Improving Cavalieri volume estimation based on non-equidistant planar sections: The trapezoidal estimator.

The Cavalieri estimator allows one to infer the volume of an object from area measurements in equidistant planar sections. It is known that applying this estimator in the non-equidistant case may inflate the coefficient of error considerably. We therefore consider a newly introduced variant, the trapezoidal estimator, and make it available to practitioners. Its typical variance behaviour for natural objects is comparable to the equidistant case. We state this unbiased estimator, describe variance estimates and explain how the latter can be simplified under rather general but realistic models for the gaps between sections. Simulations and an application to a synthetic area function based on parietal lobes of 18 monkeys illustrate the new methods.

Hippocampal volume and cell number in depression, schizophrenia, and suicide subjects.

Many studies suggest that the hippocampus is involved in the pathophysiology of psychiatric disorders, especially major depressive disorder (MDD) and schizophrenia. Especially, in vivo imaging studies indicate that the volume of hippocampus may be reduced in both disorders. Moreover, suicide may have a unique neurobiology. The aim of the present study is to investigate if depression, schizophrenia or suicide is associated with reduced postmortem volume of the hippocampal formation and/or changes in the numbers of neurons and/or glial cells in the different subregions of the hippocampus. We studied postmortem brain samples from 10 subjects with schizophrenia, 8 subjects with major depression, 11 suicide subjects with a history of depressive disorder, and 10 control subjects with no history of psychiatric or neurological diseases. The total volume and numbers of neurons and glial cells were estimated for the main hippocampal subregions using design-unbiased stereological techniques. We found the total volume and total numbers of neurons and glial cells similarly reduced by approximately 20% to 35% in depression and schizophrenia subjects relative to control subjects across all hippocampal regions. In suicide subjects, we only found increased neuron number in CA2/3 subregion. The volume and number of cells are reduced in depression and schizophrenia subjects relative to control subjects across all hippocampal regions. Our findings imply that the hippocampus may be a common site of pathophysiology in depression and schizophrenia. Community living suicide subjects seem to differ in hippocampal neurobiology compared to hospitalized subjects dying with MDD without suicide.

Evaluating the Feasibility of DNA Methylation Analyses Using Long-Term Archived Brain Formalin-Fixed Paraffin-Embedded Samples.

We here characterize the usability of archival formalin-fixed paraffin-embedded (FFPE) brain tissue as a resource for genetic and DNA methylation analyses with potential relevance for brain-manifested diseases. We analyzed FFPE samples from The Brain Collection, Aarhus University Hospital Risskov, Denmark (AUBC), constituting 9479 formalin-fixated brains making it one of the largest collections worldwide. DNA extracted from brain FFPE tissue blocks was interrogated for quality and usability in genetic and DNA methylation analyses by different molecular techniques. Overall, we found that DNA quality was inversely correlated with storage time and DNA quality was insufficient for Illumina methylation arrays; data from methylated DNA immunoprecipitation, clonal bisulfite sequencing, and pyrosequencing of BDNF and ST6GALNAC1 suggested that the original methylation pattern is indeed preserved. Proof-of-principle experiments predicting sex based on the methylation status of the X-inactivated SLC9A7 gene, or genotype differences of the Y and X chromosomes, showed consistency between predicted and actual sex for a subset of FFPE samples. In conclusion, even though DNA from FFPE samples is of low quality and technically challenging, it is likely that a subset of samples can provide reliable data given that the methodology used is designed for small DNA fragments. We propose that simple PCR-based quality control experiments at the genetic and DNA methylation level, carried out at the beginning of any given project, can be used to enrich for the best-performing FFPE samples. The apparent preservation of genetic and DNA methylation patterns in archival FFPE samples may bring along new perspectives for the identification of genetic and epigenetic changes associated with brain-manifested diseases.

Postmortem structural studies of the thalamus in schizophrenia.

In this review, we seek to answer the following question: Do findings in the current literature support the idea that thalamo-cortical dysfunction in schizophrenia is due to structural abnormalities in the thalamus? We base our review on the existing literature of design-unbiased stereological studies of the postmortem thalamus from subjects with schizophrenia. Thus, all reported results are based upon the use of unbiased principles of sampling to determine volume and/or total cell numbers of thalamus or its constituent nuclei. We found 28 such papers covering 26 studies. In a series of tables we list all positive and negative findings from the total thalamus, the mediodorsal, pulvinar and anterior nuclei, as well as less frequently studied thalamic regions. Only four studies examined the entire thalamus and the results were inconsistent. We found largely consistent evidence for structural changes (reduced volume and cell numbers) in the pulvinar located in the posterior thalamus. In contrast, findings in the mediodorsal thalamic nucleus are inconsistent, with the largest and most recent studies generally failing to support earlier reports of a lower number of neurons in schizophrenia. Thus, the current findings of stereological studies of the thalamus in schizophrenia support the idea that thalamo-cortical dysfunction in schizophrenia might be attributable, at least in part, to structural alterations in the pulvinar that could impair thalamic inputs to higher order cortical association areas in the frontal and parietal lobes. However, more studies are needed before robust conclusions can be drawn.

