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

Journal Pre-proofs Research report Hippocampal volume and cell number in depression, schizophrenia, and suicide subjects Fenghua Chen, Aksel B. Bertelsen, Ida E. Holm, Jens R. Nyengaard, Raben Rosenberg, Karl-Anton Dorph-Petersen PII: DOI: Reference:

S0006-8993(19)30600-6 https://doi.org/10.1016/j.brainres.2019.146546 BRES 146546

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Brain Research

Received Date: Revised Date: Accepted Date:

7 June 2019 29 September 2019 7 November 2019

Please cite this article as: F. Chen, A.B. Bertelsen, I.E. Holm, J.R. Nyengaard, R. Rosenberg, K-A. Dorph-Petersen, Hippocampal volume and cell number in depression, schizophrenia, and suicide subjects, Brain Research (2019), doi: https://doi.org/10.1016/j.brainres.2019.146546

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Hippocampal volume and cell number in depression, schizophrenia, and suicide subjects Fenghua Chen1,2, Aksel B. Bertelsen1, Ida E. Holm3,4, Jens R. Nyengaard2,5, Raben Rosenberg1, Karl-Anton Dorph-Petersen1,4,5,6

Translational Neuropsychiatry Unit1, Core Center for Molecular Morphology, Section for Stereology and Microscopy2, Department of Clinical Medicine3, Aarhus University, Aarhus, Denmark Department of Pathology, Randers Regional Hospital, Randers, Denmark4 Centre for Stochastic Geometry and Advanced Bioimaging5, Aarhus University, Aarhus, Denmark Translational Neuroscience Program6, Department of Psychiatry, University of Pittsburgh, Pittsburgh, PA, USA

Running title: Hippocampal volume and cell number Key words: Cell Number, Post Mortem, Schizophrenia, Depression, Suicide, Hippocampus The word count of the abstract: 245 The word count of the body of the manuscript: 5531 The number of references: 91 The number of figures: 7 The number of tables: 5

Address of correspondence: Fenghua Chen, Department of Clinical Medicine - Translational Neuropsychiatry Unit, Department of Clinical Medicine, Nørrebrogade 44, Building 2B, 8000 Aarhus C, Denmark [email protected]

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Abstract 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 seems to differ in hippocampal neurobiology compared to hospitalized subjects dying with MDD without suicide.

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1. Introduction The hippocampus has for a long time been associated with the pathophysiology of psychiatric disorders, especially major depression disorder (MDD) (Manji et al., 2001) and schizophrenia (Heckers et al., 1991; Weinberger, 1999). Substantial evidence suggests that depression and schizophrenia may be related to impairments of structural plasticity and neural cellular resilience in hippocampus (Lucassen et al., 2010). An abundance of hippocampal morphometric imaging studies showed that depressed subjects have reduced hippocampal volume in individuals with multiple depressive episodes and/or longer duration of the illness, but no differences between first episode patients and controls (Kempton et al., 2011; McKinnon et al., 2009; Schmaal et al., 2016; Sheline et al., 1996; Sheline et al., 2003; Videbech et al., 2001; Videbech et al., 2002; Videbech and Ravnkilde, 2004). Furthermore, the hippocampal morphological changes may be also modulated by disease severity, childhood maltreatment, age of onset and antidepressant medication (Frodl and O'Keane, 2013; McKinnon et al., 2009). Also, neuroimaging techniques, including structural MRI, magnetic resonance spectroscopy (MRS) and positron emission tomography (PET), have indicated evidence for abnormal hippocampal structure and function in schizophrenia (Heckers, 2001). The recent largest meta-analysis of brain MRI scans from individuals with schizophrenia found significantly smaller hippocampus compared with healthy controls (van Erp et al., 2016a; van Erp et al., 2016b). They also found that hippocampal volume deficits were more severe in samples with a higher proportion of unmedicated subjects. However, a limited number of studies have examined the structure and morphology of the postmortem human hippocampus in depression and schizophrenia (Arnold, 1997; Arnold, 2000; Benes et al., 1998; Bogerts et al., 1990; Bogerts et al., 1993; Bogerts, 1997; Falkai et al., 2016; Schmitt and Falkai, 2013). Moreover, design-based stereological postmortem studies of brain structure associated with MDD and schizophrenia offer an enhanced capacity to detect subtle

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alterations and provide accurate and precise quantitative data (Dorph-Petersen and Lewis, 2011). Only a few studies of schizophrenia have investigated total cell numbers using design-based stereological methods. Three of these studies reported no differences in neuronal numbers in all cornu ammonis subdivisions of the hippocampus between subjects with schizophrenia and controls (Heckers et al., 1991; Schmitt et al., 2009; Walker et al., 2002). A fourth and recent stereological postmortem study in schizophrenia found no differences in neuron and oligodendrocyte number in hippocampal subregions except DG (Falkai et al., 2016). However, until now, only two studies completed a stereological estimation of total numbers of neurons and glial cells in the hippocampus in MDD (Cobb et al., 2013; Malchow et al., 2015). Additionally, post-mortem studies in depression included individuals who have died by suicide, so factors specific to suicidality, rather than depression in general, can influence results (Cannon et al., 2006; Spies et al., 2015). Although suicide as a cause of death was originally viewed as an extreme outcome of MDD and then later as a potential confound in studies of the neurobiology of mood disorder, suicide is increasingly thought to have its own unique neurobiology (Oquendo et al., 2008; Oquendo and Mann, 2008). However, it is still not clear whether there may be different mechanisms that lead to distinct structural and morphological changes in hippocampus between depression with suicide and depression without suicide. The aim of the present study is to investigate if depression, schizophrenia or suicide is associated with reduced volume of the hippocampal formation and/or changes in the number of neurons and/or glial cells in the different subregions of the hippocampus. In addition, to identify if the three groups have similar or different changes in the volume and cell number of the hippocampus. To address this purpose, we used methods based upon unbiased stereological techniques to analyze postmortem brain samples.

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2. Results The results of the present study are summarized in Figs. 1-4, and Tables 1 and 2. The mean total volume of hippocampus was significantly different between the four groups (F3,35 = 15.5, p < 0.0001) (Fig. 1). The mean hippocampal volume was significantly smaller in depression (p < 0.0001; 27.4%) and schizophrenia (p < 0.0001; 23.5%) compared to control subjects, with no significant difference for the suicide group. The volumes of the subregions in the hippocampus showed similar pattern of results as the total volume of hippocampus. As shown in Fig. 2 and Table 1, the total number of neurons (N) and glial cells (G) were significantly different in all four subregions of hippocampus (the cell layer of DG, hilus, CA2/3 and CA1) with: N: F3,35 = 6.69, p = 0.0009 and G: F3,35 = 3.46, p = 0.027 in DG; N: F3,35 = 3.49, p = 0.026 and G: F3,35 = 6.02, p = 0.002 in hilus; N: F3,35 = 9.70, p < 0.0001 and G: F3,35 = 9.05, p = 0.0001 in CA2/3; N: F3,35 = 9.49, p = 0.0001 and G: F3,35 = 5.12, p = 0.0048 in CA1. The reported p-values are the “raw” results of the ANOVA analysis not taking into account the multiple tests and thus form our primary statistical model. Alternatively, if taking the multiple comparisons across two cell types (neurons and glia) and four regions (DG, hilus, CA2/3, and C1) into account, a simple but rather conservative approach would be to apply the Bonferroni correction, which would lead to a required significance level of p = 0.05/8 = 0.00625. While the Bonferroni correction is considered overly conservative in cases of positive correlated data (as in the current study, where the same pattern is observed across all tested groups) this second model still find significant changes in most of the tests only losing the significance for glial cells in DG and for neurons in Hilus. However, as stated, we consider this second model overtly conservative. Thus, in the rest of the manuscript we base our discussion on our primary model. The Bonferroni post hock tests for the four groups in each ANOVA is reported in Table 1.

