Parvalbumin interneurons in human ventromedial prefrontal cortex: a comprehensive post-mortem study of myelination and perineuronal nets in neurotypical individuals and depressed suicides with and without a history of child abuse.

Cortical parvalbumin interneurons (PV+) are major regulators of excitatory/inhibitory information processing, and their maturation is associated with the opening of developmental critical periods (CP). Recent studies reveal that cortical PV+ axons are myelinated, and that myelination along with perineuronal net (PNN) maturation around PV+ cells is associated with the closures of CP. Although PV+ interneurons are susceptible to early-life stress, their relationship between their myelination and PNN coverage remains unexplored. This study compared the fine features of PV+ interneurons in well-characterized human post-mortem ventromedial prefrontal cortex samples (n = 31) from depressed suicides with or without a history of child abuse (CA) and matched controls. In healthy controls, 81% of all sampled PV+ interneurons displayed a myelinated axon, while a subset (66%) of these cells also displayed a PNN, proposing a relationship between both attributes. Intriguingly, a 3-fold increase in the proportion of unmyelinated PV+ interneurons with a PNN was observed in CA victims, along with greater PV-immunofluorescence intensity in myelinated PV+ cells with a PNN. This study, which is the first to provide normative data on myelination and PNNs around PV+ interneurons in human neocortex, sheds further light on the cellular and molecular consequences of early-life adversity on cortical PV+ interneurons.

Chondroitin sulfate glycan sulfation patterns influence histochemical labeling of perineuronal nets: a comparative study of interregional distribution in human and mouse brain.

Perineuronal nets (PNNs) are a condensed subtype of extracellular matrix that form a net-like coverings around certain neurons in the brain. PNNs are primarily composed of chondroitin sulfate (CS) proteoglycans from the lectican family that consist of CS-glycosaminoglycan side chains attached to a core protein. CS disaccharides can exist in various isoforms with different sulfation patterns. Literature suggests that CS disaccharide sulfation patterns can influence the function of PNNs as well as their labeling. This study was conducted to characterize such interregional CS disaccharide sulfation pattern differences in adult human (n = 81) and mouse (n = 19) brains. Liquid chromatography tandem mass spectrometry was used to quantify five different CS disaccharide sulfation patterns, which were then compared to immunolabeling of PNNs using Wisteria Floribunda Lectin (WFL) to identify CS-glycosaminoglycans and anti-aggrecan to identify CS proteoglycans. In healthy brains, significant regional and species-specific differences in CS disaccharide sulfation and single versus double-labeling pattern were identified. A secondary analysis to investigate how early-life stress impacts these PNN features discovered that although early-life stress increases WFL+ PNN density, the CS-glycosaminoglycan sulfation code and single versus double PNN-labeling distributions remained unaffected in both species. These results underscore PNN complexity in traditional research, emphasizing the need to consider their heterogeneity in future experiments.

Child abuse associates with increased recruitment of perineuronal nets in the ventromedial prefrontal cortex: a possible implication of oligodendrocyte progenitor cells.

Child abuse (CA) is a strong predictor of psychopathologies and suicide, altering normal trajectories of brain development in areas closely linked to emotional responses such as the prefrontal cortex (PFC). Yet, the cellular underpinnings of these enduring effects are unclear. Childhood and adolescence are marked by the protracted formation of perineuronal nets (PNNs), which orchestrate the closure of developmental windows of cortical plasticity by regulating the functional integration of parvalbumin interneurons into neuronal circuits. Using well-characterized post-mortem brain samples, we show that a history of CA is specifically associated with increased densities and morphological complexity of WFL-labeled PNNs in the ventromedial PFC (BA11/12), possibly suggesting increased recruitment and maturation of PNNs. Through single-nucleus sequencing and fluorescent in situ hybridization, we found that the expression of canonical components of PNNs is enriched in oligodendrocyte progenitor cells (OPCs), and that they are upregulated in CA victims. These correlational findings suggest that early-life adversity may lead to persistent patterns of maladaptive behaviors by reducing the neuroplasticity of cortical circuits through the enhancement of developmental OPC-mediated PNN formation.