Loss of Microtubule-Associated Protein 2 Immunoreactivity Linked to Dendritic Spine Loss in Schizophrenia.

BACKGROUND: Microtubule-associated protein 2 (MAP2) is a neuronal protein that plays a role in maintaining dendritic structure through its interaction with microtubules. In schizophrenia (Sz), numerous studies have revealed that the typically robust immunoreactivity (IR) of MAP2 is significantly reduced across several cortical regions. The relationship between MAP2-IR reduction and lower dendritic spine density, which is frequently reported in Sz, has not been explored in previous studies, and MAP2-IR loss has not been investigated in the primary auditory cortex (Brodmann area 41), a site of conserved pathology in Sz. METHODS: Using quantitative spinning disk confocal microscopy in two cohorts of subjects with Sz and matched control subjects (Sz subjects, n = 20; control subjects, n = 20), we measured MAP2-IR and dendritic spine density and spine number in deep layer 3 of BA41. RESULTS: Subjects with Sz exhibited a significant reduction in MAP2-IR. The reductions in MAP2-IR were not associated with neuron loss, loss of MAP2 protein, clinical confounders, or technical factors. Dendritic spine density and number also were reduced in Sz and correlated with MAP2-IR. In 12 (60%) subjects with Sz, MAP2-IR values were lower than the lowest values in control subjects; only in this group were spine density and number significantly reduced. CONCLUSIONS: These findings demonstrate that MAP2-IR loss is closely linked to dendritic spine pathology in Sz. Because MAP2 shares substantial sequence, regulatory, and functional homology with MAP tau, the wealth of knowledge regarding tau biology and the rapidly expanding field of tau therapeutics provide resources for identifying how MAP2 is altered in Sz and possible leads to novel therapeutics.

Intracortical excitatory and thalamocortical boutons are intact in primary auditory cortex in schizophrenia.

Schizophrenia is associated with auditory processing impairments that could arise as a result of primary auditory cortex excitatory circuit pathology. We have previously reported a deficit in dendritic spine density in deep layer 3 of primary auditory cortex in subjects with schizophrenia. As boutons and spines can be structurally and functionally co-regulated, we asked whether the densities of intracortical excitatory or thalamocortical presynaptic boutons are also reduced. We studied 2 cohorts of subjects with schizophrenia and matched controls, comprising 27 subject pairs, and assessed the density, number, and within-bouton vesicular glutamate transporter (VGluT) protein level of intracortical excitatory (VGluT1-immunoreactive) and thalamocortical (VGluT2-immunoreactive) boutons in deep layer 3 of primary auditory cortex using quantitative confocal microscopy and stereologic sampling methods. We found that VGluT1- and VGluT2-immunoreactive puncta densities and numbers were not altered in deep layer 3 of primary auditory cortex of subjects with schizophrenia. Our results indicate that reduced dendritic spine density in primary auditory cortex of subjects with schizophrenia is not matched by a corresponding reduction in excitatory bouton density. This suggests excitatory boutons in primary auditory cortex in schizophrenia may synapse with structures other than spines, such as dendritic shafts, with greater frequency. The discrepancy between dendritic spine reduction and excitatory bouton preservation may contribute to functional impairments of the primary auditory cortex in subjects with schizophrenia.

Reduced glutamate decarboxylase 65 protein within primary auditory cortex inhibitory boutons in schizophrenia.