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As seen in Figs. 2 and 3 and table 1, the general pattern is that control and suicide groups showed similar values and likewise the depression and schizophrenia groups showed similar values. Thus, the statistical differences detected by the ANOVA were driven by lower values for the two latter groups (depression & schizophrenia) compared to the two former groups (control & suicide). However, the post hoc test was not able to detect significant differences between individual groups for glial cells in DG and neurons in hilus despite the overall significant difference between groups according to our primary statistical model (as these two comparisons lose significance in our secondary statistical model). The only exception to the general pattern is the 22% higher neuron number in CA2/3 in suicide subjects relative to controls. As indicated in Fig. 3 and Table 2, we did not detect any significant differences in the numerical density of neurons or glial cells. Volume and cell number did not correlate noticeably with age, storage time, and PMI (Fig. 4, PMI data not shown).

3. Discussion The present study found the volume and the number of neurons and glial cells were similarly reduced by approximately 20% to 35% in depression and schizophrenia subjects relative to control subjects across all hippocampal regions. In suicide subjects, the volume and the number of neurons and glial cells did not significantly differ compared to control subjects, except increased neuron number in the CA2/3 subregion.

3.1.

Limitations of the current study

Like most postmortem studies, the current study is limited by the relative small number of subjects. Furthermore, while we strived to obtain balanced study groups regarding sex and laterality, it was in the present material not possible to reach a perfect, equal distribution of male/female and right/left hippocampi.

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Interestingly, most reviews of sex differences in the human brain state that the hippocampus is larger in females (Andreano and Cahill, 2009; Cahill, 2006; Gur et al., 2010; Hines, 2010; Knickmeyer et al., 2014; Lentini et al., 2013). However, most references to a larger hippocampal volume in female cite the same few early MRI studies (Filipek et al., 1994; Giedd et al., 1996; Goldstein et al., 2001). In addition, some studies suggest that the hippocampus is larger in females, but only during childhood (Cosgrove et al., 2007) or adolescence (Neufang et al., 2009). A new meta-analysis study of male-female difference in hippocampal MRI volume found that human males of all ages exhibit a larger hippocampal volume than females, but adjusting for individual differences in total brain volume or intracranial volume results in no reliable sex difference. Thus, the human hippocampal volume is probably not sexually-dimorphic (Tan et al., 2016), at least regarding size. Anyhow, while probably minor, it is unknown to what degree—if at all—the unequal distribution of females and males in the present study influences our results. Asymmetry of the hippocampal volume has been reported previously in smaller cohorts of younger and older adults (Hasan and Pedraza, 2009; Wellington et al., 2013; Zhang et al., 2010), as well as in patients with Alzheimer´s disease (AD) and Mild Cognitive Impairment (MCI) (Sarica et al., 2018; Shi et al., 2009). A big new MRI study in generally healthy participants also showed that the right hippocampus was slightly, but significantly, larger than the left hippocampus for both men and women (Nobis et al., 2019). In our study, we found no significant differences between right and left hippocampal volumes. While our left/right ratio were similar across the four study groups, we included more right than left hippocampi in our study. Because of potential lateralization of the disease processes, this may influence our results to an unknown degree. However, the effect should be similar across the four groups and thus not bias our conclusions. The most important limitation of the current study is that the brain specimens came from two different brain banks. The tissue in the control and suicide groups came from The Research Tissue

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Bank (Forskningsvævsbanken) at Core Center for Molecular Morphology, Section for Stereology and Microscopy, Department of Clinical Medicine, Aarhus University (Dorph-Petersen, 2001). The depression and schizophrenia groups consist of tissue from the large Brain Collection until recently located at Translational Neuropsychiatry Unit, Aarhus University (Dorph-Petersen and Rosenberg, 2006). From a technical point of view, processing differences (including storage time and PMI) between the two tissue collections is unlikely to create a different number of observable cells in the thionin stain for Nissl substance. All cells appeared stained and we counted both neurons and glial cells, endothelial cells forming only a miniscule uncounted fraction of the total. Thus, our cell number estimates should be robust to this limitation. On the other hand, potential differences in initial tissue processing could cause the observed differences in hippocampal volumes. However, as the observed mean cell densities were virtually identical between groups in most regions (Fig 3), the volume changes closely matched the robust cell number changes. Therefore, the observed volume changes are most likely of biological origin. The subjects from the control and suicide groups lived in society and died in the period 1997-2001, while the subjects from the depression and schizophrenia groups died at a mental state hospital in the period 1945-1982. Thus, we cannot exclude unknown effects on the depression and schizophrenia groups of being born approximately 50 years prior to the control group as well as being hospitalized at a state mental hospital. However, we did not find any significant correlation within groups between birth year and the observed variables, and living at a state mental hospital supposedly were beneficial for the individuals many of which lived to old age. Differences in mean subject age between the two collections (Table 3) did not appear to influence our findings as no systematic within-group correlations were observed (Fig. 4).

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We cannot perform correlation analysis between symptom severity/duration of episode/medication and hippocampal volume/cell number due to incomplete information of exact number, duration and treatment of episodes in some subjects and lack of such information in suicide subjects. However, comparison between subjects with or without catatonic features in both groups did not reveal any significant differences. As most of the subjects except in the control group received treatment, we cannot rule out effects of such treatments on the observed variables. Exposure of non-human primates to antipsychotics has been associated with reduced brain volume and glial cell number (Dorph-Petersen et al., 2005; Konopaske et al., 2007; Konopaske et al., 2008), the volume reductions supported by subsequent rodent studies (Barr et al., 2013; Vernon et al., 2011; Vernon et al., 2012; Vernon et al., 2014) and seen in a large human imaging study (Ho et al., 2011). However, those studies did not report changes in neuron numbers. Rodent studies have reported that antidepressant treatment may block hippocampal atrophy by enhancing neurogenesis and synaptic plasticity (Chen et al., 2009; Chen et al., 2010), The large ENIGMA structural neuroimaging study showed no significant lower hippocampal volume after taking antidepressants (Schmaal et al., 2016). Increasing DG granule cell and glial cell number with age in antidepressant-treated subjects may reflect proliferative effects of antidepressant medication (Cobb et al., 2013). Moreover, SSRI-treated MDDs, but not tricyclictreated MDDs, had more granule neurons in DG (Boldrini et al., 2013). In our study, all depression and schizophrenia subjects were dead before SSRIs drugs were developed. Therefore, the influence of SSRI medication on the observed differences for depression and schizophrenia subjects (but not for the suicide subjects) can be excluded in the present study. On the other hand, insulin coma therapy (ICT) was extensively used in the 1940s and 1950s in Denmark, and the most severe risk of ICT was brain damage (Revitch, 1954). However, only three subjects in the schizophrenia group had ICT (Table 4).