Chondroitin sulfate glycan sulfation patterns influence histochemical labeling of perineuronal nets: a comparative study of interregional distribution in human and mouse brain.

Perineuronal nets (PNNs) are a condensed subtype of extracellular matrix that form a net-like coverings around certain neurons in the brain. PNNs are primarily composed of chondroitin sulfate (CS) proteoglycans from the lectican family that consist of CS-glycosaminoglycan (CS-GAG) side chains attached to a core protein. CS disaccharides can exist in various isoforms with different sulfation patterns. Literature suggests that CS disaccharide sulfation patterns can influence the function of PNNs as well as their labeling. This study was conducted to characterize such interregional CS disaccharide sulfation pattern differences in adult human (N = 81) and mouse (N = 19) brains. Liquid chromatography tandem mass spectrometry was used to quantify five different CS disaccharide sulfation patterns, which were then compared to immunolabeling of PNNs using Wisteria Floribunda Lectin (WFL) to identify CS-GAGs and anti-aggrecan to identify CS proteoglycans. In healthy brains, significant regional and species-specific differences in CS disaccharide sulfation and single versus double-labeling pattern were identified. A secondary analysis to investigate how early-life stress (ELS) impacts these PNN features discovered that although ELS increases WFL+ PNN density, the CS-GAG sulfation code and single versus double PNN-labeling distributions remained unaffected in both species. These results underscore PNN complexity in traditional research, emphasizing the need to consider their heterogeneity in future experiments.

Universal method for the isolation of microvessels from frozen brain tissue: A proof-of-concept multiomic investigation of the neurovasculature.

The neurovascular unit, comprised of vascular cell types that collectively regulate cerebral blood flow to meet the needs of coupled neurons, is paramount for the proper function of the central nervous system. The neurovascular unit gatekeeps blood-brain barrier properties, which experiences impairment in several central nervous system diseases associated with neuroinflammation and contributes to pathogenesis. To better understand function and dysfunction at the neurovascular unit and how it may confer inflammatory processes within the brain, isolation and characterization of the neurovascular unit is needed. Here, we describe a singular, standardized protocol to enrich and isolate microvessels from archived snap-frozen human and frozen mouse cerebral cortex using mechanical homogenization and centrifugation-separation that preserves the structural integrity and multicellular composition of microvessel fragments. For the first time, microvessels are isolated from postmortem ventromedial prefrontal cortex tissue and are comprehensively investigated as a structural unit using both RNA sequencing and Liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Both the transcriptome and proteome are obtained and compared, demonstrating that the isolated brain microvessel is a robust model for the NVU and can be used to generate highly informative datasets in both physiological and disease contexts.

Cell type specific transcriptomic differences in depression show similar patterns between males and females but implicate distinct cell types and genes.

Major depressive disorder (MDD) is a common, heterogenous, and potentially serious psychiatric illness. Diverse brain cell types have been implicated in MDD etiology. Significant sexual differences exist in MDD clinical presentation and outcome, and recent evidence suggests different molecular bases for male and female MDD. We evaluated over 160,000 nuclei from 71 female and male donors, leveraging new and pre-existing single-nucleus RNA-sequencing data from the dorsolateral prefrontal cortex. Cell type specific transcriptome-wide threshold-free MDD-associated gene expression patterns were similar between the sexes, but significant differentially expressed genes (DEGs) diverged. Among 7 broad cell types and 41 clusters evaluated, microglia and parvalbumin interneurons contributed the most DEGs in females, while deep layer excitatory neurons, astrocytes, and oligodendrocyte precursors were the major contributors in males. Further, the Mic1 cluster with 38% of female DEGs and the ExN10_L46 cluster with 53% of male DEGs, stood out in the meta-analysis of both sexes.

Widespread Decrease of Cerebral Vimentin-Immunoreactive Astrocytes in Depressed Suicides.

