Neural histology and neurogenesis of the human fetal and infant brain.

The human brain develops slowly and over a long period of time which lasts for almost three decades. This enables good spatio-temporal resolution of histogenetic and neurogenetic events as well as an appropriate and clinically relevant timing of these events. In order to successfully apply in vivo neuroimaging data, in analyzing both the normal brain development and the neurodevelopmental origin of major neurological and mental disorders, it is important to correlate these neuroimaging data with the existing data on morphogenetic, histogenetic and neurogenetic events. Furthermore, when performing such correlation, the genetic, genomic, and molecular biology data on phenotypic specification of developing brain regions, areas and neurons should also be included. In this review, we focus on early developmental periods (form 8 postconceptional weeks to the second postnatal year) and describe the microstructural organization and neural circuitry elements of the fetal and early postnatal human cerebrum.

Suppression of Smad-1 mRNA expression level by Smad-2 likely control dichotomy of NF-κB and Smads mediated activation.

The aim of this study was to find out how NF-κB and Smad-mediated signaling influenced the expression of astrogliogenic versus neurogenic markers of brain development in U4C cells which were either enriched (Tg Jak-1) or deprived in Jak-1 molecule (Jak-1 KO). Genetically modified U4C cells were transfected with NF-kB reporter plasmid in order to follow its activation when cells were cotransfected with different combinations of Smads constructs. In wild type cells no significant activation of NF-κB was observed while genetically modified cells exhibited somewhat different pattern of NF-κB activation depending on the Smad constructs combination used. The absence of NF-κB activation in Jak-1 transgenic cells transfected with Smad-1 plus Smad-3 was accompanied by the appearance of apoptotic cells as revealed by DAPI staining. Smad-1 expression was undetectable in Jak-1 transgenic cells and was downregulated in wild type cells upon transfection with Smad-2. The absence of p65 nuclear translocation in Smad-2 transfected cells and the presence of Smad-4 in nucleus of the same cells indicates dichotomy in NF-κB and Smads mediated signaling pathways. The significance of this study is that helps to elucidate the point of collaboration among three different signaling pathways - Jak-1 mediated cytokine signaling, NF-κB and Smads mediated pathways.

Does Alzheimer's disease begin in the brainstem?

Although substantial evidence indicates that the progression of pathological changes of the neuronal cytoskeleton is crucial in determining the severity of dementia in Alzheimer's disease (AD), the exact causes and evolution of these changes, the initial site at which they begin, and the neuronal susceptibility levels for their development are poorly understood. The current clinical criteria for diagnosis of AD are focused mostly on cognitive deficits produced by dysfunction of hippocampal and high-order neocortical areas, whereas noncognitive, behavioural and psychological symptoms of dementia such as disturbances in mood, emotion, appetite, and wake-sleep cycle, confusion, agitation and depression have been less considered. The early occurrence of these symptoms suggests brainstem involvement, and more specifically of the serotonergic nuclei. In spite of the fact that the Braak and Braak staging system and National Institutes of Aging - Reagan Institute (NIA-RI) criteria do not include their evaluation, several recent reports drew attention to the possibility of selective and early involvement of raphe nuclei, particularly the dorsal raphe nucleus (DRN), in the pathogenesis of AD. Based on these findings of differential susceptibility and anatomical connectivity, a novel pathogenetic scheme of AD progression was proposed. Although the precise mechanisms of neurofibrillary degeneration still await elucidation, we speculated that cumulative oxidative damage may be the main cause of DRN alterations, as the age is the main risk factor for sporadic AD. Within such a framework, beta-amyloid production is considered only as one of the factors (although a significant one in familial cases) that promotes molecular series of events underlying AD-related neuropathological changes.

Hemispheric asymmetry, modular variability and age-related changes in the human entorhinal cortex.

The verrucae areae entorhinalis (VAE) are a characteristic feature of the human brain that occupy the anterior and posterolateral parts of the parahippocampal gyri and correspond to the islands of layer II neurons. We analyzed VAE in 60 neurologically normal subjects ranging from 23 to 85 years of age using a casting method. In 10 of these subjects the total number of neurons in the entorhinal islands was estimated stereologically using the optical fractionator. The number and surface area of VAE were higher in the left hemisphere compared with the right, and this leftward asymmetry was highly significant. Regression analysis showed a negative correlation between average VAE area and age in both hemispheres, representing a rate loss of about 800 microm2 per year. The estimated number of neurons obtained with the optical fractionator showed no significant difference between the left and the right hemisphere (468,000+/-144,000 vs. 405,000+/-117,000). There was a highly significant negative correlation between neuron numbers and age in both sides. In addition, clusters of small, undifferentiated layer II neurons ('heterotopias') were frequently observed in the rostral part of the entorhinal cortex in young and elderly adults. Layer II entorhinal neurons are among the first to show neurofibrillary changes during normal aging. The present data confirm the occurrence of age-related neuron loss in the entorhinal cortex. Considering the consistent projections from ipsilateral auditory association areas that, together with Broca's motor-speech area (Brodmann areas 44 and 45), show leftward asymmetry from early infancy (such as Brodmann area 22, planum temporale, and area 52 in the long insular gyrus), we speculate that functional lateralization of the human entorhinal cortex may be associated with specialization for memory processing related to language. Due to the dependence of hippocampal formation on entorhinal projections, this finding is also consistent with the greater capacity of the left hippocampus for verbal episodic memory.

nNOS expression in reactive astrocytes correlates with increased cell death related DNA damage in the hippocampus and entorhinal cortex in Alzheimer's disease.

