Norbert Ulfig

Monoclonal antibodies SMI 311 and SMI 312 as tools to investigate the maturation of nerve cells and axonal patterns in human fetal brain

Abstract Neurofilaments, which are exclusively found in nerve cells, are one of the earliest recognizable features of the maturing nervous system. The differential distribution of neurofilament proteins in varying degrees of phosphorylation within a neuron provides the possibility of selectively demonstrating either somata and dendrites or axons. Non-phosphorylated neurofilaments typical of somata and dendrites can be visualized with the aid of monoclonal antibody SMI 311, whereas antibody SMI 312 is directed against highly phosphorylated axonal epitopes of neurofilaments. The maturation of neuronal types, the development of area-specific axonal networks, and the gradients of maturation can thus be demonstrated. Optimal immunostaining with SMI 311 and SMI 312 is achieved when specimens are fixed in a mixture of paraformaldehyde and picric acid for up to 3 days and sections are incubated free-floating. Neurons, with their dendritic domains immunostained by SMI 311 in a Golgi-like manner, can be completely visualized in relatively thick sections. The limitations of Golgi-preparations, such as glia-labeling, artifacts, and the staining of only a small non-representative percentage of existing neurons, are not apparent in SMI preparations, which additionally provide the possibility of selectively staining axonal networks. The results achieved in normal fetal brain provide the basis for studies of developmental disturbances.

Ontogeny of the human amygdala.

Abstract: Data on the fetal development of the human amygdala is reviewed with special reference to major ontogenetic events. In the fifth gestational month, the inferior portion of the amygdala reveals cell‐dense columns merging with the ganglionic eminence (proliferative zone) in Nissl‐stained sections. These columns contain vimentin‐positive fibers and can therefore be regarded as migrational routes. In the sixth and seventh months, distinct reorganization of the cytoarchitectonics takes place. The sequential occurrence of afferens can be visualized using anti‐GAP‐43; moreover, outgrowing axons appear to reach the periphery of the ganglionic eminence. The latter may thus represent an intermediate target for growing axons using anti‐calbindin and anti‐calretinin. Migrating and immature amygdaloid neurons can be shown in the fifth month. From the eighth month onwards, various nonpyramidal neurons and pyramidal neurons are immunolabeled. Transient expression of calretinin in pyramidal neurons is observed. When punctate calbindin and calretinin immunostaining in the fifth and eighth months is compared, distinct redistribution is observed. On the whole, it is apparent that the amygdala has reached a high degree of maturity in the eighth month. At this developmental stage, AKAP79, being enriched in postsynaptic densities, shows a characteristic nuclear‐specific distribution pattern. The latter largely corresponds to the expression pattern of NMDAR1. Thus, AKAP79 may have a preference for anchoring enzymes to glutamate receptors. The aforementioned results provide a basis for investigations on subtle changes in pathologically altered material, such as hemorrhage, in the ganglionic eminence of preterm infants.

Normal and Abnormal Development of the Human Cerebral Cortex

The functional organization of the developing human brain does not only differ substantially from that of the mature brain, but it also undergoes continuous changes. During fetal brain development, transient neuronal circuitries are formed which are essential for the subsequent development of mature projections. Transitory connections are linked to transient structures which are particularly prominent in the human fetal brain and are susceptible to damage under pathological circumstances. The First International Symposium on Normal and Abnormal Development of the Human Fetal Brain aimed to gather basic information on these transitory organizations and the major developmental events that occur in the fetal brain. This paper summarizes a roundtable discussion at the symposium on transient characteristics and injuries in the developing human cerebral cortex. An overview of cortical development is first presented and then the development of several fetal structures, including the subventricular zone, ganglionic eminence, marginal zone, subplate and cortical plate, is discussed with regard to their important contribution to the formation of the correct fiber projections in the adult. The last section focuses on the anomalies that are commonly found in the premature fetal brain on the one hand and on the other hand are related to the transitory characteristics of the developing brain.

Ganglionic eminence of the human fetal brain—new vistas

This review deals with recent findings concerning the complex functions of the ganglionic eminence (GE), which represents a conspicuous domain of the telencephalic proliferative zone and persists nearly throughout fetal life. The GE not only contains precursor neurons of the basal ganglia, it also contributes significantly to the population of interneurons in the cerebral cortex and to a population of thalamic neurons. The latter migrate through a distinct transient structure, the gangliothalamic body (GTB). The GE also represents an intermediate target for growing thalamic axons (on their way to the cerebral cortex) and cortical axons (on their way to the thalamus). In developmental neuropathology the GE plays an important role in prematurely born infants. The pathogenesis of GE bleedings is discussed with regard to the abundant expression of interleukin‐6 (IL‐6) receptors on GE cells. The consequences of such bleedings are discussed in view of cellular responses, such as the induction of leukemia inhibitory factor (LIF) expression in GE cells after hemorrhage. Anat Rec 267:191–195, 2002.