Post-mortem investigations have implicated cerebral astrocytes immunoreactive (-IR) for glial fibrillary acidic protein (GFAP) in the etiopathology of depression and suicide. However, it remains unclear whether astrocytic subpopulations IR for other astrocytic markers are similarly affected. Astrocytes IR to vimentin (VIM) display different regional densities than GFAP-IR astrocytes in the healthy brain, and so may be differently altered in depression and suicide. To investigate this, we compared the densities of GFAP-IR astrocytes and VIM-IR astrocytes in post-mortem brain samples from depressed suicides and matched non-psychiatric controls in three brain regions (dorsomedial prefrontal cortex, dorsal caudate nucleus and mediodorsal thalamus). A quantitative comparison of the fine morphology of VIM-IR astrocytes was also performed in the same regions and subjects. Finally, given the close association between astrocytes and blood vessels, we also assessed densities of CD31-IR blood vessels. Like for GFAP-IR astrocytes, VIM-IR astrocyte densities were found to be globally reduced in depressed suicides relative to controls. By contrast, CD31-IR blood vessel density and VIM-IR astrocyte morphometric features in these regions were similar between groups, except in prefrontal white matter, in which vascularization was increased and astrocytes displayed fewer primary processes. By revealing a widespread reduction of cerebral VIM-IR astrocytes in cases vs. controls, these findings further implicate astrocytic dysfunctions in depression and suicide.

Single-nucleus transcriptomics of the prefrontal cortex in major depressive disorder implicates oligodendrocyte precursor cells and excitatory neurons.

Major depressive disorder (MDD) has an enormous impact on global disease burden, affecting millions of people worldwide and ranking as a leading cause of disability for almost three decades. Past molecular studies of MDD employed bulk homogenates of postmortem brain tissue, which obscures gene expression changes within individual cell types. Here we used single-nucleus transcriptomics to examine ~80,000 nuclei from the dorsolateral prefrontal cortex of male individuals with MDD (n = 17) and of healthy controls (n = 17). We identified 26 cellular clusters, and over 60% of these showed differential gene expression between groups. We found that the greatest dysregulation occurred in deep layer excitatory neurons and immature oligodendrocyte precursor cells (OPCs), and these contributed almost half (47%) of all changes in gene expression. These results highlight the importance of dissecting cell-type-specific contributions to the disease and offer opportunities to identify new avenues of research and novel targets for treatment.

Characterization of Vimentin-Immunoreactive Astrocytes in the Human Brain.

Astrocytes are commonly identified by their expression of the intermediate filament protein glial fibrillary acidic protein (GFAP). GFAP-immunoreactive (GFAP-IR) astrocytes exhibit regional heterogeneity in density and morphology in the mouse brain as well as morphological diversity in the human cortex. However, regional variations in astrocyte distribution and morphology remain to be assessed comprehensively. This was the overarching objective of this postmortem study, which mainly exploited the immunolabeling of vimentin (VIM), an intermediate filament protein expressed by astrocytes and endothelial cells which presents the advantage of more extensively labeling cell structures. We compared the densities of vimentin-immunoreactive (VIM-IR) and GFAP-IR astrocytes in various brain regions (prefrontal and primary visual cortex, caudate nucleus, mediodorsal thalamus) from male individuals having died suddenly in the absence of neurological or psychiatric conditions. The morphometric properties of VIM-IR in these brain regions were also assessed. We found that VIM-IR astrocytes generally express the canonical astrocytic markers Aldh1L1 and GFAP but that VIM-IR astrocytes are less abundant than GFAP-IR astrocytes in all human brain regions, particularly in the thalamus, where VIM-IR cells were nearly absent. About 20% of all VIM-IR astrocytes presented a twin cell morphology, a phenomenon rarely observed for GFAP-IR astrocytes. Furthermore VIM-IR astrocytes in the striatum were often seen to extend numerous parallel processes which seemed to give rise to large VIM-IR fiber bundles projecting over long distances. Moreover, morphometric analyses revealed that VIM-IR astrocytes were more complex than their mouse counterparts in functionally homologous brain regions, as has been previously reported for GFAP-IR astrocytes. Lastly, the density of GFAP-IR astrocytes in gray and white matter were inversely correlated with vascular density, but for VIM-IR astrocytes this was only the case in gray matter, suggesting that gliovascular interactions may especially influence the regional heterogeneity of GFAP-IR astrocytes. Taken together, these findings reveal special features displayed uniquely by human VIM-IR astrocytes and illustrate that astrocytes display important region- and marker-specific differences in the healthy human brain.

