Mature astrocytes in the adult human neocortex express the early neuronal marker doublecortin.

Doublecortin (DCX) is a microtubule-associated protein expressed by migrating neuroblasts and is considered to be a reliable marker of neurogenesis. DCX has been used to study the relation between neurogenesis in adult human brain and neurological and neurodegenerative disease processes in the search for putative therapeutic strategies. Using autopsy and surgically resected tissue from a total of 60 patients, we present evidence that DCX is present in several cellular compartments of differentiated astrocytes in the adult human neocortex. One of these compartments consisted of peripheral processes forming punctate envelopes around mature neuronal cell bodies. Markers of glial activation, such as GFAP and HLA, were not associated with DCX immunoreactivity, however, the presence of cytoarchitectural alterations tended to correlate with reduced DCX staining of astrocytic somata. Interestingly, local Alzheimer pathology that showed no relation with cytoarchitectural abnormalities appeared to correlate negatively with the expression of DCX in the astrocytic somata. In combination with the literature our data support the view that DCX in the adult human neocortex may have a function in glia-to-neuron communication. Furthermore, our results indicate that in the adult human neocortex DCX is neither a reliable nor a selective marker of neurogenesis.

Prefrontal alterations in GABAergic and glutamatergic gene expression in relation to depression and suicide.

People that committed suicide were reported to have enhanced levels of gene transcripts for synaptic proteins in their prefrontal cortex (PFC). Given the close association of suicide with major depressive disorder (MDD), we here assessed whether these changes are related to suicide or rather to depression per se. We used quantitative PCR to determine mRNA levels of 32 genes encoding for proteins directly involved in glutamatergic or GABAergic synaptic transmission in postmortem samples of the anterior cingulate cortex (ACC) and the dorsolateral PFC (DLPFC). Seventy-two brain samples from 3 groups of subjects were derived from the Stanley Medical Research Institute (SMRI): i) patients with MDD who committed suicide (MDD-S), ii) MDD patients who died of non-suicidal causes (MDD-NS) and iii) age-matched, non-psychiatric control subjects. In the ACC, a significantly enhanced expression of genes related to glutamatergic or GABAergic synaptic transmission was found only in MDD-S patients, whereas in MDD-NS patients, decreased levels for these transcripts were found. Moreover, in the DLPFC, expression of these genes was decreased in MDD-S, relative to MDD-NS patients, whereas both groups showed increased expression compared to control subjects. In conclusion, our findings indicate that MDD is associated with increases in GABA and glutamate related genes in the DLPFC (irrespective of suicide), while in the ACC, the increase in GABA and glutamate related genes may relate to suicide, rather than to MDD per se.

Prefrontal changes in the glutamate-glutamine cycle and neuronal/glial glutamate transporters in depression with and without suicide.

There are indications for changes in glutamate metabolism in relation to depression or suicide. The glutamate-glutamine cycle and neuronal/glial glutamate transporters mediate the uptake of the glutamate and glutamine. The expression of various components of the glutamate-glutamine cycle and the neuronal/glial glutamate transporters was determined by qPCR in postmortem prefrontal cortex. The anterior cingulate cortex (ACC) and the dorsolateral prefrontal cortex (DLPFC) were selected from young MDD patients who had committed suicide (MDD-S; n = 17), from MDD patients who died of non-suicide related causes (MDD-NS; n = 7) and from matched control subjects (n = 12). We also compared elderly depressed patients who had not committed suicide (n = 14) with matched control subjects (n = 22). We found that neuronal located components (EAAT3, EAAT4, ASCT1, SNAT1, SNAT2) of the glutamate-glutamine cycle were increased in the ACC while the astroglia located components (EAAT1, EAAT2, GLUL) were decreased in the DLPFC of MDD-S patients. In contrast, most of the components in the cycle were increased in the DLPFC of MDD-NS patients. In conclusion, the glutamate-glutamine cycle - and thus glutamine transmission - is differentially affected in depressed suicide patients and depressed non-suicide patients in an area specific way.