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A strength of the current study is the clean subject groups with no drug or alcohol abuse and no suicide in the depression and schizophrenia groups. The two latter groups hospitalized at a state mental hospital at time of death likely represent more severe manifestations of the diseases compared to subjects living in society. Interestingly, reviewing postmortem studies of thalamus in schizophrenia, studies of hospital-acquired samples are significantly more likely to find structural brain changes compared to studies of community-based samples (Dorph-Petersen and Lewis, 2017) possibly reflecting the higher fidelity of studying “cleaner” disease groups.

3.2.

Comparison to previous studies

In line with our results, a reduced volume of hippocampus is found in neuroimaging studies of subjects with depression (Bremner et al., 2000; Sheline et al., 1996; Sheline et al., 1999) and schizophrenia (Heckers, 2001). However, they reported that the reduced hippocampal volume in MDD only appeared in individuals with multiple depressive episodes and/or longer duration of the illness. Moreover, the total hippocampal volume decreased with duration of depressive illness in one postmortem study (Cobb et al., 2013). In our present study, half of the depressed subjects had single depressive episodes and of shorter duration. However, there was no difference between the two halves in the dependent measures. Thus, our findings indicate that the reduced hippocampal volume also may appear in subjects with a single depressive episode. Our results are consistent with other postmortem studies that the volume of hippocampus is reduced in schizophrenia (Bogerts et al., 1990; Bogerts et al., 1993; Bogerts, 1997; Schmitt and Falkai, 2013). Previous postmortem morphometric studies have observed moderate apoptosis and atrophy of neurons in the dentate gyrus and the CA1 and CA4 regions of the hippocampus of subjects with major depression (Campbell and MacQueen, 2004; Fuchs et al., 2004; Lucassen et al., 2006). Furthermore, a recent stereological postmortem study in schizophrenia found a decreased neuron number in DG (Falkai et al., 2016). Our results show that neuronal number in DG and CA1 is significantly reduced in

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depression and schizophrenia, and this also holds true for hilus and CA2/3 subregions. The reduced neuronal number in DG may reflect decreased hippocampal neurogenesis in both MDD and schizophrenia. However, a newer post-mortem study (Sorrells et al., 2018) suggest that recruitment of young neurons to the primate hippocampus decreases rapidly during the first years of life, and that neurogenesis in the DG does not continue in adult humans. Their findings do not support the notion that adult neurogenesis continue in the human hippocampal DG. In the same time, another newer study of human autopsy hippocampi from healthy human individuals ranging from 14 to 79 years of age showed that human neurogenesis in DG persists throughout aging (Boldrini et al., 2018). While the Sorrells et al study included surgically resected tissue from 22 patients with epilepsy, the Boldrni et al study only used tissue from healthy subjects. This difference may contribute to the conflicting findings. Thus, controversy over the presence or absence of adult neurogenesis is likely to continue for the foreseeable future. We found glial cells number in hilus, CA2/3 and CA1 significantly reduced in depression, and a lower number in the DG subregion. In the schizophrenia group, the glial cells number was significantly decreased in hilus and CA2/3 subregions and showed no significant differences in DG and CA1 subregions. In agreement with our results, two schizophrenia studies found reduced oligodendrocyte numbers in CA4 (hilus) (Falkai et al., 2016; Schmitt et al., 2009). Most glial cells can easily be distinguished by visual inspection of their morphology based on a thionin staining (Nissl stain) according to their smaller size and lack of a nucleolus and stained cytoplasm. However, microglia are not discernable with a Nissl stain while oligodendroglia appear as small dark nuclei with dark chromatin, and it is difficult to reliably distinguish between astrocytes and microglia with a Nissl stain (Hamidi et al., 2004). Thus, all glial cells were combined for counting

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in the present. In order to identify different subtypes of glial cells, special immunohistochemical staining markers will be used in the future studies. Our observations suggest that the decreased volume was at least partly due to a lower number of neurons and glial cells in the subregions. Possibly, the reduced glial cell number may be a primary event destroying the trophic support of their local neurons, and then causing a reduced neuron number as a secondary event. Therefore, our findings support the hypothesis that the hippocampal volume loss might be the result of remodelling of key cellular elements, involving neuronal loss/death (apoptosis) and retraction of dendrites, axonal, and synaptic components, decreased neurogenesis in the dentate gyrus, neuronal apoptosis and loss of glial cells (Czéh et al., 2001; Lucassen et al., 2001; Rajkowska, 2000). This finding is in line with our previous animal studies that the basal levels of the hippocampal volume and neuron number were significantly smaller in the FSL rats (“depressed” rats) compared to the FRL rats (control rats) (Chen et al., 2010; Kaae et al., 2012). Our observation is further supported in stress-induced experimental animals and animal models of depression (Fuchs et al., 2001; Paizanis et al., 2007). However, only few postmortem studies are in agreement with our present results, which showed fewer granular neuron number in DG of MDDs compared to controls (Boldrini et al., 2013), a larger DG volume and a higher number of dividing cells in pharmacologically treated subjects (Boldrini et al., 2009; Boldrini et al., 2012). Moreover, there was no significant difference between MDD and controls in total number or density of neurons or glial cells in hippocampal subregions in one postmortem study (Cobb et al., 2013). No changes of cell density in our present study (Fig. 3 and Table 2) is in line with the study by Cobb et al (Cobb et al., 2013). Conversely, the study by Stockmeier et al. (Stockmeier et al., 2004) found that the density of neurons and glial cells in the hippocampal subregions were significantly increased by 30-35% in

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MDD. One possible explanation for this discrepancy may be methodological differences as only our study was based upon design-unbiased stereological principles of sampling.

3.3.

Biological basis of reduced volume and cell number

The precise mechanisms underlying the volume loss and decreases of cell number in the hippocampus of depression are still poorly understood. Recent studies have tried to explain these changes by diminished neurotrophic factor signaling, particularly brain-derived neurotrophic factor (BDNF) (Castrén et al., 2007; Castrén and Rantamäki, 2010; D Kuipers and Bramham, 2006). Reduced nerve growth factor (NGF) and BDNF concentrations are related to medication-naïve early psychotic subjects and in medicated chronic schizophrenic subjects (Martinotti et al., 2012). Moreover, brain BDNF levels have been found to be reduced in postmortem samples from depressed subjects, and antidepressant therapy restores brain BDNF levels to the normal range (Otsuki et al., 2008). Other factors underlying this cellular remodeling, including decreasing monoamine neurotransmitters, stress and abnormalities in the hypothalamic-pituitary-adrenal (HPA) axis, biological abnormalities, may be due to activation of distinct signal transduction pathways, which biochemical and molecular changes potentially make the neuron morphologically unresponsive to stimuli, inhibit dendritic arborisation, decrease neurogenesis in the DG, loose glial cells, and finally reduce volume in the hippocampus (Cameron and McKay, 1999; Duman, 2002; Duman, 2004; Duman and Li, 2012; Frodl and O'Keane, 2013; Gerritsen et al., 2011; Sapolsky, 2000). However, it is not known whether these modifications really contribute to the development of depression and schizophrenia (Paizanis et al., 2007).