Moderate decline in select synaptic markers in the prefrontal cortex (BA9) of patients with Alzheimer's disease at various cognitive stages.

Synaptic loss, plaques and neurofibrillary tangles are viewed as hallmarks of Alzheimer's disease (AD). This study investigated synaptic markers in neocortical Brodmann area 9 (BA9) samples from 171 subjects with and without AD at different levels of cognitive impairment. The expression levels of vesicular glutamate transporters (VGLUT1&2), glutamate uptake site (EAAT2), post-synaptic density protein of 95 kD (PSD95), vesicular GABA/glycine transporter (VIAAT), somatostatin (som), synaptophysin and choline acetyl transferase (ChAT) were evaluated. VGLUT2 and EAAT2 were unaffected by dementia. The VGLUT1, PSD95, VIAAT, som, ChAT and synaptophysin expression levels significantly decreased as dementia progressed. The maximal decrease varied between 12% (synaptophysin) and 42% (som). VGLUT1 was more strongly correlated with dementia than all of the other markers (polyserial correlation = -0.41). Principal component analysis using these markers was unable to differentiate the CDR groups from one another. Therefore, the status of the major synaptic markers in BA9 does not seem to be linked to the cognitive status of AD patients. The findings of this study suggest that the loss of synaptic markers in BA9 is a late event that is only weakly related to AD dementia.

Association of a History of Child Abuse With Impaired Myelination in the Anterior Cingulate Cortex: Convergent Epigenetic, Transcriptional, and Morphological Evidence.

OBJECTIVE: Child abuse has devastating and long-lasting consequences, considerably increasing the lifetime risk of negative mental health outcomes such as depression and suicide. Yet the neurobiological processes underlying this heightened vulnerability remain poorly understood. The authors investigated the hypothesis that epigenetic, transcriptomic, and cellular adaptations may occur in the anterior cingulate cortex as a function of child abuse. METHOD: Postmortem brain samples from human subjects (N=78) and from a rodent model of the impact of early-life environment (N=24) were analyzed. The human samples were from depressed individuals who died by suicide, with (N=27) or without (N=25) a history of severe child abuse, as well as from psychiatrically healthy control subjects (N=26). Genome-wide DNA methylation and gene expression were investigated using reduced representation bisulfite sequencing and RNA sequencing, respectively. Cell type-specific validation of differentially methylated loci was performed after fluorescence-activated cell sorting of oligodendrocyte and neuronal nuclei. Differential gene expression was validated using NanoString technology. Finally, oligodendrocytes and myelinated axons were analyzed using stereology and coherent anti-Stokes Raman scattering microscopy. RESULTS: A history of child abuse was associated with cell type-specific changes in DNA methylation of oligodendrocyte genes and a global impairment of the myelin-related transcriptional program. These effects were absent in the depressed suicide completers with no history of child abuse, and they were strongly correlated with myelin gene expression changes observed in the animal model. Furthermore, a selective and significant reduction in the thickness of myelin sheaths around small-diameter axons was observed in individuals with history of child abuse. CONCLUSIONS: The results suggest that child abuse, in part through epigenetic reprogramming of oligodendrocytes, may lastingly disrupt cortical myelination, a fundamental feature of cerebral connectivity.

Effects of neuregulin-1 administration on neurogenesis in the adult mouse hippocampus, and characterization of immature neurons along the septotemporal axis.