The immunocytochemical distribution of the neuronal form of nitric oxide synthase (nNOS) was compared with neuropathological changes and with cell death related DNA damage (as revealed by in situ end labeling, ISEL) in the hippocampal formation and entorhinal cortex of 12 age-matched control subjects and 12 Alzheimer's disease (AD) patients. Unlike controls, numerous nNOS-positive reactive astrocytes were found in AD patients around beta-amyloid plaques in CA1 and subiculum and at the places of clear and overt neuron loss, particularly in the entorhinal cortex layer II and CA4. This is the first evidence of nNOS-like immunoreactivity in reactive astrocytes in AD. In contrast to controls, in all but one AD subject, large numbers of ISEL-positive neuronal nuclei and microglial cells were found in the CA1 and CA4 regions and subiculum. Semiquantitative analysis showed that neuronal DNA fragmentation in AD match with the distribution of nNOS-expressing reactive astroglial cells in CA1 (r = 0.74, P < 0.01) and CA4 (r = 0.58, P < 0.05). A portion of the nNOS-positive CA2/CA3 pyramidal neurons was found to be spared even in the most affected hippocampi. A significant inverse correlation between nNOS expression and immunoreactivity to abnormally phosphorylated tau proteins (as revealed by AT8 monoclonal antibody) in perikarya of these CA2/3 neurons (r = -0.85, P < 0.01) suggests that nNOS expression may provide selective resistance to neuronal degeneration in AD. In conclusion, our results imply that an upregulated production of NO by reactive astrocytes may play a key role in the pathogenesis of AD.

Ultrastructural analysis and TUNEL demonstrate motor neuron apoptosis in Werdnig-Hoffmann disease.

Werdnig-Hoffmann disease (WHD) is the most severe clinical type of spinal muscular atrophy characterized by loss of lower motor neurons and paralysis. We examined the hypothesis that disease pathogenesis is based on an inappropriate persistence of normally occurring motor neuron programmed cell death. The diagnosis of WHD was made on the basis of clinical findings, electromyoneurography, and biopsy, and further confirmed by mutation analysis of the survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes using PCR. We used ultrastructural analysis as well as TUNEL and ISEL methods to assess DNA fragmentation, and immunocytochemistry to identify expression of the apoptosis-related proteins bcl-2 and p53. A significant number of motor neurons in the spinal cord of children with WHD were shown to die by apoptosis. As revealed by TUNEL, dying neurons in WHD patients comprised 0.2%-6.4% of the neuron numbers counted. This finding contradicts earlier studies that failed to find such evidence and suggests that early blockade of prolonged motor neuron apoptosis may be a potential therapeutic strategy for WHD.

Nitrinergic neurons in the developing and adult human telencephalon: transient and permanent patterns of expression in comparison to other mammals.

A subpopulation of cerebral cortical neurons constitutively express nitric oxide synthase (NOS) and, upon demand, produce a novel messenger molecule nitric oxide (NO) with a variety of proposed roles in the developing, adult, and diseased brain. With respect to the intensity of their histochemical (NADPH-diaphorase histochemistry) and immunocytochemical (nNOS and eNOS immunocytochemistry) staining, these nitrinergic neurons are generally divided in type I and type II cells. Type I cells are usually large, intensely stained interneurons, scattered throughout all cortical layers; they frequently co-express GABA, neuropeptide Y, and somatostatin, but rarely contain calcium-binding proteins. Type II cells are small and lightly to moderately stained, about 20-fold more numerous than type I cells, located exclusively in supragranular layers, and found almost exclusively in the primate and human brain. In the developing cerebral cortex, nitrinergic neurons are among the earliest differentiating neurons, mostly because the dominant population of prenatal nitrinergic neurons are specific fetal subplate and Cajal-Retzius cells, which are the earliest generated neurons of the cortical anlage. However, at least in the human brain, a subpopulation of principal (pyramidal) cortical neurons transiently express NOS proteins in a regionally specific manner. In fact, transient overexpression of NOS-activity is a well-documented phenomenon in the developing mammalian cerebral cortex, suggesting that nitric oxide plays a significant role in the establishment and refinement of the cortical synaptic circuitry. Nitrinergic neurons are also present in human fetal basal forebrain and basal ganglia from 15 weeks of gestation onwards, thus being among the first chemically differentiated neurons within these brain regions. Finally, a subpopulation of human dorsal pallidal neurons transiently express NADPH-diaphorase activity during midgestation.

Nucleus subputaminalis (Ayala): the still disregarded magnocellular component of the basal forebrain may be human specific and connected with the cortical speech area.

