Size of hippocampal pyramidal neurons in schizophrenia

Background Meta-analyses of hippocampal size have indicatedthat this structure is smaller in schizophrenia. This couldreflect a reduction in the size of constituent neurons or areduced number of neurons.

Aims To measure the size of hippocampal pyramidal neurons inthe brains of people with and without schizophrenia.

Method Pyramidal neuron size in hippocampal subfields was estimated stereologically from sections taken at 5 mm intervals throughoutthe whole length of right and left hippocampi from the brainsof 13 people with schizophrenia and 16 controls. Results wereassessed using repeated-measures analysis of covariance lookingfor a main effect of diagnosis and gender, and interactionsof these with side.

Results We were unable to detect significant differences relatedto diagnosis, gender or side for any hippocampal subfield forthis series of cases.

Conclusions For this series of brains, hippocampal cell sizeis unchanged in schizophrenia.

INTRODUCTION

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INTRODUCTION

The hippocampus has interested investigators into schizophreniafor many decades. Experimental and human studies have shownits undoubted importance for memory function, which is selectivelyimpaired in people with schizophrenia (Gruzelier et al, 1988;Saykin et al, 1991; Gur et al, 1998). Furthermore, some structuralimaging studies of living patients, as well as post-mortemstudies, report reductions in hippocampal size in schizophrenia(Bogerts et al, 1985; Falkai & Bogerts, 1986; Jeste & Lohr, 1989;Nelson et al, 1998) – although not all studiesconfirm this, perhaps because the numbers of individuals studiedwere sometimes too low to detect the quite subtle reductionsdescribed (Altshuler et al, 1990; Bruton et al, 1990; Heckerset al, 1990, 1991). The meta-analysis of in vivo magneticresonance imaging (MRI) studies by Nelson et al (1998) indicateda reduction in hippocampal size of approximately 4%.

Reduction in size of a brain structure may reflect a reducedsize of the constituent glial cells and neurons and their processesas well as (or as an alternative to) a reduced number of neurons.Thus, it is important to document estimates of cell size aswell as cell number in brain structures that are of interestin schizophrenia. Here we present our findings with respectto pyramidal cell volume, estimated stereologically, in thehippocampus on both sides of brains taken from 13 people withschizophrenia and 16 controls.

METHOD

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METHOD

The brains studied in this investigation were a subset of 29samples from a collection that has been described elsewhere (Highley et al, 1999; McDonald et al, 2000; Walker et al,2002). In brief, brains were collected post mortem from patientswith schizophrenia and a control group, and fixed by suspensionin 10% formalin solution. The case notes of the patients andcontrols were assessed by a psychiatrist (T.J.C. or Dr StephenJ. Cooper from Queen’s University, Belfast) to ensurethat they either fulfilled DSM–IV criteria for schizophreniaor schizoaffective disorder (American Psychiatric Association, 1994),or were free of psychopathological disorder. The nextof kin gave consent for use of brain tissue for research. Allbrains were examined by a neuropathologist (B.M.), masked todiagnosis and gender, who confirmed them as being free fromsignificant neuropathological changes. In particular, therewas no evidence of cerebrovascular disease, Alzheimer’sdisease or Parkinson’s disease. All measures were made (by M.A.W.) masked to diagnosis and gender. The demographicdetails of the brain donors in this study are given in Table 1.

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Table 1 Preparation and donor characteristics of the brain samples used in this study

The temporal lobes were dissected away from the rest of thebrain, and sliced into 5 mm coronal slices throughout theirentire length, such that the entirety of the hippocampus wasavailable for histological examination. Each slice was embeddedin paraffin wax, and a 25 µm section was cut from its anterior face, mounted on a coated slide, stained with cresylviolet and luxol fast blue, and coverslipped. The outlinesof four cytoarchitecturally defined hippocampal subfields weredelineated in the manner described by West & Gundersen(1990):

the hilus (CA4)
an amalgamation of the CA2 and CA3 subfields(hereafter CA2/3)
the CA1 subfield
the subiculum.