Adult hippocampal neurogenesis is associated with learning and affective behavioural regulation. Its diverse functionality is segregated along the septotemporal axis from the dorsal to ventral hippocampus. However, features distinguishing immature neurons in these regions have yet to be characterized. Additionally, although we have shown that administration of the neurotrophic factor neuregulin-1 (NRG1) selectively increases proliferation and overall neurogenesis in the mouse ventral dentate gyrus (DG), likely through ErbB3, NRG1's effects on intermediate neurogenic stages in immature neurons are unknown. We examined whether NRG1 administration increases DG ErbB3 phosphorylation. We labeled adultborn cells using BrdU, then administered NRG1 to examine in vivo neurogenic effects on immature neurons with respect to cell survival, morphology, and synaptogenesis. We also characterized features of immature neurons along the septotemporal axis. We found that neurogenic effects of NRG1 are temporally and subregionally specific to proliferation in the ventral DG. Particular morphological features differentiate immature neurons in the dorsal and ventral DG, and cytogenesis differed between these regions. Finally, we identified synaptic heterogeneity surrounding the granule cell layer. These results indicate neurogenic involvement of NRG1-induced antidepressant-like behaviour is particularly associated with increased ventral DG cell proliferation, and identify novel distinctions between dorsal and ventral hippocampal neurogenic development.

High Resolution Dissection of Reactive Glial Nets in Alzheimer's Disease.

Fixed human brain samples in tissue repositories hold great potential for unlocking complexities of the brain and its alteration with disease. However, current methodology for simultaneously resolving complex three-dimensional (3D) cellular anatomy and organization, as well as, intricate details of human brain cells in tissue has been limited due to weak labeling characteristics of the tissue and high background levels. To expose the potential of these samples, we developed a method to overcome these major limitations. This approach offers an unprecedented view of cytoarchitecture and subcellular detail of human brain cells, from cellular networks to individual synapses. Applying the method to AD samples, we expose complex features of microglial cells and astrocytes in the disease. Through this methodology, we show that these cells form specialized 3D structures in AD that we refer to as reactive glial nets (RGNs). RGNs are areas of concentrated neuronal injury, inflammation, and tauopathy and display unique features around ß-amyloid plaque types. RGNs have conserved properties in an AD mouse model and display a developmental pattern coinciding with the progressive accumulation of neuropathology. The method provided here will help reveal novel features of the healthy and diseased human brain, and aid experimental design in translational brain research.

Phenotypic Alterations in Hippocampal NPY- and PV-Expressing Interneurons in a Presymptomatic Transgenic Mouse Model of Alzheimer's Disease.

Interneurons, key regulators of hippocampal neuronal network excitability and synchronization, are lost in advanced stages of Alzheimer's disease (AD). Given that network changes occur at early (presymptomatic) stages, we explored whether alterations of interneurons also occur before amyloid-beta (Aß) accumulation. Numbers of neuropeptide Y (NPY) and parvalbumin (PV) immunoreactive (IR) cells were decreased in the hippocampus of 1 month-old TgCRND8 mouse AD model in a sub-regionally specific manner. The most prominent change observed was a decrease in the number of PV-IR cells that selectively affected CA1/2 and subiculum, with the pyramidal layer (PY) of CA1/2 accounting almost entirely for the reduction in number of hippocampal PV-IR cells. As PV neurons were decreased selectively in CA1/2 and subiculum, and given that they are critically involved in the control of hippocampal theta oscillations, we then assessed intrinsic theta oscillations in these regions after a 4-aminopyridine (4AP) challenge. This revealed increased theta power and population bursts in TgCRND8 mice compared to non-transgenic (nTg) controls, suggesting a hyperexcitability network state. Taken together, our results identify for the first time AD-related alterations in hippocampal interneuron function as early as at 1 month of age. These early functional alterations occurring before amyloid deposition may contribute to cognitive dysfunction in AD.

Cellular and Molecular Inflammatory Profile of the Choroid Plexus in Depression and Suicide.