3.4.

Unique neurobiology in suicide

In suicide subjects with depressive symptoms, our results showed that the volume and the number of neurons and glial cells showed no differences in suicided subjects compared to control subjects,

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except increased 21.7% neuron number and 7.1% glial cells in CA2/3 subregion. Interestingly, a postmortem study showed no significant differences in neuron number or neuron density in MDD subjects compared to healthy controls, but the density of oligodendrocytes in MDD was higher in CA2/3 and hilus (Malchow et al., 2015). Their results are almost consistent with our findings in the suicide subjects with depressive symptoms, because seven of eight subjects in the MDD group died by suicide in their study (Malchow et al., 2015). Our results showed significant differences between suicide subjects with depressive symptoms compared with depression subjects without suicide. Suicide is thought to be an extreme act of subjects with a psychiatric disorder compounded by one or more adverse life events and a biological predisposition or vulnerability (Underwood and Arango, 2011). Recent biological studies have mainly found differences in neurotransmitter receptors, neurons, glial cells, and/or white matter in the prefrontal cerebral cortex (Rajkowska, 2000). Our findings support the hypothesis that the pathobiology behind suicide differs from depression in general.

3.5.

Pathomorphology of depression and schizophrenia

Neuroimaging and postmortem studies have found similarities and differences in the pathomorphology of depression and schizophrenia (Baumann and Bogerts, 1999; Brisch et al., 2008; Busatto, 2013). These differences show especially that schizophrenia mainly affects the heteromodal association cortex and mesiotemporal structures, and MDD primary affect the nucleus accumbens. Conversely, some overlaps in structural findings, such as hippocampus, may be consistent with shared psychotic symptoms in both diseases. It is clear that there is potential overlap in the pathophysiology and/or etiology of depression and schizophrenia and thus the hippocampus has become an important focus for studies of structural and functional pathology in both disorders. This hypothesis is supported by our present findings that a consistent feature of depression and schizophrenia is a small hippocampus.

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Postmortem studies in humans suggest that reduced hippocampal neural stem cell proliferation (NSP) plays a role in the pathophysiology of schizophrenia, but fail to confirm a contribution in depression (Reif et al., 2006; Reif et al., 2007). In particular, hippocampal volume loss is correlated with illness duration of MDD. Moreover, they show that atrophy of the hippocampus in major depression worsened with repeated episodes. Therefore, decreased hippocampal volume may be a consequence of MDD. Conversely, the hippocampal volume reduction has no correlation to disease duration in schizophrenia subjects (Bogerts et al., 1985; Bogerts et al., 1990; Bogerts et al., 1993; Marsh et al., 1994). These studies support a hypothesis that the structural changes in hippocampus may be acquired before the onset of the psychotic symptoms and then remain static. Nonetheless, these hypotheses need to be studied further in neuroimaging and postmortem studies in the future. In conclusion, we found that hippocampal volume and its number of neurons and glial cells are reduced in a similar way by 20% to 35% in depressed and schizophrenia subjects relative to control subjects across all regions. Thus, the smaller hippocampal volume in MDD and schizophrenia is likely due to the smaller number of hippocampal neurons and glial cells. As such, the present study supports the structural and neuro-plasticity hypothesis of depression and schizophrenia. Future postmortem studies should ideally be designed based upon larger samples to study the interaction between classification according to clinical characteristics, severity of depression and treatment. Also, it would be relevant in future studies to test the specificity of the current findings by stereological investigation of other brain regions such as the thalamus or the cerebral cortex in the same material.

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4. Experimental Procedure 4.1.

Human postmortem brains

The study was based on a total of 39 brain tissue samples from four groups of individuals groupmatched for sex and age: 1) ten control subjects with no history of psychiatric or neurological diseases; 2) eleven suicide subjects with a history of depressive disorder; 3) eight subjects with major depression; and 4) ten subjects with schizophrenia. The brain samples came from two Danish tissue archives—see discussion. Demographic and treatment details are listed in Tables 3 and 4. The subjects were Scandinavian Caucasians and none of the subjects had a history of drug or alcohol abuse or any neurological or psychiatric comorbidities. The MDD and schizophrenia subjects in the study were diagnostically reassessed according to DSM-IV criteria (Diagnostic and Statistical Manual of Mental Disorders, 4th Ed.) and ICD-10 (The International Statistical Classification of Diseases and Related Health Problems 10th Ed.) by trained psychiatrists (ABB, RR) based on patient records and autopsy reports. The suicide and control subjects in the study were diagnostically reassessed based on autopsy reports and police reports. The control subjects were screened in the Danish Psychiatric Central Research Register to verify control status. The information of insulin coma therapy (ICT) and electroconvulsive therapy (ECT) is listed in Table 4. In addition, a trained neuropathologist (IEH) examined all hippocampal sections confirming the absence of any signs of neurodegenerative diseases. All 11 suicide subjects had at least one depressive episode, and we excluded death by suicide for the subjects in the three non-suicide groups. We aimed to include a balanced and matched number of left and right hippocampi—one per brain—from each group, the side dictated by tissue availability in the depression and schizophrenia groups. The study was approved by The Central Denmark Region Committees on Biomedical Research Ethics and was conducted blind to diagnostic status.

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4.2.

Tissue preparation (Fig. 5)

All tissue processing were run in sets of four brains at a time—one brain from each group. In each set, the same hemisphere (right or left) was selected from all four brains. For the control and suicide groups the brainstem and cerebellum were detached from the brains, which were otherwise complete. For these brains, the two cerebral hemispheres were separated by a midsagittal cut through the corpus callosum, and the selected hemisphere submerged in melted 5% agarose and, using a cutting guide, cut systematic, uniformly random into 1-cm coronal slabs. Brains from the depression and schizophrenia groups were previously sliced, after fixation, by hand into slabs during the original neuropathological examination. From the slabs of each selected hemisphere, we removed the hippocampus with a rim of surrounding tissue and placed it in a coded jar to ensure blinding of the groups. To cryoprotect the tissue, the hippocampal blocks were then for ~2 weeks washed in a graded series of 12%, 16%, and 18% sucrose in PBS. Subsequently, the hippocampal blocks were reassembled in their original position using a 7% agarose solution. Each reassembled hippocampus was embedded in 7% agarose and, using a cutting guide, cut into systematic, uniformly random slabs (2.5 mm thick) oriented roughly perpendicular to the posterior-anterior longitudinal axis of the hippocampus. About 20-26 slabs were generated. One 80-µm-thick section was cut from each slab using a calibrated Microm cryo-star HM560 cryostat (Microm, Walldorf, Germany). The cryostat block advance (BA) was checked on a regular basis and varied less than 1% when set on 80 µm. The cut sections were store individually in 4% paraformaldehyde and kept at 4C until staining. The sections were mounted on gelatin-coated glass slides, air-dried at room temperature 1 hour, and then stained for Nissl substance using thionin. About 12-16 section hit the hippocampus. In order to estimate the shrinkage due to processing the volume of each block was measured by a high precision implementation of the Archimedean method before and after cryoprotection (Dorph-

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Petersen et al., 2005). The mean tissue shrinkage due to the cryoprotection was 0.07%. Furthermore, the average area shrinkage in the X-Y-direction of the cryostat sections was 0.37%, checked across all sections.