Brain macrophages and microglia in human fetal hydrocephalus

Whereas several studies have addressed the activation of microglia (the resident mononuclear phagocytes of the brain) and macrophages within the nervous system in experimental animal models of congenital and induced hydrocephalus, little is known of their state of activation or regional distribution in human fetal hydrocephalus. This investigation aimed to address such questions. Ten human fetal cases [20-36 gestational weeks (GW) at postmortem] previously diagnosed with hydrocephalus on ultrasound examination in utero, and 10 non-hydrocephalic controls (22-38 GW at postmortem) were assessed immufcnohistochemically with antibodies directed against MHC class II and CD68 antigens, and lectin histochemistry with Lycopersicon esculentum (tomato lectin). Adjacent sections were also immunoreacted with an antiserum to laminin to detect cerebral blood vessels. Eight out of the 10 hydrocephalus cases showed numerous CD68 and tomato lectin-positive macrophages located at focal regions along the ependymal lining of the lateral ventricles (particularly within the occipital horn). However, only five of these cases demonstrated MHC class II positive macrophages associated with the ventricular lining. Microglial reactivity within periventricular regions could also be identified using the lectin in four cases, two of which were also immunoreactive with CD68 (but not with MHC class II). By comparison, in control cases five out of 10 fetal brains (aged between 20 and 24 GW) showed few or no ependymal or supraependymal macrophages. One case at 28 GW, and cases at 32 and 38 GW (two of which were diagnosed with intrauterine hypoxic-ischemia) did, however, show some MHC class II (CD68 negative) cells located at the ependymal surface. Nevertheless, these were not as numerous or intensely immunoreactive as in the hydrocephalus cases. Microglia interspersed throughout the intermediate zone and circumscribing the basal ganglia were within normal confines in all cases examined. Hydrocephalic cases additionally showed focal regions of hypovascularization or alterations in the structure and orientation of capillaries within periventricular areas, compared to controls. The macrophage response detected at the ependymal lining of the ventricles and within the periventricular area in hydrocephalus may be related both to the severity of hydrocephalus and the age of the fetus.

Mean cell spacing abnormalities in the neocortex of patients with schizophrenia

It has been postulated that the prefrontal cortices of schizophrenic patients have significant alterations in their interneuronal (neuropil) space. The present study re-examines this finding based on measurements of mean cell spacing within the cell minicolumn. The population studied consisted of 13 male schizophrenic patients (DSM-IV criteria) and 13 age-matched controls. Photomicrographs of Brodmanns areas 9, 4 (M1), 3b (S1), and 17 (V1) were analyzed with computerized image analysis to measure parameters of minicolumnar morphometry, i.e., columnarity index (CI), minicolumnar width (CW), dispersion of minicolumnar width (V(CW)), and mean interneuronal distance (MCS). The results indicate alterations in the mean cell spacing of schizophrenic patients according to both the lamina and cortical area examined. The lack of variation in the columnarity index argues in favor of a defect postdating the formation of the cell minicolumn.

Microvessel length density, total length, and length per neuron in five subcortical regions in schizophrenia

Recent studies (Prabakaran et al. in Mol Psychiat 9:684–697, 2004; Hanson and Gottesman in BMC Med Genet 6:7, 2005; Harris et al. in PLoS ONE 3:e3964, 2008) have suggested that microvascular abnormalities occur in the brains of patients with schizophrenia. To assess the integrity of the microvasculature in subcortical brain regions in schizophrenia, we investigated the microvessel length density, total microvessel length, and microvessel length per neuron using design-based stereologic methods in the caudate nucleus, putamen, nucleus accumbens, mediodorsal nucleus of the thalamus, and lateral nucleus of the amygdala in both hemispheres of 13 postmortem brains from male patients with schizophrenia and 13 age-matched male controls. A general linear model multivariate analysis of variance with diagnosis and hemisphere as fixed factors and illness duration (patients with schizophrenia) or age (controls), postmortem interval and fixation time as covariates showed no statistically significant differences in the brains from the patients with schizophrenia compared to the controls. These data extend our earlier findings in prefrontal cortex area 9 and anterior cingulate cortex area 24 from the same brains (Kreczmanski et al. in Acta Neuropathol 109:510–518, 2005), that alterations in microvessel length density, total length, and particularly length per neuron cannot be considered characteristic features of schizophrenia. As such, compromised brain metabolism and occurrence of oxidative stress in the brains of patients with schizophrenia are likely caused by other mechanisms such as functional disruption in the coupling of cerebral blood flow to neuronal metabolic needs.