The inflammatory hypothesis of depression is one of the main theories that endeavors to explain and describe the underlying biological mechanisms of depression and suicide. While mounting evidence indicates altered peripheral and central inflammatory profiles in depressed patients and suicide completers, little is known about how peripheral and central inflammation might be linked in these contexts. The choroid plexus (ChP), a highly vascularized tissue that produces cerebrospinal fluid (CSF) and lacks a blood-brain-barrier, is an interface between peripheral and central immune responses. In the present study, we investigated the cellular and molecular inflammatory profile of the ChP of the lateral ventricle in depressed suicides and psychiatrically healthy controls. Gene expression of macrophages, pro- and anti-inflammatory cytokines, and various factors implicated in immune cell trafficking were measured; and density of ionized calcium-binding adaptor molecule 1-positive (Iba1+) macrophages associated with the ChP epithelial cell layer (ECL) was examined. Significant downregulations of the genes encoding interleukin 1ß (IL1ß), a pro-inflammatory acute-phase protein; intercellular cell adhesion molecule 1 (ICAM1), a protein implicated in immune cell trafficking in the ChP; and IBA1, a monocyte/macrophage marker; were detected in depressed suicides as compared to controls. No difference in the density of Iba1+ macrophages associated with the ChP ECL was observed. While interpretation of these findings is challenging in the absence of corroborating data from the CSF, peripheral blood, or brain parenchyma of the present cohort, we hypothesize that the present findings reflect a ChP compensatory mechanism that attenuates the detrimental effects of chronically altered pro-inflammatory signaling caused by elevated levels of pro-inflammatory cytokines, such as IL-1ß, peripherally and/or centrally. Together, these findings further implicate neuroimmune processes in the etiology of depression and suicide.

Increased doublecortin (DCX) expression and incidence of DCX-immunoreactive multipolar cells in the subventricular zone-olfactory bulb system of suicides.

Postmortem studies have confirmed the occurrence of adult hippocampal neurogenesis in humans and implicated this process in antidepressant response, yet neurogenesis in other regions remains to be examined in the context of depression. Here we assess the extent of subventricular zone-olfactory bulb (SVZ-OB) neurogenesis in adult humans having died by suicide. Protein expression of proliferative and neurogenic markers Sox2, proliferating cell nuclear antigen, and doublecortin (DCX) were examined in postmortem SVZ and OB samples from depressed suicides and matched sudden-death controls. In the SVZ, DCX-immunoreactive (IR) cells displayed phenotypes typical of progenitors, whereas in the olfactory tract (OT), they were multipolar with variable size and morphologies suggestive of differentiating cells. DCX expression was significantly increased in the OB of suicides, whereas SVZ DCX expression was higher among unmedicated, but not antidepressant-treated, suicides. Although very few DCX-IR cells were present in the control OT, they were considerably more common in suicides and correlated with OB DCX levels. Suicides also displayed higher DCX-IR process volumes. These results support the notion that OB neurogenesis is minimal in adult humans. They further raise the possibility that the differentiation and migration of SVZ-derived neuroblasts may be altered in unmedicated suicides, leading to an accumulation of ectopically differentiating cells in the OT. Normal SVZ DCX expression among suicides receiving antidepressants suggests a potentially novel mode of action of antidepressant medication. Given the modest group sizes and rarity of DCX-IR cells assessed here, a larger-scale characterization will be required before firm conclusions can be made regarding the identity of these cells.

Regional and sub-regional differences in hippocampal GABAergic neuronal vulnerability in the TgCRND8 mouse model of Alzheimer's disease.

Hippocampal network activity is predominantly coordinated by γ-amino-butyric acid (GABA)ergic neurons. We have previously hypothesized that the altered excitability of hippocampal neurons in Alzheimer's disease (AD), which manifests as increased in vivo susceptibility to seizures in the TgCRND8 mouse model of AD, may be related to disruption of hippocampal GABAergic neurons. In agreement, our previous study in TgCRND8 mice has shown that hippocampal GABAergic neurons are more vulnerable to AD-related neuropathology than other types of neurons. To further explore the mechanisms behind the observed decrease of GABAergic neurons in 6 month-old TgCRND8 mice, we assessed the relative proportion of somatostatin (SOM), neuropeptide Y (NPY) and paravalbumin (PV) sub-types of GABAergic neurons at the regional and sub-regional level of the hippocampus. We found that NPY expressing GABAergic neurons were the most affected, as they were decreased in CA1-CA2 (pyramidal-, stratum oriens, stratum radiatum and molecular layers), CA3 (specifically in the stratum oriens) and dentate gyrus (specifically in the polymorphic layer) in TgCRND8 mice as compared to non-transgenic controls. SOM expressing GABAergic neurons were decreased in CA1-CA2 (specifically in the stratum oriens) and in the stratum radiatum of CA3, whereas PV neurons were significantly altered in stratum oriens sub-region of CA3. Taken together, these data provide new evidence for the relevance of hippocampal GABAergic neuronal network disruption as a mechanism underlying AD sequelae such as aberrant neuronal excitability, and further point to complex hippocampal regional and sub-regional variation in susceptibility to AD-related neuronal loss.