The volume density (V_v) of these subfields was measured onboth sides of the brain, using stereological point-counting techniques (Howard & Reed, 1998). Volume density in thisstudy refers to the proportion of each hippocampal subfieldthat is occupied by pyramidal neuronal cell bodies.

The prepared slides were examined using a 660 objective andan Olympus BX50 microscope mounted with a JVC TK-C1380 colourvideo camera and stage motor, which in turn were controlledand viewed on a computer running the Olympus Cast-Grid 2.0stereology sampling software. On each slide, each subfield was examined at specific points positioned in a raster search patternarray which covered the entirety of the subfield (Fig. 1).The search pattern was 0.5x0.5 mm² for the hilus and CA2/3subfields, 1x1mm² for the CA1 subfield, and 0.75x0.75 mm² forthe subiculum. Each subfield appeared on an average of 6.7slides per case (range 3–15; in a few cases the hippocampuswas cut obliquely, which meant it appeared in few sections, although this did not alter the intensity of the sampling).Neuron density (N_v) was counted in a mean 57 dissectors and volume density (V_v) was assessed with a mean 88 frames percase; a mean of 89 neurons were counted for N_v, and a meanof 135 points were counted for V_v estimation per case. At eachsuch point a plane within the section was brought into focus,and an array of 36 random test points thrown over the microscopeimage. The number of test points that fell over pyramidal cellbodies was counted, and the mean number of point counts ([notdef]) per image calculated. Pyramidal cellswere identified on the basis of their position, orientationshape, presence of an apical dendrite and prominent, single nucleolus. The volume density was calculated for each subfieldby V_v = /36.

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Fig. 1 Hippocampal region CA2/3 with superimposed raster sampling pattern.

In a previous study, the neuron density (number of cells perunit volume, N_v) within each hippocampal subfield had been estimated (Walker et al, 2002; further details available fromthe author upon request). Using these data, the mean pyramidalcell body volume (V_N) was calculated using the formula V_N= V_v/N_v.

Statistical methods
The cell volume for each subfield was assessed by repeated-measures analysis of covariance (ANCOVA), with diagnosis and genderas between-subject factors and side as a within-subject factor,using SAS version 6.12 for Macintosh. As there was a significantdifference between the brains from the control and schizophreniagroups in the duration spent in formalin prior to histologicalpreparation, this variable was entered as a covariate in the ANCOVA model. The main effect of diagnosis, gender, and interactionsof these with side, gender, and gender and side together weretested for. Given the number of effects tested for each measure, ${alpha}$ was set at 0.01. Thus, for an effect to be significant, ithad to generate a value for P of 0.01 or below.

RESULTS

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RESULTS

Quality of measures
The observed coefficient of error (OCE) for the individual estimatesof subfield volume, cell density and cell number were calculatedfor 10 cases in the manner described by Gundersen & Jensen (1987) and West & Gundersen (1990). This coefficientis a measure of the variability of measures of a specific structureacross different slices; possible values are 0 to 1. It is increased by irregularly shaped structures and by inaccurate and unreliablemeasurement. For the measures of V_N, the mean OCE values were all $>=$ 0.121. For the measures of V_v, mean OCE valueswere all $>=$ 0.061. For N_v, the mean values of OCEwere all $>=$ 0.1001. The values of OCE can be usedto estimate the percentage of observed relative variance, (s.d./[notdef])², of each measure which is accounted forby true inter-individual variance, as opposed to the stereologicalvolume estimate (West & Gundersen, 1990; West, 1999).Ideally, this should be greater than 50%. This was true for all measures of V_N (all 580.2%). The conclusion to be drawnis that the measures are of adequate reliability and accuracy.

