P.J. Lucassen

Neither major depression nor glucocorticoid treatment affects the cellular integrity of the human hippocampus

In major depression, decreased hippocampal volume has been attributed to hypercortisolemia, a frequent sign of the disorder, because in animals an excess of corticosteroids has led to dendritic atrophy, astrogliosis and loss of neurons in this brain region. The present study is the first to investigate the structural integrity of the human hippocampus in major depression and following glucocorticoid treatment. Post‐mortem hippocampal tissue from 15 patients who had had major depression or bipolar affective disorder, 10 patients who had been treated with glucocorticoids and 16 controls was assessed using haematoxylin‐eosin, Nissl and Bodian staining. The patterns of reactive astrogliosis (glial fibrillary acidic protein, GFAP), synaptic density (synaptophysin), synaptic reorganization (growth‐associated protein B‐50) and early signs of Alzheimers disease (Alz‐50) were examined immunocytochemically. Multivariate analysis, with the patients age, tissue fixation time and postmortem delay as covariates, was performed. There was no evidence of neuronal cell loss or other major morphological alterations in any of the groups, nor was there a significant change in the distribution pattern of synaptophysin or Alz‐50. Changes in B‐50 and GFAP staining were observed in the steroid‐treated and depressed patients in areas CA1 and CA2 only. The human hippocampus in major depression and after glucocorticoid treatment does not reveal any major morphological changes or signs of neuronal cell death, but does show subtle alterations in B‐50 and GFAP expression in selected parts of the pyramidal cell layer.

Chronic psychosocial stress differentially affects apoptosis in hippocampal subregions and cortex of the adult tree shrew.

We studied the effect of chronic psychosocial stress on cell death and volume changes in the tree shrew hippocampus. In situ end labelling (ISEL) identified low frequent but convincing apoptosis in many hippocampal subregions. Also in entorhinal cortex, apoptosis was found, generally at higher frequencies. After 28u2003days of chronic stress, apoptosis was significantly reduced in the CA1 stratum radiatum, whereas an increase was observed in the hilus (Pu2003<u20030.04). With all subregions taken together, the hippocampus showed a decrease, whereas in the cortex, an increase in apoptosis was found after stress (Pu2003<u20030.04). In a parallel and similar chronic stress study, post mortem morphometry of the same brain regions was performed, revealing mild decreases (7.6%) in entire hippocampal volume. We conclude that (i) low frequent apoptosis occurs throughout the adult tree shrew brain, and (ii) 28u2003days of chronic stress differentially affects its occurrence in distinct hippocampal subregions and entorhinal cortex. As previous stereological investigations failed to detect any loss in the principal neuronal layers, psychosocial stress, therefore, must affect other (structural) parameters like dendritic tree, interneurons, neurogenesis, or glia.

DNA damage distribution in the human brain as shown by in situ end labeling; area-specific differences in aging and Alzheimer disease in the absence of apoptotic morphology.

DNA damage has been proposed to underlie neuronal degeneration in aging and Alzheimer disease (AD). To determine the histological distribution of DNA damage, in situ end labeling (ISEL) was applied as a marker for DNA breaks on 4 differentially affected brain areas. Occipital cortex showed considerable variation between cortical layers and between patients. Temporal cortex displayed little ISEL-labeling in controls, and in AD, surprisingly. In the hippocampus, which is strongly affected in AD, many ISEL-positive nuclei and glialike cells were found in AD as compared with controls. The hypothalamic supraoptic and paraventricular nuclei showed little DNA-damage, whereas the nucleus basalis was often, but not always, labeled by ISEL. In contrast to others, no apoptotic morphology was observed, only necrotic morphology. Our results in relation to postmortem delay indicate that, area dependent, increased DNA vulnerability may occur in AD. Furthermore, the distribution of DNA damage in cortex differs from that of plaques and tangles, suggesting that these 3 phenomena are, in principle, independent. Whether the enhanced level of hippocampal DNA breaks in AD underlies, or rather is a consequence of, previous degenerative changes in this brain area remains to be established.

Severe learning deficits in apolipoprotein E-knockout mice in a water maze task.

Recent studies on apolipoprotein E (apoE) have stressed the importance of this protein in neuronal viability, especially in the hippocampal area. In the present study, we used the Morris water maze to assess spatial learning and memory in 6-month-old homozygous apoE-deficient and heterozygous control mice. The apoE status was checked by genotyping and immunocytochemistry. ApoE-knockout mice were not able to learn the task at all, developed neither spatial nor other strategies to locate the platform, but rather an unusual repetitive behavioral pattern of wall bumping. Heterozygous control mice did not experience any difficulty with the task. Swimming ability and general locomotor activity of both groups were comparable. These results indicate that absence of apoE in these animals might be critical for spatial learning and memory abilities.

