. No correlations were observed between age and Purkinje cell size in either group or between other potentially confounding variables and either morphometric feature. No significant difference in Purkinje cell density was detected between the two groups.

The average cross-sectional areas of Purkinje cells of the schizophrenia group (mean=343 ², SD=34) were 8.3% smaller than those of the comparison group (mean=374 ², SD=34) (t=2.37, df=25, p<0.03; Mann-Whitney U=44, p=0.02). This finding is consistent with a preliminary observation in a separate set of six subjects with schizophrenia and six comparison subjects in which we found a 16.5% reduction in Purkinje cell cross-sectional cytoplasmic areas labeled with a type 1 inositol 1,4,5-triphosphate receptor transmembrane cDNA probe in an in situ hybridization study (unpublished data).

Significant correlations were observed between Purkinje cell size and Mini-Mental State scores (r=0.67, df=12, p<0.02), BPRS scores (r=–0.57, df=12, p<0.04), and antipsychotic drug dose (r=0.59, df=12, p<0.05) in the schizophrenia group. Correlation between Purkinje cell size and duration of illness approached significance (r=–0.53, df=12, p<0.07). No significant correlations were observed with age at onset, SANS score, SAPS score, or AIMS score.

DISCUSSION

Although data from volumetric neuroimaging reports of the cerebellar vermis in schizophrenia have conflicted (2), metabolic imaging studies have been more consistent, indicating abnormal cerebellar blood flow (5), glucose metabolism (6), and activation with cognitive challenges (7). To our knowledge, there have been only two previous Purkinje cell morphometric studies. Reyes and Gordon (8) observed significantly reduced Purkinje cell linear density, and Lohr and Jeste (9) found no differences in Purkinje cell linear density but did see a trend toward Purkinje cell size reduction in schizophrenia. In our study, a significant decrease in Purkinje cell size was observed with normal Purkinje cell linear density in elderly, chronically hospitalized patients with schizophrenia. Possible reasons for the discrepancies between studies might include differences in paradigms for selecting fields and Purkinje cells, diagnostic uncertainties in previous retrospective studies, and older methods for measuring neuronal profiles. To address these challenges, we used random systematic sampling of a large number of fields and neu­rons, a prospective study group of schizophrenia patients in whom we have demonstrated diagnostic reliability, and computer-assisted neuronal morphometry.

We found a significant positive correlation between antipsychotic dose and Purkinje cell size, and we speculate that treatment may promote enlargement of Purkinje cells in the cerebellar vermis in schizophrenia. Smaller neuron size also correlated with global cognitive impairment and psychopathology, but not age at onset or other specific symptom profiles. In addition, there was a nonsignificant suggestion of smaller Purkinje cell size with increased duration of illness. Since these are single correlations and our study group was small and limited to elderly, “poor outcome” patients, care must be taken in interpreting and extrapolating these results. In addition, we did not study a nonschizophrenic psychiatric comparison group and so cannot say if the reduced Purkinje cell size is specific for schizophrenia or not.

Smaller neuron size also has been observed in schizophrenia in the hippocampus, entorhinal cortex, dorsolateral prefrontal cortex, substantia nigra, and locus ceruleus (10). It is interesting that although much attention has rightfully focused on these regions, which have long been associated with higher cognitive and emotional functioning in schizophrenia, data such as ours are increasing to indicate abnormalities throughout the nervous system. Our data are particularly interesting in the light of the more complex, cognitive functions of the cerebellum that are being increasingly recognized (1). The molecular neuropathological basis for such changes in the cerebellum and elsewhere is yet to be revealed.

Received Dec. 4, 1997; revision received April 22, 1998; accepted May 5, 1998. From the Center for Neurobiology and Behavior, Department of Psychiatry; the Smell and Taste Center, Department of Otorhinolaryngology: Head and Neck Surgery; and the Department of Neurology, University of Pennsylvania School of Medicine.. Address reprint requests to Dr. Arnold, Department of Psychiatry, University of Pennsylvania School of Medicine, 142 Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104-4283; [email protected] (e-mail). Supported by a grant from the Scottish Rite Benevolent Foundation"s Schizophrenia Research Program, grants MH-43880 and MH-55199 from NIMH, and grant DC-00161 from the National Institute on Deafness and Other Communication Disorders.The authors thank the residents and staff of the Penn Schizophrenia Mental Health Clinical Research Center and the Department of Pathology and Laboratory Medicine; Li-Ying Han and Catherine Choi for their assistance; and all the patients and their families whose generosity made this research possible.