BACKGROUND: Schizophrenia is associated with perceptual and physiological auditory processing impairments that may result from primary auditory cortex excitatory and inhibitory circuit pathology. High-frequency oscillations are important for auditory function and are often reported to be disrupted in schizophrenia. These oscillations may, in part, depend on upregulation of gamma-aminobutyric acid synthesis by glutamate decarboxylase 65 (GAD65) in response to high interneuron firing rates. It is not known whether levels of GAD65 protein or GAD65-expressing boutons are altered in schizophrenia. METHODS: We studied two cohorts of subjects with schizophrenia and matched control subjects, comprising 27 pairs of subjects. Relative fluorescence intensity, density, volume, and number of GAD65-immunoreactive boutons in primary auditory cortex were measured using quantitative confocal microscopy and stereologic sampling methods. Bouton fluorescence intensities were used to compare the relative expression of GAD65 protein within boutons between diagnostic groups. Additionally, we assessed the correlation between previously measured dendritic spine densities and GAD65-immunoreactive bouton fluorescence intensities. RESULTS: GAD65-immunoreactive bouton fluorescence intensity was reduced by 40% in subjects with schizophrenia and was correlated with previously measured reduced spine density. The reduction was greater in subjects who were not living independently at time of death. In contrast, GAD65-immunoreactive bouton density and number were not altered in deep layer 3 of primary auditory cortex of subjects with schizophrenia. CONCLUSIONS: Decreased expression of GAD65 protein within inhibitory boutons could contribute to auditory impairments in schizophrenia. The correlated reductions in dendritic spines and GAD65 protein suggest a relationship between inhibitory and excitatory synapse pathology in primary auditory cortex.

Stereological approaches to identifying neuropathology in psychosis.

The challenges involved in identifying the neuropathological substrates of the clinical syndrome recognized as schizophrenia are well known. Stereological sampling provides a means to obtain accurate and precise quantitative estimates of components of neural circuits and thus offers promise of an enhanced capacity to detect subtle alterations in brain structure associated with schizophrenia. In this review, we 1) consider the importance and rationale for robust quantitative measures of brain abnormalities in postmortem studies of schizophrenia; 2) provide a brief overview of stereological methods for obtaining such measures; 3) discuss the methodological details that should be reported to document the robustness of a stereological study; 4) given the constraints of postmortem human studies, suggest how to approach the limitations of less robust designs; and 5) present an overview of methodologically sound stereological estimates from postmortem studies of schizophrenia.

Design-based stereology: introduction to basic concepts and practical approaches for estimation of cell number.

In regulatory toxicology studies, qualitative histopathological evaluation is the reference standard for assessment of test article-related morphological changes. In certain cases, quantitative analysis may be required to detect more subtle morphological changes, such as small changes in cell number. When the detection of subtle test article-related morphological changes is critical to the decision-making process, sensitive quantitative methods are needed. Design-based stereology provides the tools for obtaining accurate, precise quantitative structural data from tissue sections. These tools have the sensitivity necessary to detect small changes by combining statistical sampling principles with geometric analysis of the tissue microstructure. It differs from other morphometric methods based on tissue section analysis by providing estimates that are statistically valid, truly three-dimensional, and referent to the entire organ. Further, because the precision of the stereological analysis procedure can be predicted, studies can be designed and powered to detect subtle, potentially toxicologically significant changes. Although stereological methods have not been widely applied in toxicologic pathology, recent advances have made it feasible to implement these methods in a regulatory toxicology setting, particularly methods for estimation of total cell number.

Pyramidal neuron number in layer 3 of primary auditory cortex of subjects with schizophrenia.

Individuals with schizophrenia demonstrate impairments of sensory processing within primary auditory cortex. We have previously identified lower densities of dendritic spines and axon boutons, and smaller mean pyramidal neuron somal volume, in layer 3 of the primary auditory cortex in subjects with schizophrenia, all of which might reflect fewer layer 3 pyramidal neurons in schizophrenia. To examine this hypothesis, we developed a robust stereological method based upon unbiased principles for estimation of total volume and pyramidal neuron numbers for each layer of a cortical area. Our method generates both a systematic, uniformly random set of mapping sections as well as a set of randomly rotated sections cut orthogonal to the pial surface, within the region of interest. We applied our approach in twelve subjects with schizophrenia, each matched to a normal comparison subject. Primary auditory cortex volume was assessed using Cavalieri's method. The relative and absolute volume of each cortical layer and, within layer 3, the number and density of pyramidal neurons were estimated using our novel approach. Subject groups did not differ in regional volume, layer volumes, or pyramidal neuron number, although pyramidal neuron density was significantly greater in subjects with schizophrenia. These findings suggest that previously observed lower densities of dendritic spines and axon boutons reflect fewer numbers per neuron, and contribute to greater neuronal density via a reduced neuropil. Our approach represents a powerful new method for stereologic estimation of features of interest within individual layers of cerebral cortex, with applications beyond the current study.

Volume and neuron number of the lateral geniculate nucleus in schizophrenia and mood disorders.