4.3.

Microscope setup

The histological sections were analyzed using an Olympus BX50 light microscope (Olympus, Denmark) modified for stereology with a digital camera (Olympus DP70, Denmark), a motorized microscope stage (Prior H138 with controller H29, Cambridge, United Kingdom), and a microcator (Heidenhain MT-12, Traunreut, Germany) to measure the z-axis movement. A computer with the newCAST software package (version: 4.4.5.0), Visiopharm, Hørsholm, Denmark) was interfaced to the digital camera superimposing stereological probes (points and counting frames) on the live images. The microscopy analysis was performed by one person (FC) blind to the diagnosis of individual cases. Within each section, the specific areas of interest were delineated as shown in Fig. 6 using a 4× objective (Olympus, UPlanApo, NA=0.16). 4.4.

Definition of anatomical areas (Fig. 6)

Within each section, the specific areas of interest were delineated as shown in Fig. 6 using a 4× objective (Olympus, UPlanApo, NA=0.16) at a final on-screen magnification of 156×. In this study, the hippocampal formation included the granule cell layer of the dentate gyrus (DG-C), the hilus of the dentate gyrus (DG), the subdivisions of the cornu ammonis (CA), and subiculum. The numbers of neurons and glial cells were estimated in the following anatomical divisions: 1) the granule cell layer of the DG, including also the subgranular zone; 2) the hilus of the DG; the pyramidal cell layers of 3) CA2/3 and 4) CA1, respectively (Fig. 6). The DG-C appeared as a C-shaped layer, with the soma of the neurons being small in size and very tightly packed. It is readily identified in all sections in which it is present because of the intensity

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with which it stains and the fact that it is not continuous with the cellular layers of the other subregions. Between the blades of the DG-C is the hilus, a polymorphic region mainly composed by loosely packed inhibitory mossy cells, mossy fibers from granule cells, and glial cells. In a Nisslstained section of the hippocampus, the hilus appear light, because of the low number of cell bodies. The midpoint of the inner border of the hilus abuts the medial end of the well-defined pyramidal layer of CA2/3. The boundary between the pyramidal cell layer of CA3 and hilus can be readily identified as the point at which the tightly package layer of CA3 pyramidal cells appears to bend back upon itself and to become more diffusely organized as the inner hilar cell layer. The pyramidal cell layer in CA3 is both wider and more diffusely organized than in CA2. Field CA2 of the hippocampus has the narrowest and most densely packed pyramidal cell layer of hippocampal fields. Field CA1 of the hippocampus has a wider pyramidal cell layer than fields CA3 or CA2 and cells tend to be smaller and more widely separated. The border of CA1 with CA2 is not sharp because some CA2 pyramidal cells appear to extend over the emerging CA1 pyramidal cell layer. The boundary of CA1 can be placed, however, shortly after the pyramidal cell layer starts to broaden. The border with the subiculum is equally difficult to place unless one is prepared to indicate a rather oblique border zone. The pyramidal cell layer of CA1 overlaps that of the subiculum for a considerable distance. A narrow cell-free zone often corresponds to the oblique border between CA1 and the subiculum. The pyramidal cell layer of the subiculum is often wider than of CA1. As the CA2 region is very small and only differs from the CA3 in the way that the pyramidal cells are not receiving any mossy fibers, the CA2 and CA3 in this study were combined in one region named the CA2/3 in concordance with earlier stereological studies of the human hippocampus (West and Gundersen, 1990). Pyramidal cell layers of CA2/3 are easily distinguished from the stratum radiatum (including the stratum lucidum of CA3) internally placed and the stratum oriens towards the outside (CA2/3-O).

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The stratum radiatum of CA2/3 and CA1, respectively, were defined by the location inside the right adjacent pyramidal cell layers. The inner border of the CA2/3 stratum radiatum (CA2/3-R) was defined by a decrease in cell density, whereas the inner border of CA1 stratum radiatum (CA1-R) was defined by the stratum lacunosum moleculare (CA1-LM), which has a higher density of cells and a distinct course of the fibers. The molecular layer of the dentate gyrus (DG-M) is towards the outside of the granule cell layer of the dentate gyrus.

4.5.

Estimating the volume using the Cavalieri estimator

The total volume of the hippocampus, as well as the individual volumes of all layers of the DG, CA subdivisions and subiculum (Fig. 6), were estimated using the Cavalieri estimator (Gundersen and Jensen, 1987) by point counting with the above-mentioned 4× objective. Thus, the volumes were estimated as the product of the intersection distance (T = 2.5 mm) and the area per point a multiplied by the sum of the number of points Pi hitting the respective hippocampal subregion in each section:

V : T  a   Pi Here and elsewhere:= indicates that the quantity at left is estimated by the formula at the right. Table 5 lists the respective area per point and number of counted points for each region.

4.6.

Estimating neuron and glial cell number using the optical fractionator

We used the shrinkage robust optical fractionator based upon the number-weighted mean section thickness (Dorph-Petersen et al., 2001) to estimate the number of neurons and glial cells in four hippocampal subregions: granule cell layer, hilus, CA1, and CA2/3 (Fig. 6). For neurons, we used the nucleolus as the counting unit—one distinct nucleolus was observed in each neuronal nucleus. For glial cells the nucleus was used as counting unit. Endothelial cells only formed a minor fraction of the total amount of cells and were not included in the study (Fig 7). Glial