ApoE epsilon4 genotype is accompanied by lower metabolic activity in nucleus basalis of Meynert neurons in Alzheimer patients and controls as indicated by the size of the Golgi apparatus.

We previously found apolipoprotein (apoE) epsilon4-dependent lower metabolic activity in nucleus basalis of Meynert (NBM) neurons in Alzheimer disease (AD). In the present study we examined the metabolic activity in the NBM of 39 mentally intact control subjects with different APOE genotype. The control subjects had either no AD pathology (Braak stage 0) or the very beginning of AD pathology (Braak stage I-II). We used the Golgi apparatus (GA) size as a measure of neuronal metabolic activity. Control subjects carrying an apoE epsilon4 allele showed reduced neuronal metabolism; they had significantly more neurons with smaller GA sizes compared to control subjects not carrying an apoE epsilon4 allele. Only control subjects not carrying an apoE epsilon4 allele had increased neuronal metabolism in Braak I-II subjects. They had more neurons with larger GA sizes compared to Braak 0 subjects, which may reflect a compensatory mechanism. Our data indicate that APOE epsilon4 may act by a lower neuronal metabolism as a risk factor for cognitive impairment in normal aging and early prodromal AD. As the disease progresses into later stages of AD (Braak V-VI) neuronal metabolism strongly diminishes, resulting in neurons with extremely small GA sizes, irrespective of APOE genotype.

Postnatal development of amygdaloid projections to the prefrontal cortex in the rat studied with retrograde and anterograde tracers.

The prefrontal cortex (PFC) and the amygdala are involved in a number of common functions, such as emotional and social behavior, stress, visceral functions, ingestive behavior, self-stimulation, and certain aspects of learning and memory. The amygdala massively projects to the PFC and may play a role in the developmental plasticity reported for several of these functions. We have studied the normal postnatal development of the amygdaloid projections to the rat prefrontal cortex by using the retrogradely transported fluorescent dye fast blue and the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). Shortly after birth some fibers were observed in the frontal pole of the rat brain. These fibers were scattered throughout all prefrontal cortical areas. The majority of the amygdaloid cells contributing to this pattern at that stage of development were located in the anterior and ventral basolateral nuclei, whereas a minority were located in the posterior basolateral nucleus. The transition from a diffuse fiber distribution to a characteristic bilaminar pattern occurred around postnatal day 12 in the lateral and rostral medial PFC. The PHA-L injections confirmed the existence of a topographical organization of the amygdalo-prefrontocortical projections. Our observations suggest that the development of amygdala innervation of the PFC parallels the emergence of PFC cytoarchitectural organization.

Age-related increase in the total number of corticotropin-releasing hormone neurons in the human paraventricular nucleus in controls and Alzheimer's disease: comparison of the disector with an unfolding method.

It has been hypothesized that the corticotropin-releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN) become hyperactive with age, and even more so in Alzheimer's disease. This hyperactivity could be due to an increased production of CRH per neuron, or an increased number of PVN neurons producing CRH, or both. As a first step in elucidating which of these biological mechanisms might be operative, we have estimated the absolute number of CRH immunoreactive neurons in the PVN of 10 human control subjects between 36 and 91 years of age and 10 Alzheimer patients between 40 and 97 years of age. CRH neurons were immunocytochemically detected in 6 microns paraffin sections with the aid of a highly specific monoclonal antibody to CRH. The antibody signal was amplified by the biotin-streptavidin and alkaline phosphatase methods. The absolute number of CRH neurons in the PVN was obtained by multiplying the number of CRH neurons in a unit volume (NV) by the total volume of the PVN. Two different methods were used to estimate the NV: an unfolding method and a disector method (about three times more time-consuming). Compared to the disector, the unfolding method consistently yielded a lower cell number for all patients by 38% (+/- 2.8%; mean +/- SEM). However, both methods yielded an increase in the absolute number of CRH neurons in control and Alzheimer patients with age. No statistically significant difference in the absolute number of CRH neurons was found between control and Alzheimer patients with both methods. The age-dependent increase in the absolute number of CRH neurons within the PVN of both control and Alzheimer patients is interpreted as a sign of activation of the CRH neurons with age.