Subjects with schizophrenia show deficits in visual perception that suggest changes predominantly in the magnocellular pathway and/or the dorsal visual stream important for visiospatial perception. We previously found a substantial 25% reduction in neuron number of the primary visual cortex (Brodmann's area 17, BA17) in postmortem tissue from subjects with schizophrenia. Also, many studies have found reduced volume and neuron number of the pulvinar--the large thalamic association nucleus involved in higher-order visual processing. Here, we investigate if the lateral geniculate nucleus (LGN), the visual relay nucleus of the thalamus, has structural changes in schizophrenia. We used stereological methods based on unbiased principles of sampling (Cavalieri's principle and the optical fractionator) to estimate the total volume and neuron number of the magno- and parovocellular parts of the left LGN in postmortem brains from nine subjects with schizophrenia, seven matched normal comparison subjects and 13 subjects with mood disorders. No significant schizophrenia-related structural differences in volume or neuron number of the left LGN or its major subregions were found, but we did observe a significantly increased total volume of the LGN, and of the parvocellular lamina and interlaminar regions, in the mood group. These findings do not support the hypothesis that subjects with schizophrenia have structural changes in the LGN. Therefore, our previous observation of a schizophrenia-related reduction of the primary visual cortex is probably not secondary to a reduction in the LGN.

Effect of chronic antipsychotic exposure on astrocyte and oligodendrocyte numbers in macaque monkeys.

BACKGROUND: Both in vivo and postmortem studies suggest that oligodendrocyte and myelination alterations are present in individuals with schizophrenia. However, it is unclear whether prolonged treatment with antipsychotic medications contributes to these disturbances. We recently reported that chronic exposure of macaque monkeys to haloperidol or olanzapine was associated with a 10%-18% lower glial cell number in the parietal grey matter. Consequently, in this study we sought to determine whether the lower glial cell number was due to fewer oligodendrocytes as opposed to lower numbers of astrocytes. METHODS: With fluorescent immunocytochemical techniques, we optimized the visualization of each cell type throughout the entire thickness of tissue sections, while minimizing final tissue shrinkage. As a result, we were able to obtain robust stereological estimates of total oligodendrocyte and astrocyte numbers in the parietal grey matter with the optical fractionator method. RESULTS: We found a significant 20.5% lower astrocyte number with a non-significant 12.9% lower oligodendrocyte number in the antipsychotic-exposed monkeys. Similar effects were seen in both the haloperidol and olanzapine groups. CONCLUSIONS: These findings suggest that studies investigating glial cell alterations in schizophrenia must take into account the effect of antipsychotic treatment.

Primary visual cortex volume and total neuron number are reduced in schizophrenia.

A number of studies that assessed the visual system in subjects with schizophrenia found impairments in early visual processing. Furthermore, functional imaging studies suggested changes in primary visual cortex activity in subjects with schizophrenia. Interestingly, postmortem studies of subjects with schizophrenia reported an increased density of neurons in the primary visual cortex (Brodmann's area 17, BA17). The observed changes in visual processing may thus be reflected in structural changes in the circuitry of BA17. To characterize the structural changes further we used stereological methods based on unbiased principles of sampling (Cavalieri's principle and the optical fractionator) to estimate the total volume and neuron number of BA17 in postmortem brains from 10 subjects with schizophrenia and 10 matched normal comparison subjects. In addition, we assessed cortical thickness. We found a marked and significant reduction in total neuron number (25%) and volume (22%) of BA17 in the schizophrenia group relative to the normal comparison subjects. In contrast, we found no changes in neuronal density or cortical thickness between the two groups. Subjects with schizophrenia therefore have a smaller cortical area allocated to primary visual perception. This finding suggests the existence of a schizophrenia-related change in cortical parcellation.

Effect of chronic exposure to antipsychotic medication on cell numbers in the parietal cortex of macaque monkeys.

Both in vivo and post-mortem investigations have demonstrated smaller volumes of the whole brain and of certain brain regions in individuals with schizophrenia. It is unclear to what degree such smaller volumes are due to the illness or to the effects of antipsychotic medication treatment. Indeed, we recently reported that chronic exposure of macaque monkeys to haloperidol or olanzapine, at doses producing plasma levels in the therapeutic range in schizophrenia subjects, was associated with significantly smaller total brain weight and volume, including an 11.8-15.2% smaller gray matter volume in the left parietal lobe. Consequently, in this study we sought to determine whether these smaller volumes were associated with lower numbers of the gray matter's constituent cellular elements. The use of point counting and Cavalieri's principle on Nissl-stained sections confirmed a 14.6% smaller gray matter volume in the left parietal lobe from antipsychotic-exposed monkeys. Use of the optical fractionator method to estimate the number of each cell type in the gray matter revealed a significant 14.2% lower glial cell number with a concomitant 10.2% higher neuron density. The numbers of neurons and endothelial cells did not differ between groups. Together, the findings of smaller gray matter volume, lower glial cell number, and higher neuron density without a difference in total neuron number in antipsychotic-exposed monkeys parallel the results of post-mortem schizophrenia studies, and raise the possibility that such observations in schizophrenia subjects might be due, at least in part, to antipsychotic medication effects.