NMDA and Kainate Induce Internucleosomal DNA Cleavage Associated With Both Apoptotic and Necrotic Cell Death in the Neonatal Rat Brain

Injection of N‐methyl‐D‐aspartate (NMDA) or kainate in the striatum of 7‐day‐old rats induced massive cell loss in the ipsilateral striatum, hippocampus and inner cortical layers. In order to examine whether apoptosis contributes to cell death in this model of excitotoxic injury we examined the progression of internucleosomal DNA fragmentation and changes in cellular ultrastructure. Agarose gel electrophoresis of DNA extracted from the ipsilateral striatum, cerebral cortex and hippocampus clearly showed breakdown of DNA into oligonucleosome‐sized fragments, indicative of apoptosis, 12 h post‐NMDA injection. In addition, an increase between 12 and 24 h was observed as well as a continuous presence 5 days later. Kainate induced a similar time course of oligonucleosomal DNA fragmentation, but the intensity of the ethidium bromide stained bands was less compared with that observed for NMDA. DNA fragmentation was not detected in animals intrastriatally injected with Tris‐HCl or in animals treated with MK‐801 [(+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohept‐5,10‐imine hydrogen maleate, 1 mg/kg] 30 min after NMDA injection. MK‐801 had no effect on DNA fragmentation induced by kainate. In addition to agarose gel electrophoresis, terminal deoxynucleotidyltransferase‐mediated dUTP‐biotin nick end labelling (TUNEL) was used for detection of DNA fragmentation in sections. A gradual increase in the density of both apoptotic and non‐apoptotic TUNEL nuclei was found in the anterior cingulate (ACC) and retrosplenial (RSC) areas of the cortex, the striatum, and the CA1 area and dentate gyrus of the hippocampus over the first 24 h post‐NMDA or kainate injection. In the contralateral hemisphere hardly any TUNEL nuclei were present and their density was comparable with that in animals injected with vehicle only. In the ipsilateral mammillary nucleus (MN), which showed no signs of acute cell swelling after intrastriatal injection with NMDA, internucleosomal DNA fragmentation was found 24 and 48 h after intrastriatal NMDA injection. Here, the density of TUNEL cells with apoptotic morphology was high at 12 and 24 h post‐NMDA injection but returned to control levels by 5 days. Electron microscopy showed cells with a clearly apoptotic morphology in the ACC and RSC and in the MN 24 h after NMDA injection. In the CA1 area of the hippocampus a necrotic, rather than an apoptotic, ultrastructure prevailed, indicating that the TUNEL method stained both apoptotic and necrotic cells. Based on biochemical and morphological criteria this study provides strong evidence that both apoptosis and necrosis are involved in NMDA‐ or kainate‐induced excitotoxic cell death in the neonatal rat brain.

Functional neuroanatomy and neuropathology of the human hypothalamus

The human hypothalamus is involved in a wide range of functions in the developing, adult and aging subject and is responsible for a large number of symptoms of neuroendocrine, neurological and psychiatric diseases. In the present review some prominent hypothalamic nuclei are discussed in relation to normal development, sexual differentiation, aging and a number of neuropathological conditions.The suprachiasmatic nucleus, the clock of the brain, shows seasonal and circadian variations in its vasopressin neurons. During normal aging, but even more so in Alzheimers disease, the number of these neurons decreases. In homosexual men this nucleus is larger than in heterosexual men.The difference between the sexually dimorphic nuclei of men and women arises between the ages of 2–4 to puberty. In adult men this nucleus is twice as large as in adult women. In the process of aging, a sex-dependent decrease in cell number occurs. The vasopressin and oxytocin cells of the supraoptic and paraventricular nucleus are present in adult numbers as early as mid-gestation. Lower oxytocin neuron numbers are found in Prader-Willi syndrome, AIDS and Parkinsons disease. Familial hypothalamic diabetes insipidus is based upon a point mutation in the vasopressin-neurophysin-glycopeptide gene.Parvicellular corticotropin-releasing hormone-containing neurons in the paraventricular nucleus increase in number and are activated during the course of aging.In post-menopausal women, the infundibular or arcuate nucleus contains hypertrophie neurons containing oestrogen receptors. These neurons may be involved in the initiation of menopausal flushes.The nucleus tuberalis lateralis may be involved in feeding behaviour and metabolism. In Huntingtons disease the majority of its neurons is lost; in Alzheimers disease it shows very strong cytoskeletal alterations.Tuberomammillary nucleus neurons contain, e.g., histamine or galanine, and project to the cortex. Strong cytoskeletal changes, as well as plaques and tangles are found in this nucleus in Alzheimers disease.The various hypothalamic nuclei are probably involved in many functions and symptoms of which only a minority has been revealed.

Decreased neuronal activity in the nucleus basalis of Meynert in Alzheimer's disease as suggested by the size of the Golgi apparatus

In order to study changes in neuronal activity in the nucleus basalis of Meynert in aging and Alzheimers disease, we applied a polyclonal antibody directed against the Golgi apparatus on formalin-fixed, paraffin-embedded material. Subsequently, an image analysis system was used to measure the size of the Golgi apparatus in (i) all nucleus basalis neurons and also separately in (ii) the remaining large cells (perikaryonal diameter > 30 microns). A significant reduction of 49% in the size of the Golgi apparatus was found in the entire population of nucleus basalis neurons in Alzheimers disease. Furthermore, although there was no significant decrease in the size of the persisting large neurons in the nucleus basalis of Meynert, a significantly decreased size of the Golgi apparatus was found in these neurons in Alzheimers disease. These results suggest that the overall activity of nucleus basalis neurons is severely decreased in Alzheimers disease. Furthermore, these data support the idea that atrophy and decreased activity are the main phenomena in the nucleus basalis in Alzheimers disease; they also indicate that the size of the Golgi apparatus is a sensitive parameter to follow this process.