Azathioprine and prednisolone immunosuppression versus maintenance triple therapy including cyclosporine for orthotopic liver transplantation. A comparative biochemical and histologic study in one center.

Improvement of graft survival after orthotopic liver transplantation is often attributed to cyclosporine. In order to assess the effects on liver function and histology, we compared the results of conventional immunosuppression (azathioprine/prednisolone = group I) and a triple drug regimen, which included CsA (group II) during the first year after transplantation. Group I consisted of 33 patients; group II of 18 patients. Significant differences are present in favor of the CsA regimen with regard to transaminases and cholestatic parameters. Liver synthesis function was slightly better, though already very good under conventional immunosuppression. One week after transplantation, normal histology was not observed in group I, while 90% of the patients showed acute rejection. In group II, 53% of the patients showed normal histology; only 40% of the patients in group II showed acute rejection (P less than 0.0002). One year after transplantation, liver histology was normal in 57% of the conventionally treated patients and in 90% of the CsA-treated patients. Also, less rejection occurred in group II during the first year after transplantation. One-year graft survival was 67% in group I and in group II 75%, which is not statistically different. Creatinine clearance did not differ in both groups. However compared with pretransplantation creatinine clearances, kidney function in the CsA-treated patients decreased with approximately 20 ml/min. These results show that liver synthesis and liver function are better under the CsA-containing triple-drug-maintenance regimen, which is supported by the far better liver histology. Kidney function is reduced, even under low dose CsA treatment.

Synaptic characteristics of identified pyramidal and multipolar non-pyramidal neurons in the visual cortex of young and adult rabbits. A quantitative Golgi-electron microscope study.

The visual cortex of 20 day old rats and rabbits has been considered as mature on the basis of the observations that the dendritic arborization and the overall synaptic population have almost reached their adult stage in these animals. In the present study we have investigated the visual cortex of 20 day and 7 month old (adult) rabbits in order to determine whether this apparent adult appearance also holds for the synaptic organization of individual neurons. Neurons mainly located in layers III and IV of the primary visual cortex (area 17) were Golgi-impregnated, gold toned and deimpregnated and were then, after embedding in plastic, sectioned serially. The number and length of synaptic profiles, and the length of the neuronal boundaries were analysed in every tenth section. From these counts and measurements the size distribution of the synaptic discs, the number of synapses per 100 micron2 neuronal surface and the receptive surface expressed as the percentage of the total neuronal surface covered with synaptic contacts were estimated using stereological methods. At both ages studied, the density of synapses was significantly higher for the non-pyramidal neurons than for the pyramidal neurons. Differences in the amount of receptive surface were parallel to the differences observed for the number of synapses per 100 micron2. At day 20 the receptive surface of the non-pyramidal neurons was significantly larger than that of the pyramidal neurons. The receptive surface of the non-pyramidal neurons in the adult stage was not only larger than that of the pyramidal neurons in the adults, but also larger than that of the day 20 non-pyramidal neurons. From our results the following conclusions can be drawn: (1) The synaptic input received by the pyramidal neurons is mainly established at day 20 of postnatal life, i.e. prior to the establishment of adult visual behaviour. (2) The non-pyramidal neurons complete their maturation in a later stage than the pyramidal neurons. (3) Medium to large sized synaptic contacts are newly formed after day 20 and are mainly added to the synaptic population on dendrites of non-pyramidal neurons. (4) The specific increase in the number of synapses on non-pyramidal neurons is discussed in relation to intracortical inhibition which is thought to be important for the fine regulation of visual function during development.