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cells can be distinguished from neurons based on a thionin staining for Nissl substance according to their smaller size and lack of a nucleolus and stained cytoplasm. Most glial cells can easily be distinguished by visual inspection of their morphology. Oligodendrocytes display more compact, darker-stained nuclei eccentrically located in one end of a polar cell body. Astrocytes have larger and lighter-stained nuclei, and lack of stained cytoplasm. Microglia are characterized by irregular shape, dark stained nuclei and patches of condensed chromatin with little cytoplasmic staining (Fitting et al., 2008; Fitting et al., 2010). However, it is difficult to reliably distinguish between astrocytes and microglia in sections stained for Nissl substance (Hamidi et al., 2004). Therefore, we combined all glial cells in our counting. Each of the four subregions was sampled systematic, uniformly randomly by unbiased counting frames (Gundersen, 1977). We used two concentric counting frames of different size per field of view—one for glia of area a(glia) and one for neurons of area a(neuron). Frames were distributed in a square grid with a grid area A(grid), depending on the region. See Table 5 for details on the sampling parameters. The analysis was done by focusing down through the tissue, and a cell was counted when the respective sampling item came into focus wholly or partially inside the unbiased counting frame, without touching the exclusion lines of the frame. For all cell counts, we used a disector height h of 20 μm with an upper guard zone of 15 μm. The optimal disector height and required size of upper guard zone were determined from an initial calibration study where neurons and glial cells were sampled systematic, uniformly random with unbiased counting frames in the full thickness of nine sections from one brain. For each sampled cell, the z-position (i.e., the distance from the section surface) and the local section thickness (measured centrally in the frame) was recorded. Z-plots of the calibration data revealed the cell density to be constant in the depth from 15 µm to 45 µm corresponding to full stain penetration and linear shrinkage of the section in

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the thickness. See e.g. Fig. 7 in Dorph-Petersen et al. (Dorph-Petersen et al., 2009) and corresponding text for further discussion of z-calibration for cell counting. The total number, N, of neurons or glial cells was estimated as:

N :

1 1 1 T A tQ      Q      Q ssf asf hsf BA a h

where ssf is the section sampling fraction, asf the area sampling fraction, hsf the height sampling fraction,

Q



the total number of sampled cells (neurons or glial) in the region across all sections

hitting the hippocampus, T the mean distance between sections (i.e. the slab thickness), BA the cryostat block advance, A the area of the basic tile of the sampling grid, a the area of the counting frame, h the disector height, and t Q  the number-weighted mean section thickness.

 t  q  : q 

t Q

i

i

 i

where ti and q i are the section thickness and the cell count, respectively, for the ith counting frame. See Dorph-Petersen et al. (Dorph-Petersen et al., 2001) for further details.

4.7.

Evaluation of the precision of the estimates

To assess the precision of the stereological estimates, we determined the corresponding coefficients of error (CE) using the methods described by Gundersen et al. (Gundersen and Jensen, 1987; Gundersen et al., 1999). We used the equations based on smoothness class m = 1 in the terminology of Gundersen et al. (Gundersen et al., 1999). The calculated mean CEs ranged from 0.005 to 0.11, which was less than half of the observed coefficient of variation (CV) (0.06 to 0.28) for the corresponding variables. Thus, the major contributor of the group variance was the inherent biological variability among the subjects, indicating the sampling design was robust.

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4.8.

Statistical procedures

We used one-way analysis of variance (ANOVA) to evaluate differences between the four groups. If the ANOVA revealed significant group differences, post hoc tests (Bonferroni) were carried out to elucidate the pattern of group differences. Pearson r correlations were performed between dependent variables and age at death and PMI. Two sided P-values less than 0.05 were considered statistically significant. Statistical analyses and graphical representations of the findings were carried out using SPSS11 (SPSS Corp, Chicago, IL, USA) and Sigmaplot 10 (SYSTAT Inc, San Jose, CA, USA) software.

Acknowledgments María García-Amado Sancho & Leticia Ramírez-Lugo are thanked for technical and scientific assistance in the initial part of the project. Eva B. Vedel Jensen and Ute Hahn are thanked for statistical consulting. Maj-Britt Lundorf, Per Fuglsang Mikkelsen, Nadia Gadeberg Knudsen, & Julie Damgaard Jensen are thanked for skillful technical assistance. Anne E. Stürup is thanked for work in the Brain Collection database needed for the selection of subjects.

Disclosure of potential conflict of interest Fenghua Chen, Aksel Bertelsen, Ida E. Holm, Jens Randel Nyengaard, Raben Rosenberg, and KarlAnton Dorph-Petersen report no biomedical financial interests or potential conflicts of interest.

Funding This study was supported by The Danish Council for Independent Research, Medical Sciences, Denmark; Pulje til Styrkelse af Psykiatrisk Forskning; Aarhus University Research Foundation;

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Savværksejer Jeppe Juhl og hustru Ovita Juhls Mindelegat; and Læge Gerhard Linds Legat. Centre for Stochastic Geometry and Advanced Bioimaging is supported by Villum Foundation.

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Figure Legends Fig. 1. Estimated total hippocampal volume in the control (C), suicide (Su), depression (D), and schizophrenia (Sc) groups of subjects. The horizontal bars indicate group means. Lettering above plots indicates significance. Groups not sharing the same letter are significantly different at p < 0.05. Fig 2. Estimated total number of neurons and glial cells in the four main subregions of the hippocampus in the control (C), suicide (Su), depression (D), and schizophrenia (Sc) groups of subjects. The horizontal bars indicate group means. Lettering above plots indicates significance. Groups not sharing the same letter are significantly different at p < 0.05. Notice, the ANOVA indicated statistically significant differences among the four groups for all eight plots. However, the Bonferroni post hoc test did not have the power to resolve the pairwise differences for the glial cells in DG or the neurons in hilus. Fig 3. Estimated densities of neurons and glial cells in the four main subregions of the hippocampus in the control (C), suicide (Su), depression (D), and schizophrenia (Sc) groups of subjects. The horizontal bars indicate group means. As noted in Table 2, there is no statistically significant differences among the four groups for all eight plots. Fig 4. Scatter plots of volume and total neuron and glial cell number estimates vs. age and storage time, respectively. Control – filled circles, Suicide – thin open circles, Depression – thick open circles, and Schizophrenia – half-filled circles. Fig. 5. Summary of tissue preparation procedures: A: The hippocampus is dissected from each block with a surrounding rim of tissue. B: After cryo protection in sucrose solutions, the tissue pieces are glued together in place using agarose. C: The reassembled hippocampus is embedded in a brick of agarose and cut systematic, uniformly random into 2.5-mm slabs orthogonal to the anterior-posterior (i.e. longitudinal) axis. D: The 2.5-mm slab indicated by the arrow in C. E: Corresponding 80-µm cryo section stained with thionin. Notice the fine horizontal gap in the section corresponding to the original blocking. Fig. 6. Delineation for volume and cell counts. A: 80-µm thionin stained section through the hippocampus. B: Delineation of the section above. The volume is estimated for each of the five cell layers: the granule cell layer of the dentate gyrus (DG-C), Hilus, cornu ammonis subdivisions 2/3 and 1 (CA2/3-C and CA1-C), and subiculum, and for each of the white matter layers: The molecular layer of the dentate gyrus (DG-M), stratum lacunosum moleculare of CA1 and CA2/3 (CA1-LM and CA2/3-LM), stratum radiatum of CA1 and CA2/3 (CA1-R, CA2/3-R), stratum oriens of CA1 and CA2/3 (CA1-O and CA2/3-O), and Alveus. The total number of neurons and glial cells is estimated for each of the four cell layers: DG-C, Hilus, CA2/3-C, and CA1-C. Fig. 7. Disector sampling of cells with the optical fractionator in hilus. Two neurons with one distinct nucleolus each (arrows) are sampled by the large unbiased counting frame. One glial cell (arrowhead) is sampled by the smaller unbiased counting frame.