Automated analysis of global ischemia-induced CA1 neuronal death using terminal UTP nick end labeling (TUNEL).

In the brain, DNA fragmentation is associated with apoptotic cell death following ischemic/excitotoxic damage. Fragmented DNA can be detected in situ by labeling the 3'OH termini of the internucleosomal generated fragments with deoxynucleotides, through a process known as terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling, or TUNEL. TUNEL is frequently being used to assess neuronal death following cerebral ischemia in a number of animal models. However, conventional techniques for TUNEL can be time consuming, and are often subjective and thus can lead to inconsistencies among investigators. Moreover, the lack of tools for its quantification and standardization limits the use of this technique in assessing the magnitude of cell death. In the present report, we describe an improved higher throughput technique for TUNEL staining at room temperature on a BioGenex automated stainer, and its subsequent quantitative analysis using NORTHERN ECLIPSE, an imaging analysis program. Its implementation allows us to effectively quantify TUNEL positive cells in the CA1 region of the hippocampus following global forebrain ischemia in rats. We conclude that this general histological technique can be applied to the study of cell death in numerous other experimental models.

Explant-cell culture of primary mammary tumors from MMTV-c-Myc transgenic mice.

We have established an explant-cell culture system for mammary gland tumors from c-myc oncogene-expressing transgenic mice and potentially other transgenic strains. By coating culture dish surfaces with fetal bovine serum and using culture media supplemented with low serum and growth factors, the mammary tumor specimens could be maintained in culture for over 3 mo. Throughout the culture period, the explants produced abundant outgrowths of epithelial cells. As the outgrowths of epithelial cells filled the dishes, the explants were serially transferred from one dish to another-a process that could be repeated at least six times, thus providing a continuous supply of primary tumor cells. This culture system provides a useful tool for studying the biology of mouse mammary gland tumors and possibly tumors from other organ sites.

Amygdalar expression of proteins associated with neuroplasticity in major depression and suicide.

INTRODUCTION: Doublecortin (DCX) and polysialilated neural cell adhesion molecule (PSA-NCAM), two proteins associated with immature neuronal phenotypes and elevated neuroplasticity in the adult brain, have recently been identified in the mammalian amygdala, and evidence from rodent studies suggests that stress may modify their expression in this brain region. The purpose of the present study was to investigate whether the expression of proteins involved in neuroplasticity is altered in the amygdala of individuals with depression. METHODS: Basolateral amygdala (BLA) and central amygdala (CeA) postmortem human brain samples were collected from individuals with a history of depression (n = 22 and 25, respectively) and psychiatrically healthy controls (CTRL; n = 14). Proteins associated with neuroplasticity were quantified using Western blotting. RESULTS: Immunoblots revealed that depressed subjects displayed increased expression of DCX (p = 0.033) and PSA-NCAM (p = 0.027) in the BLA as compared to CTRLs. Subsequent analyses revealed that this effect was due primarily to a subset of depressed subjects who had not died by suicide (DNS). DNS subjects displayed higher DCX than CTRLs (p < 0.001) and depressed suicides (p = 0.001), and higher PSA-NCAM than CTRLs (p = 0.007). Conversely, within the CeA, DNS subjects displayed a tendency toward lower DCX expression than CTRLs (p = 0.062), and higher BDNF levels than DS subjects (p = 0.045). CONCLUSION: These results suggest that the BLA and CeA display contrasting patterns of neuroplasticity in depression, and that greater impairment of amygdalar neuroplasticity may be associated with increased risk of suicide.