Prediction of uneventful cardioversion and maintenance of sinus rhythm from direct-current electrical cardioversion of chronic atrial fibrillation and flutter.

The present study was undertaken to reassess prospectively the immediate and long-term results of direct-current electrical cardioversion in chronic atrial fibrillation or atrial flutter, and to determine factors predicting clinical outcome of the arrhythmia after direct-current cardioversion. Two-hundred forty-six patients underwent direct-current electrical cardioversion and were followed during a mean of 260 days. Multivariate analysis was used to identify factors predicting short- and long-term arrhythmia outcome. Cardioversion was achieved in 70% of patients with atrial fibrillation and in 96% of patients with atrial flutter. Stepwise logistic regression analysis revealed that arrhythmia duration (p less than 0.001), type of arrhythmia (fibrillation vs flutter, p less than 0.02) and age (p less than 0.05) independently influenced conversion rate. On an actuarial basis, 42 and 36% of patients remained in sinus rhythm during 1 and 2 years, respectively. Multivariate regression analysis revealed that the type of arrhythmia (p = 0.0008), low precardioversion functional class (p = 0.002) and the presence of nonrheumatic mitral valve disease (p = 0.03) independently increased the length of the arrhythmia-free episode. Rheumatic heart disease shortened this period (p = 0.03). In conclusion, patients having a high probability of conversion together with a prolonged post-shock arrhythmia-free episode can be identified. This may improve the cost-benefit ratio of cardioversion.

Post-mortem brain tissue cultures from elderly control subjects and patients with a neurodegenerative disease.

Aging may be viewed as a progressive loss of normal biological function. Due to complex genetic and environmental interactions, the sequence of functional impairment shows a high degree of individual variability. In humans life style and health care have an additional influence on the aging process. To study aging and age-related disorders of the human nervous system, brain tissue that has undergone aging and pathological alterations can provide valuable study material. Recently, we have shown that adult human postmortem brain tissue can be cultured and experimentally manipulated. This approach permits the study of cellular aspects of human neuronal aging and neurodegenerative processes and complements those existing research methods such as in vivo imaging (MRI, PET, etc.) and fixed or frozen postmortem brain tissue examination.

The magnocellular neurons of the hypothalamo-neurohypophyseal system display remarkable neuropeptidergic phenotypes leading to novel insights in neuronal cell biology.

For decades the magnocellular neurons of the hypothalamo-neurophypophyseal system (HNS), in which either vasopressin or oxytocin are produced and released into the bloodstream, have been playing a pivotal role in fundamental discoveries in the nervous system. The primary structure of vasopressin and oxytocin was the first of all neuropeptides to be published, i.e., in the 1950s by the Nobel prize laureate Du Vigneaud. Moreover, many trend-setting discoveries have their origin in the HNS, which abundantly expresses vasopressin and oxytocin, clearly displays its function and is relatively easily to manipulate. Examples are the phenomenon of coexpression of neuropeptides, patch-clamping of nerve endings, axonal transport of RNA, neuroglia interactions and the behavioral effects. An extraordinarily intriguing example is the homozygous Brattleboro rat, which lacks vasopressin by a germ-line mutation, and has disclosed many of the fundamental characteristics of peptidergic neurons, and neurons in general. In this chapter we will discuss a few of them, in particular the recent data on mutations in vasopressin RNA. It is to be expected that the HNS will retain its informative role in the next decades.

Reduced neuronal activity and reactivation in Alzheimer's disease.