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Volume of hippocampus (cm³) Figure 1

4

3

2

1

0 a a b b

C

Su

D

Sc

Neuron numbers

Figure 2 a

ab

bc

Glial cell numbers c 2.5

16

N glial cells (106)

DG

N neurons (106)

2.0

12

8

4

1.0

0.5

C

Su

D

Sc

0.0

3.0

14

2.5

12

N glial cells (106)

Hilus

N neurons (106)

0

1.5

2.0 1.5 1.0 0.5 0.0

C

Su

D

Sc

a

ab

c

bc

C

Su

D

Sc

a

a

b

b

C

Su

D

Sc

a

ab

b

ab

C

Su

D

Sc

10 8 6 4 2

C

Su

D

Sc

ab

a

b

b

0

4

12 10

N glial cells (106)

CA2/3

N neurons (106)

3

2

8 6 4

1 2

0

C

Su

D

Sc

a

a

b

b

0

14

50

12 10

N glial cells (106)

CA1

N neurons (106)

40

8 6 4

30

20

10

2 0

C

Su

D

Sc

0

Neuron densities

Figure 3

Glial cell densities 40

200

NV glial cells (1000 mm-3)

DG

NV neurons (1000 mm-3)

250

150

100

50

0

C

Su

D

Sc

30

20

10

0

16

C

Su

D

Sc

C

Su

D

Sc

C

Su

D

Sc

C

Su

D

Sc

70

NV glial cells (1000 mm-3)

Hilus

NV neurons (1000 mm-3)

60

12

8

4

50 40 30 20 10

0

C

Su

D

Sc

0

80

30

NV glial cells (1000 mm-3)

CA2/3

NV neurons (1000 mm-3)

25 20 15 10 5 0

C

Su

D

Sc

60

40

20

0

20

70

NV glial cells (1000 mm-3)

CA1

NV neurons (1000 mm-3)

60

15

10

5

50 40 30 20 10

0

C

Su

D

Sc

0

Volume of hippocampus (cm3)

Figure 4

4

4

3

3

2

2

1

1

0

0

Neuron number (106)

0

20

40

60

80

100

40

40

30

30

20

20

10

10

0

20

30

40

50

60 70 80

20

30

40

50

60 70 80

20

30

40

50

60 70 80

0

0

Glial cell number (106)

10

20

40

60

80

100

10

60

60

50

50

40

40

30

30

20

20

10

10

0

0

0

20

40

60

Age (years)

80

100

10

Storage time (years)

Figure 5

Figure 6

B

Alveus

CA1-O

CA1-C

Sub

CA1-R

iculu

m

CA1-L

M

DG-M

Hilus

CA 2/ CA2 /3-L M CA2/3-R

DG-C 3-

C /3

2 CA

-O us

e Alv

2 mm

Figure 7

Table 1

Table 1. Volume and total number of neurons and glial cells in subregions of hippocampus in different groups

Volumes

(cm3) a

CV

Total number of neurons (x 106) b

CE

DG

CV

CE

Hilus

CV

CE

CV

CE

CA1

CV

CE

DG

CV

CE

Hilus

CV

CE

CV

CE

CA1

CV

CE

Control

2.84

0.162

0.007

12.69

0.224

0.052

2.05

0.235

0.065

2.31

0.217

0.070

10.11

0.200

0.051

1.44

0.227

0.068

9.07

0.256

0.061

6.33

0.272

0.080

24.03

0.285

0.063

Suicide

2.83

0.111

0.009

11.39

0.238

0.056

1.95

0.154

0.065

2.81

0.165

0.062

9.77

0.161

0.052

1.46

0.232

0.069

8.27

0.157

0.063

6.78

0.208

0.073

22.27

0.224

0.064

Depression

2.06

0.153

0.008

8.77

0.228

0.062

1.52

0.303

0.076

1.94

0.168

0.074

6.72

0.191

0.063

1.08

0.384

0.083

5.68

0.423

0.080

4.14

0.275

0.095

14.82

0.448

0.082

Schizophrenia

2.17

0.06

0.007

8.39

0.24

0.063

1.60

0.287

0.073

1.89

0.218

0.075

7.54

0.206

0.060

1.09

0.310

0.079

6.72

0.209

0.070

4.34

0.277

0.095

17.30

0.254

0.076

F3,35 p p (Con vs. Sui)

CA2/3

Total number of glial cells (x 106) CA2/3

15.50

6.69

3.49

9.70

9.49

3.46

6.02

9.05

5.12

< 0.0001

0.0009

0.026

< 0.0001

0.0001

0.027

0.002

0.0001

0.0048

1

↓10.2%

1.00

↓4.7%

0.77

↑21.7%

1.00

↓3.4%

1.00

↑1.4%

1.00

↓8.8%

1.00

↑7.1%

1.00

↓7.3%

p (Con vs. dep)

< 0.0001

1

↓0.3% ↓27.4%

0.011

↓30.9%

0.08

↓26.0%

0.51

↓15.9%

0.001

↓33.6%

0.24

↓24.7%

0.003

↓37.4%

0.013

↓34.7%

0.011

↓38.3%

p (Con vs. Sch)

< 0.0001

↓23.5%

0.002

↓33.9%

0.15

↓21.8%

0.23

↓18.2%

0.008

↓25.4%

0.19

↓24.5%

0.05

↓25.9%

0.019

↓31.4%

0.077

↓28.0%

p (De vs. Sch)

1

1

1.00

p (De vs. Sui)

< 0.0001

0.162

p (Sui vs. Sch)

< 0.0001

0.049

a: the observed coefficient of variation across the subjects in a group b: the root mean square (quadratic mean) of CEs within a group

1.00

1

1.00

1

1.00

1.00

0.208

0.001

0.256

< 0.0001

0.002

0.17

0.032

0.002

0.051

0.023

0.092

0.252

0.002

0.215

Table 2

Table 2. Density of neurons and glial cells in the subregions of hippocampus in the different groups

Control Suicide Depression Schizophrenia F3,35 P

Density of neurons (1000 mm-3) DG Hilus CA2/3 CA1 139 9.22 17.3 14.5 139 10.3 17.8 15.1 144 9.36 17.9 14.4 135 9.60 16.9 15.1 0.10 0.58 0.29 0.24 0.96 0.63 0.83 0.87

Density of glial cells (1000 mm-3) DG Hilus CA2/3 CA1 16.0 41.1 47.8 34.3 18.0 43.2 43.4 34.6 18.0 35.4 38.0 32.2 17.2 40.7 39.0 35.5 0.31 0.98 1.66 0.14 0.82 0.41 0.19 0.93

Table 3

Table 3. The current study is based on hippocampi from four groups of subjects matched according to sex, age, side, postmortem interval and storage time.