Mapping auditory core, lateral belt, and parabelt cortices in the human superior temporal gyrus.

The goal of the present study was to determine whether the architectonic criteria used to identify the core, lateral belt, and parabelt auditory cortices in macaque monkeys (Macaca fascicularis) could be used to identify homologous regions in humans (Homo sapiens). Current evidence indicates that auditory cortex in humans, as in monkeys, is located on the superior temporal gyrus (STG), and is functionally and structurally altered in illnesses such as schizophrenia and Alzheimer's disease. In this study, we used serial sets of adjacent sections processed for Nissl substance, acetylcholinesterase, and parvalbumin to identify the distinguishing cyto- and chemoarchitectonic features of the core, lateral belt, and parabelt in monkey. These criteria were evaluated in postmortem tissue from a human subject, leading to the identification of additional criteria specific to human. The criteria were validated in an additional set of eight human subjects. Regions were delineated and their volumes estimated using the Cavalieri method in these subjects, and the sources of methodologic contribution to variability of the estimates was assessed. Serial reconstructions of the auditory cortex in humans were made showing the location of the lateral belt and parabelt with respect to gross anatomical landmarks. Architectonic criteria for the core, lateral belt, and parabelt were readily adapted from monkey to human. Additionally, we found evidence for an architectonic subdivision within the parabelt, present in both species. Variability of regional volume estimates was readily constrained using a multifaceted approach to reduce potential sources of variability in regional delineation.

The influence of chronic exposure to antipsychotic medications on brain size before and after tissue fixation: a comparison of haloperidol and olanzapine in macaque monkeys.

It is unclear to what degree antipsychotic therapy confounds longitudinal imaging studies and post-mortem studies of subjects with schizophrenia. To investigate this problem, we developed a non-human primate model of chronic antipsychotic exposure. Three groups of six macaque monkeys each were exposed to oral haloperidol, olanzapine or sham for a 17-27 month period. The resulting plasma drug levels were comparable to those seen in subjects with schizophrenia treated with these medications. After the exposure, we observed an 8-11% reduction in mean fresh brain weights as well as left cerebrum fresh weights and volumes in both drug-treated groups compared to sham animals. The differences were observed across all major brain regions (frontal, parietal, temporal, occipital, and cerebellum), but appeared most robust in the frontal and parietal regions. Stereological analysis of the parietal region using Cavalieri's principle revealed similar volume reductions in both gray and white matter. In addition, we assessed the subsequent tissue shrinkage due to standard histological processing and found no evidence of differential shrinkage due to drug exposure. However, we observed a pronounced general shrinkage effect of approximately 20% and a highly significant variation in shrinkage across brain regions. In conclusion, chronic exposure of non-human primates to antipsychotics was associated with reduced brain volume. Antipsychotic medication may confound post-mortem studies and longitudinal imaging studies of subjects with schizophrenia that depend upon volumetric measures.

Stereological analysis of the mediodorsal thalamic nucleus in schizophrenia: volume, neuron number, and cell types.

The mediodorsal thalamic nucleus (MD) is the principal relay nucleus for the prefrontal cortex, a brain region thought to be dysfunctional in schizophrenia. Several, but not all, postmortem studies of the MD in schizophrenia have reported decreased volume and total neuronal number. However, it is not clear whether the findings are specific for schizophrenia nor is it known which subtypes of thalamic neurons are affected. We studied the left MD in 11 subjects with schizophrenia, 9 control subjects, and 12 subjects with mood disorders. Based on morphological criteria, we divided the neurons into two subclasses, presumably corresponding to projection neurons and local circuit neurons. We estimated MD volume and the neuron number of each subclass using methods based on modern unbiased stereological principles. We also estimated the somal volumes of each subclass using a robust, but biased, approach. In addition, we investigated the left MD in four cynomolgus monkeys chronically exposed to haloperidol and in four control monkeys in order to assess the possible effects of antipsychotic medications. The three human subject groups did not differ in any of the measures. In addition, no differences were observed between the two groups of monkeys. Thus, these findings do not support the hypothesis that the MD is a locus of pathology in schizophrenia, although they cannot rule out important functional or structural changes in parameters not measured. Like other studies, this investigation is subject to the limitations involved in sampling from a heterogeneous population emphasizing the need to continue to improve the application of robust, unbiased techniques to quantitative studies of this complex brain disorder.