1. Alzheimer's disease is a multifactorial disease in which age and APOE-epsilon 4 are important risk factors. Various mutations and even viral infections such as herpes simplex (Itzhaki et al., 1997) may play an additional role. 2. The neuropathological hallmarks of Alzheimer's disease (AD), i.e. amorphous plaques, neuritic plaques (NPs), pretangles, neurofibrillary tangles (NFT) and cell death are not part of a single pathogenetic cascade but are basically independent phenomena. 3. Pretangles can occur in neurons from which the metabolic rate is not altered. However, in brain areas where classical AD changes, i.e. NPs and NFTs, are present, such as the CA1 area of the hippocampus, the nucleus basalis of Meynert and the tuberomamillary nucleus, a decreased metabolic rate is found. Decreased metabolic rate appears to be an independent phenomenon in Alzheimer's disease. It is not induced by the presence of pretangles, NFT or NPs. 4. Decreased metabolic rate may precede cognitive impairment and is thus an early occurring hallmark of Alzheimer's disease, which, in principle, may be reversible. The observation that the administration of glucose or insulin enhances memory in Alzheimer patients also supports the view that Alzheimer's disease is basically a metabolic disease. Moreover, several observations indicate that activated neurons are better able to withstand aging and AD, a phenomenon paraphrased by us as "use it or lose it". It is, therefore, attractive to direct the development of therapeutic strategies towards restimulation of neuronal metabolic rate in order to improve cognition and other symptoms in Alzheimer's disease. A number of pharmacological and non-pharmacological studies support the concept that activation of the brain indeed has beneficial effects on several aspects of cognition and other central functions.

Brain aging and Alzheimer's disease; use it or lose it.

(1) Alzheimer's disease is a multifactorial disease in which age and APOE-epsilon 4 are important risk factors. (2) The neuropathological hallmarks of AD, i.e. amorphous plaques, neuritic plaques (NPs), pretangles, neurofibrillary tangles (NFT) and cell death are not part of a single pathogenetic cascade but may occur independently. (3) In brain areas where classical AD changes, i.e. NPs and NFTs, are present, such as the CA1 area of the hippocampus, the nucleus basalis of Meynert and the tuberomamillary nucleus, a decreased metabolic rate is found. The decreased metabolic rate appears not to be induced by the presence of pretangles, NFT or NPs. (4) Decreased metabolic rate may precede cognitive impairment and is thus an early occurring hallmark of AD, which, in principle, may be reversible. The observation that the administration of glucose or insulin enhances memory in AD patients also supports the view that AD has a metabolic basis. (5) Moreover, several observations in postmortem brain indicate that activated neurons are better able to withstand aging and AD, a phenomenon paraphrased by us as 'use it or lose it'. (6) It is, therefore, attractive to direct the development of therapeutic strategies towards restimulation of neuronal metabolic rate in order to improve cognition and other symptoms in AD. A number of pharmacological and non-pharmacological studies support the concept that activation of the brain has beneficial effects and may, to a certain degree, restore several aspects of cognition and other central functions. For instance, the circadian system may be restimulated in AD patients by exposing them to more light or transcutaneous nerve stimulation. A procedure has been developed to culture human postmortem brain tissue that allows testing of the efficacy of putative stimulatory compounds such as neurotrophins.

Successful pregnancy after orthotopic liver transplantation.

A successful pregnancy occurred in the first year after liver transplantation. The patient was treated with prednisolone, azathioprine, and low-dose cyclosporine A. A healthy girl of 2260 g (fifth to tenth percentile) was born at 38 weeks.

Descriptive and comparative analysis of geometrical properties of neuronal tree structures.

The morphology of neurons is an important factor for the identification and the study of the changes that occur in the nervous system during development or as a result of disease or an experimental treatment. A number of methods to describe the topological aspects of neuronal morphology is discussed. Furthermore it is illustrated how different groups of neurons can be compared. Although both topological and metrical aspects are considered in the comparative sections emphasis is put on counting instead of measuring. Our intention is to present quick and easy methods that are applicable to camera lucida drawings.

A new method for the topological analysis of neuronal tree structures.