Effects of diagnosis, gender and side
A bar chart of mean cell volume is given in Fig. 2. The meancell volumes (standard deviations in parentheses) for the subfieldswere as follows:

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Fig. 2 Pyramidal cell volumes for hippocampal subregions (bars show means and standard error of the mean). Cases have been subdivided according to gender and disease category.

hilus: 4.08610⁷⁶ (0.84610⁷⁶) for controls and 3.82610⁷⁶ (1.23610⁷⁶)mm³ for patients;
CA2/3: 3.88610⁷⁶ (1.09610⁷⁶)mm³ for controls and 3.45610⁷⁶(1.056 10⁷⁶)mm³ for patients;
CA1:2.54610⁷⁶ (0.59610⁷⁶)mm³ for controls and 2.60610⁷⁶ (0.67610⁷⁶)mm³ for patients;
subiculum: 2.52610⁷⁶ (0.576 10⁷⁶)mm³for controls and 2.25610⁷⁶ (0.46610⁷⁶)mm³ for patients.

There was no significant effect for diagnosis, gender or sidefor any subfield. Thus, for the hilus, all F_(1,24) $<=$ 1.22, P $>=$ 0.2021; for the CA2/3 subfield, all F_(1,24) $<=$ 3.25, P $>=$ 0.0842; for the CA1 subfield, all F_(1,23) $<=$ 1.35, P $>=$ 0.2574; for the subiculum, all F_(1,23) $<=$ 2.19, P $>=$ 0.1522.

DISCUSSION

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DISCUSSION

The main finding in this study is an absence of size changein hippocampal pyramidal neurons in schizophrenia. There havebeen five earlier studies of this parameter of which we areaware (Christison et al, 1989; West & Gundersen, 1990;Benes et al, 1991, 1998; Arnold et al, 1995; Zaidel et al,1997; West, 1999); two of them found no change, and threefound a decrease in size in schizophrenia. All had comparablenumbers of cases of schizophrenia to the number in the present study. Control case numbers were similar to our study in fourstudies but were larger in one other negative study. All studiesused the Nissl stain. Only one previous study in addition toours looked at both sides of the brain, and only our studysampled the hippocampus throughout its full extent.

We addressed the potential of regional specificity of changesin schizophrenia by dividing the hippocampus into four subfields.We did not further divide our hippocampal subfields into anteriorand posterior halves. It is thus possible that changes in onehalf (anterior or posterior) of a subfield might have beenmasked or ‘diluted’ by variance in the other half.In a meta-analysis of hippocampal volumes in schizophrenia assessedby MRI it was found that inclusion of the amygdala, abuttingon the anterior hippocampus, in the region of interest significantlyincreased the size of the reduction in volume seen in schizophrenia.The recommendation was made that in future research relativealterations in anterior and posterior hippocampus in schizophreniashould be assessed separately (Nelson et al, 1998). It isalso possible that our study might have failed to detect a ‘true’reduction in cell size in some hippocampal subfields becauseof the small sample size (type II error).

Decreases in neuronal size have been reported for other regionsof the brain in schizophrenia – the dorsolateral prefrontalcortex, anterior cingulate cortex, cerebellar Purkinje cells,substantia nigra and locus caeruleus – but not in themotor cortex or calcarine cortex (reviewed by Harrison, 1999).Further studies will be needed before the primacy of thesechanges in the disease can be judged.

Clinical Implications and Limitations

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Clinical Implications and...

CLINICAL IMPLICATIONS

Memory impairments in schizophrenia cannot be attributed toalterations in size of hippocampal neurons.
Reduction insize of the hippocampus in schizophrenia is notexplained by reduced hippocampal neuron size.

LIMITATIONS

The sample sizes are relatively small.
There is the possibilityof a type II error (failure to detecta true difference betweenthe brains of people with and withoutschizophrenia).
Thehippocampus was analysed as a whole, so changes in a part,suchas the anterior half, might have been missed.

ACKNOWLEDGMENTS

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ACKNOWLEDGMENTS

This work was funded by grants from the UK Medical ResearchCouncil and the Wellcome Trust. We thank Drs S. J. Cooper andB. Herron for assistance with clinical assessment and post-mortembrain removal respectively for some of the cases included inthis study.

REFERENCES

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