Control # 1 2 3 4 5 6 7 8 9 10 Mean CV*

Diagnosis Control Control Control d Control Control Control Control Control d,e Control Control

Sex F F F F F M M M M M 5F/5M

Age (yrs) 30.6 36.7 39.6 53.6 65.5 26.8 47.9 58.1 69.9 82.2 51.1 0.35

Birth Year

1946.2

Height (cm) 168 154 168 165 151 167 174 179 180 167 167.3 0.06

Side

a

L L R R R R L R L R 6R/4L

b

PMI (hrs) 54 80 77 24 30 40 66 42 106 24 54.3 0.51

Storage time (yrs) 13.1 14.3 13.6 14.3 10.6 14.6 15.0 15.0 14.9 26.3 15.2 0.27

b

Storage time (yrs) 13.3 15.0 14.0 12.1 13.7 15.4 16.1 16.0 15.0 15.9 16.0 14.8 0.09

c

Cause of Death

Pulmonary embolism ASCVD ASCVD ASCVD ASCVD Car accident ASCVD ASCVD ASCVD ASCVD

Suicide # 1 2 3 4 5 6 7 8 9 10 11 Mean CV*

Diagnosis f

Suicide g Suicide h Suicide f Suicide h Suicide h,i Suicide f Suicide f Suicide j Suicide f,k Suicide g Suicide

Sex F F F F F M M M M M M 5F/6M

Age (yrs) 28.7 35.2 38.7 53.7 69.2 22.8 49.4 54.1 57.5 67.1 88.0 51.3 0.38

Birth Year

1946.4

Height (cm) 165 169 164 168 165 186 172 188 175 173 155 170.9 0.06

1

Side

a

L L R R R R L R R L R 7R/4L

PMI (hrs) 38 95 9 48 30 29 38 160 85 41 48 56.5 0.75

c

Cause of Death

Jumping Suffocating l Drug overdose Knife lesion Drowning Suffocating Hanging Drowning Knife lesion Car exhaust poisoning Hanging

Table 3 (cont.)

Depression #

Diagnosis

Sex

1 2 3 4 5 6 7 8 Mean CV*

Major depression Major depression Major depression m Major depression Major depression Major depression Major depression Major depression

F F F F F F M M 6F/2M

Age (yrs) 54.2 63.1 66.6 77.5 80.0 81.8 32.6 73.9 66.2 0.25

Birth Year

Height (cm) 167 161 160 154 140

1895.8

172 165 159.9 0.07

Side

a

L R R L R R R R 6R/2L

b

PMI (hrs)

Storage time (yrs)

17 9 24 24

61.1 43.7 65.8 42.8 46.1 37.1 63.3 42.9 50.4 0.22

25 24 54 25.3 0.55

c

Cause of Death

Bronchopneumonia Pulmonary embolism Bronchopneumonia Pneumonia Pneumonia Pulmonary embolism Bronchopneumonia ASCVD

Schizophrenia #

Diagnosis

Sex

1 2 3 4 5 6 7 8 9 10 Mean CV*

Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia o Schizophrenia Schizophrenia Schizophrenia

n

F F F F F F M M M M 6F/4M

Age (yrs) 62.2 65.8 69.3 73.7 81.0 81.4 33.3 60.3 78.9 79.9 68.6 0.21

Birth Year

1896.0

Height (cm) 168 159 165 163 156 167 183 173 163 166.3 0.05

* Coefficient of variation CV = SD/mean. a L: Left hippocampus, R: Right hippocampus b PMI: Post Mortem Interval. c ASCVD: Atherosclerotic Coronary Vascular Disease. d Hypertension. e Non-insulin-dependent diabetes mellitus. f Antidepressant therapy in periods (but without a formal psychiatric diagnosis). g Depressive episode.

Side

a

L R R R L R L L R R 6R/4L

b

PMI (hrs) 60 12 11 43 40 19 2 31 17 26.1 0.72

Storage time (yrs) 54.2 48.7 50.5 43.3 45.2 48.1 38.3 55.9 51.8 42.4 47.8 0.12

c

Cause of Death

ASCVD Pulmonary embolism ASCVD Kidney infection Gastrointestinal infection ASCVD Respiratory failure Pulmonary embolism ASCVD Bronchopneumonia

h Major depression. i Marijuana abuse in periods. j Bipoloar affective disorder. k Alcohol abuse as young, no abuse in many years. l Overdose by Saroten (Amitriptyline). m Insulin-dependent diabetes mellitus (in mild form). n Pulmonary tuberculosis as young. In complete remission. o Non-metastatic colon cancer at time of death.

2

Table 4

Table 4. Information for the two groups of hospitalized subjects (depression and schizophrenia) in Table 1a. Depression #

Sex

Age (yrs)

1 2 3 4 5 6 7 8

F F F F F F M M

54.2 63.1 66.6 77.5 80.0 81.8 32.6 73.9

Length of Age of onset Durationa Hospitalizationb (yrs) (mo) (mo) 42 4.3 2 16 Years 4.2 (7.2) 66 (38)f 6.1 4 (5) Early youth Years 27 79 (75)g 3.1 1.2 (1.3) 80 7 3.7 32 1 0.5 72 16 1.9 (2.2)

S/R episodec

Treatment typesd

R R S (R)f R S (R)g R S S

ECT, H, S AD, AP, ECT, H, S H, S AD, AP, ECT, H, S AD, AP, ECT, H AD, AP, ECT, H, S S AD, AP, ECT, H, S

ECTe (times) 8 6 31 6 13+ 6

Schizophrenia #

Sex

Age (yrs)

1 2 3 4 5 6 7 8 9 10

F F F F F F M M M M

62.2 65.8 69.3 73.7 81.0 81.4 33.3 60.3 78.9 79.9

Length of Age of onset Durationh Hospitalizationb (yrs) (yrs) (yrs) 42 20 3.6 47 18 13.4 37 32 22.7 40 33 6.1 48 33 29.2 47 34 32.0 13 20 19.4 34 26 24.3 32 46 40.6 22 57 55.0

a Total accumulated duration of depression b Total time in psychiatric care, time in parenthesis is time in psychiatric as well as somatic care due to psychiatric disease c S: Single episode, R: Recurrent episodes d AD: Antidepressants, AP: Antipsychotics, APa: Anti Parkinson treatment, ECT: Electro convulsive therapy, H: Hypnotics, ICT: Insulin convulsive therapy, S:Sedatives. e Number of single ECT treatments

Treatment typesd S, ICT AP, ICT AP, APa, S AP AP AP, ECT, ICT H, S S AP

ECTe (times)

ICTi (times)

1

Yes 69

39

86

f Probably depr. episode at age 38, thus possibly recurrent g Probably depr. episode at age 75, thus possibly recurrent h Total duration of psychosis i Number of single ICT treatments

Table 5

Table 5. Details of the stereological counting procedure

2

a(point)(µm ) Mean ∑(P-i) A(grid) (µm2) a(neuron)(µm2) a(glial) (µm2) h(disector) (µm) Mean ∑(Q-neuron) Mean ∑(Q-glia)

DG 179,000 110 90000 318 1,589 20 364 221

hilus 179,000 264 90000 1,589 397 20 255 272

CA2/3 179,000 186 62500 1,589 318 20 259 183

CA1 179,000 885 160000 1,589 318 20 377 238