Differential expression of calbindin and calretinin in the human fetal amygdala.

The distribution patterns of the calcium‐binding proteins calbindin and calretinin, both expressed early during development within the various amygdaloid nuclei and areas, have been investigated. Anti‐calbindin as well as anti‐calretinin mark immature, partly migrating neurons in the 5th gestational month; the number of calretinin‐immunoreactive neurons is distinctly higher. In the 8th month, calbindin and calretinin are found in a small proportion of presumed pyramidal cells and in various types of non‐pyramidal neurons. Small and large bipolar and small and large multipolar neurons are shown to express calbindin and calretinin. Double‐labellings show that calbindin and calretinin are largely contained in different subsets of these neuronal types, which are considered to represent interneurons. These nerve cell classes are widespread within the amygdala with mainly moderate to high packing densities. Diffuse immunoreactive structures, which are found in different intensities in the various amygdaloid nuclei, display distinct redistribution during fetal development. The results show that during early fetal development calbindin and particularly calretinin may be involved in the regulation of neuronal migration. In later development, definite subsets of interneurons, which are likely to be functionally different, are marked by anti‐calbindin and ‐calretinin. Different diffuse immunolabelling at various developmental stages probably indicates the sequential arrival of afferent input from brain areas containing calbindin‐ or calretinin‐immunoreactive nerve cells. With the exception that calretinin may be transiently expressed in pyramidal neurons, the distribution of calbindin‐ and calretinin‐immunoreactive structures to a large degree corresponds to that in the adult. Thus, little reorganisation is to be expected during proceeding development. Microsc. Res. Tech. 46:1–17, 1999.

Expression of calbindin and calretinin in the human ganglionic eminence

The conspicuous ganglionic eminence representing a part of the telencephalic proliferative zone contains neuroblasts of the striatum. Recently it has been found to contribute significantly to the class of interneurons in the cerebral cortex. Subpopulations of cortical interneurons contain calretinin and calbindin. The expression of calretinin and calbindin in the ganglionic eminence and adjacent areas has been investigated immunocytochemically during fetal development using the brains of 10 infants ranging in age from 16 and 26 weeks gestation. Between 16 and 20 weeks gestation, numerous calretinin-immunreactive nerve cells are found in the ganglionic eminence, particularly in the mantle and the intermediate zones. The number of calretinin-immunoreactive cells decreases gradually from 21 weeks gestation onwards. Larger calbindin-immunoreactive cells are seen in the ganglionic eminence, and their number increases from 20 weeks gestation in the mantle zone. These results may indicate that calretinin-immunoreactive precursor cells, found in the ganglionic eminence, tangentially migrate toward the cortex. Moreover, the mantle zone displaying a specific calretinin and calbindin immunolabeling may represent an intermediate target for outgrowing axons. The findings are discussed with regard to central nervous system complications in preterm infants involving the ganglionic eminence.

Distribution patterns of vimentin-immunoreactive structures in the human prosencephalon during the second half of gestation

Neuronal migration is guided by long radially oriented glial fibres. During late stages of development radial glial cells are transformed into astrocytes. A predominant intermediate filament protein within radial glial cells and immature astrocytes is vimentin. In this study fetal brain sections were used to demonstrate the transient features of vimentin‐positive radial glia. In the lower half of the cerebral wall of the 6th gestational month bundles, curvature, and crossing of vimentin‐positive fibres are regularly seen. Moreover, fibres terminating on vessels are observed. In the upper half fibres are radially oriented; when ascending towards the pial surface the number and diameter of fibres appears conspicuously decreased. Radially aligned fibres display numerous varicosities. In the 8th month the bulk of vimentin‐positive fibres is encountered next to the ganglionic eminence and below isocortical cerebral fissures. The dentate gyrus is conspicuous due to its high amount of immunolabelled fibres. Furthermore, densely packed fibres are visible within the internal and external capsule and in the vicinity of the anterior commissure. Radial glial somata are found in the proliferative areas as well as in the adjacent white matter. In the latter location bipolar, monopolar and stellate vimentin‐positive cells are present. The results demonstrate an area‐specific distribution pattern of vimentin‐positive structures which can be correlated with migrational events. Areas maturing late in development for instance, reveal dense immunolabelling in the 8th month. The orientation and position of radial fibres point to an additional developmental role of these fibres, i.e. their involvement in the guidance of growing axons. Moreover, the arrangement and morphology of vimentin‐positive fibres, such as retraction of fibres or occurrence of varicosities, are indicative of degenerative events. Accordingly, a transformation of radial glial somata, their displacement towards the white matter and finally the growth of stellate processes can clearly be demonstrated.