Microwave-enhanced in situ end-labeling of fragmented DNA: parametric studies in relation to postmortem delay and fixation of rat and human brain.

In situ end-labeling (ISEL) identifies DNA fragmentation in apoptotic or necrotic nuclei in tissue sections. However, application of ISEL on human brain requires conservation of DNA integrity during the postmortem delay (PMD) and good accessibility of fragmented DNA after (prolonged) tissue fixation. We therefore investigated ISEL in relation to PMD and fixation in rat and human brain. Application on a unilateral lesion model in perfused rat brain revealed that prolonged post-fixation strongly diminished ISEL results. However, microwave pre-treatment can counteract these masking effects without inducing nonspecific labeling contralaterally. On the other hand, in briefly post-fixed, perfused brain or immersion-fixed rat and human PMD brain, microwave pre-treatment was deleterious and induced strong nonspecific labeling. In young rat brain, PMD did not influence the low numbers of apoptotic nuclei until 24 hr PMD, when massive nuclear labeling occurred. In human cortex, DNA fragmentation patterns were independent of duration of fixation or PMD and were already present from 4.25 hr PMD onwards. Our data suggest that ISEL on human brain represents antemortem DNA damage rather than PMD artifacts. Furthermore, microwave pre-treatment appears beneficial only in particular fixation conditions.

Activation of the human supraoptic and paraventricular nucleus neurons with aging and in Alzheimer's disease as judged from increasing size of the Golgi apparatus.

The supraoptic (SON) and paraventricular nucleus (PVN) of the human hypothalamus produce vasopressin (AVP) and oxytocin (OXT). Since in these nuclei no cells are lost during aging or Alzheimers Disease (AD), factors are searched for which may be responsible for this remarkable stability. Earlier work in both rat and human indicated that the peptide synthesis of these neurons was activated in the oldest age groups as judged from increased neuronal and nuclear size and AVP plasma levels. The size of the Golgi Apparatus (GA) has proved to be a very sensitive parameter for the synthetic activity of these neurosecretory cells in animal experiments. In order to determine changes in the GA during aging and in Alzheimers Disease, we applied a polyclonal antiserum against immunoaffinity purified MG-160, a sialoglycoprotein of the medial cisternae of the GA, on formalin-fixed and paraffin-embedded sections of the SON and PVN of patients ranging in age from 29 to 97 years. However, our standard fixation procedure masked antigenic sites resulting in a minimal immunocytochemical staining in most of the tissues examined. It appeared to be possible, however, to retrieve the antigen and to obtain an excellent staining of the GA by heating sections in a microwave oven before immunostaining. Following this procedure, an increase in size and intensity of the GA became apparent in individuals from about 70 years and older. In AD patients a similar increase in size and intensity of the immunostained GA was observed. Taken together, these results indicate that SON and PVN neurons are activated during the course of aging and also in AD and that this activation takes place at an earlier age than observed previously by other cellular parameters.

Reduced neuronal activity and reactivation in Alzheimer's disease.

Publisher Summary Alzheimers disease (AD) is a multifactorial disease in which APOE-ɛ4 and age are important risk factors. In addition, various mutations and even viral infections, such as herpex simplex may play a role. AD is characterized histopathologically by the presence of large numbers of neuritic plaques (NPs) and cytoskeletal changes that are present as pretangles after staining— that is, by Alz-50 or AT8, or as neurofibrillary tangles (NFT) after silver staining. NFT are present in the cell bodies of affected neurons, while the same cytoskeletal changes are called neuropil threads when present outside neuritic plaques or dystrophic neurites when they are the neuritic components of neuritic plaques (NPs). Dystrophic neurites or neuropil threads are short, thickened, curly, coiled, or sometimes hooked fibres. To a lesser degree, NPs and cytoskeletal changes can also be observed in aged, nondemented control subjects. This chapter provides evidence in favor of the hypotheses that (1) the neuropathological Alzheimer changes cannot all be explained by a cascade starting with amyloid (β/A4) deposits as suggested example by Selkoe, but that (2) the neuropathological hallmarks of Alzheimers disease are basically independent phenomena, and that (3) cell death in Alzheimers disease is not a major generally occurring phenomenon, but is restricted to a few brain areas. A long struggle has been going on about the question what is more important for the development of dementia: cytoskeletal changes or amyloid. The answer is probably neither. As it is argued in this chapter, (4) reduced neuronal activity is most probably one of the major characteristics of AD and may underlie the clinical symptoms of dementia. Experiments are discussed that indicate that it is attractive to direct therapeutic strategies towards restimulation of neuronal metabolism and repair mechanisms in order to improve cognitive and behavioral symptoms of AD.