Statistical analysis of the frequencies of observed branching patterns of neuronal arborescences is an important means of studying neuronal growth and of characterizing axonal or dendritic populations. We recently derived simple formulae for the exact probabilities of occurrence of types of neuronal trees for both segmental and terminal growth. Additionally, the existence of a natural ordering of the neuronal tree types enables the application of the Kolmogorov goodness-of-fit test. In the present report it is illustrated how these facilities can be incorporated in the analysis of neuronal arborizations. Interesting features are that very large neuronal arborizations can be analyzed completely and that only small sample sizes are required for the estimation of the critical level corresponding to the growth hypothesis. Further, it is indicated how populations of neuronal tree structures may be compared with each other without reference to a particular growth theory.

Application of growth models to the topology of neuronal branching patterns.

The variation in topological structure of branching patterns may contain essential information with respect to the way these branching patterns have grown. For the understanding of how growth modes finally result in a particular variety in topological patterns, model studies may provide indispensible tools. These studies imply the mathematical formulation of growth models and the development of statistical procedures to compare model predictions with observed data. Recent literature shows two main approached in these model studies, viz. subtree partition analysis (SPA) and vertex analysis. This paper will briefly review the current status with respect to SPA and will apply the model approach to sets of dendritic trees taken from pyramidal, multipolar non-pyramidal and from Purkinje cells. The results show that the topological properties of many dendrites are not in agreement with the hypothesis of random terminal growth and that substantial branching of intermediate segments and/or branching dependent of the position of segments in the tree (topological distance from the cell body) must be assumed. Only two parameters are required to incorporate these assumptions in the model. In all cases up to now it is possible to find parameter values such that the model predictions of topological properties are in agreement with the observations.

A simple statistical test for the vertex ratio using Monte Carlo simulation.

The vertex ratio is the crucial quantity in vertex analysis, which is a method to characterize the mode of growth of neuronal tree structures (i.e. dendrites and axons). In this report we propose the use of the Monte Carlo test to calculate a level of significance for the vertex ratio. As a result the vertex ratio can be used to analyse neuronal trees with respect to a range of growth hypotheses, including terminal and segmental growth.

Sex differences in effects of environmental stimulation on brain weight of previously undernourished rats.

Neonatally undernourished male and female rats were housed under differential (enriched vs standard) environmental conditions during refeeding from 30 days post-partum (dpp). Animals were killed at 170 dpp and whole brain, forebrain and cerebellar weights compared with those of normally-fed, differentially housed control animals. A sex difference was observed in the response to environmental enrichment. Enriched females showed significantly increased brain weights, irrespective of previous nutritional history. Enriched males failed to show any significant alteration in brain weight. Autopsy results indicated that the enriched condition was stressful to males but not to females. The results cast doubt on the notion that infantile undernutrition may limit later effects of differential experience on the brain.

Spatial delayed alternation of rats in a T-maze: effects of neurotoxic lesions of the medial prefrontal cortex and of T-maze rotations.

The medial prefrontal cortex (mPFC) is usually considered to be a brain area important for working memory processes. In rats this statement is evidenced by their diminished performance in delay-type tasks following mPFC damage, notably in spatial delayed alternation (SDA) in a T-maze. This study has addressed two questions. First, to examine whether the functional deficiency in SDA, observed in rats with (usually large) mPFC damage, can be ascribed to an anatomically defined subarea of mPFC, the dorsal anterior cingulate area (ACd). Small, bilateral, NMDA-induced lesions were made, restricted to the dorsal part of mPFC. The performance of such animals in a T-maze paradigm, using delays of 0 and 15 s, was compared with sham-operated animals. Although these small lesions resulted in an increased number of perseverative errors, this effect was not delay-dependent, and, moreover, by the end of the training group differences had disappeared. The second aim was to study whether or not spatial (extra-maze) cues are important for the performance of this task. This was achieved by subjecting the well-trained sham-operated animals to a series of systematic trial-to-trial variations in the position of the maze in the experimental room. These spatial manipulations severely impaired the performance of the SDA task, indicating that extra-maze information is required to solve this task. In animals with ACd lesions, subjected to the same manipulations, the deficiency was comparable to that of the sham-operated animals.