The small magnocellular group located within the rostrolateral extension of the basal forebrain was named and described as the nucleus subputaminalis in the human and chimpanzee brain by Ayala. Analysis of cytoarchitectonic and cytochemical characteristics of this cell group has been largely disregarded in both classical and more current studies. We examined the nucleus subputaminalis in 33 neurologically normal subjects (ranging from 15 weeks of gestation to 71 years-of-age) by using Nissl staining, choline acetyltransferase immunohistochemistry, acetyl cholinesterase histochemistry and nerve growth factor receptor immunocytochemistry. In addition, we applied reduced nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry and calbindin-D28k immunocytochemistry in three neurologically normal subjects. At the most rostrolateral levels we describe the previously poorly characterized component of the lateral (periputaminal) subdivision of the subputaminal nucleus, which may be human specific since it is not described in non-human primates. Moreover, we find the human subputaminal nucleus best developed at the anterointermediate level, which is the part of the basal nucleus that is usually much smaller or missing in monkeys. The location of subputaminal cholinergic neurons within the frontal lobe, the ascension of their fibers through the external capsule towards the inferior frontal gyrus, the larger size of the subputaminal nucleus on the left side at the most rostral and anterointermediate levels and the most protracted development among all magnocellular aggregations within the basal forebrain strongly suggest that they may be connected with the cortical speech area. These findings give rise to many hypotheses about the possible role of the subputaminal nucleus in various neurodegenerative, neurological and psychiatric disorders, particularly Alzheimer's disease and primary progressive aphasia. Therefore, future studies on the basal forebrain should more carefully investigate this part of the basal nucleus.

Postnatal development of calcium-binding proteins calbindin and parvalbumin in human visual cortex.

In adult primate visual cortex, the calcium-binding proteins calbindin (CB) and parvalbumin (PV) are localized in different subsets of GABAergic neurons with a characteristic laminar distribution. However, the emergence and development of CB and PV in relation to the periods of functional maturation of the human visual cortex are not known. Therefore, we examined (i) postnatal changes in the distribution of immunoreactivity (ir) for CB and PV in the visual cortex; (ii) the pattern of changes in immunoreactivity in relation to the synaptic maturation; and (iii) differences in the maturation of CB and PV immunoreactivity between areas 17 and 18. We found a consistently high expression of CB in neonatal visual cortex, particularly in layer IV and infragranular layers. However, despite an early appearance of PV, its peak in development occurred only after 2 months of age, characterized by a transient overexpression in the thalamo-recipient layer IV and a continuous inside-out maturation in supragranular layers. The neonatal pattern of high CB-ir in layers IV-VI was transformed during infancy and childhood into an adult pattern of high CB-ir in layer II, but low CB-ir in layer IV and infragranular layers. There was no difference in pattern and tempo of maturation of calcium-binding proteins between area 17 and 18, indicating simultaneous development of cortical inhibitory circuits among cytoarchitectonically and functionally distinct cortical areas. In addition, the reorganization of CB/PV expression temporally and spatially coincides with the course of cortical synaptogenesis, and delineates the major stages of maturation of the human visual cortex.

C-pathway polysialogangliosides are transiently expressed in the human cerebrum during fetal development.

Gangliosides are assumed to play a crucial role in processes of cellular recognition and interaction important for neural development. They are designated as cytochemical markers of neuronal maturation, as striking changes in the ganglioside pattern parallel the nervous system development. Of particular interest to us are numerous studies that reported during migration of postmitotic neurons and axon formation in developing avian and mammalian brains a transient accumulation of highly sialylated c-pathway gangliosides. However, it has thus far been thought that c-pathway gangliosides do not appear in the human cerebrum; their absence could be somehow interpreted in the light of an evolutionary trend in the pattern of brain gangliosides: by increasing the phylogenetic scale this pattern changes by an accretion of less sialylated gangliosides and switches from c- via b- to a-series, respectively. The present study presents both biochemical and immunocytochemical evidence for the existence of c-pathway gangliosides in the human cerebrum during prenatal life, and their localization in discrete neuronal populations and growing axonal pathways.

Transient patterns of organization of the human fetal brain.

Fetal development of the human brain is characterized by continuous transformations and reorganization of the fetal telencephalic wall which consists of transient, cytoarchitectonically defined cellular compartments, the so-called embryonic/fetal zones. The cellular and fiber content of these zones is permanently changing, so that fetal neuronal circuitry elements (afferent fibers, synapses, and postsynaptic neurons) display transient patterns of areal, laminar, and modular organization. In the late human fetus and preterm infant, transient patterns of structural and physiological organization form the basis of transient behavioral states and patterns of activity. The transient subplate zone is a key compartment for transient fetal neuronal circuitry, and competitive cellular interactions within the subplate zone are crucial for the areal specification of the cerebral cortex and the formation of cortical connectivity. The subplate zone may also have a key role in cortical repair and plasticity after perinatal brain lesions.

War victims in need of physical rehabilitation in Croatia.

A Rehabilitation Information System was created in July 1993 in order to register war victims in need of physical rehabilitation all over Croatia. The system is currently operating and presented data covers the period from July 1991 to July 1995. Approximately 15,000 questionnaires had been completed and returned from medical institutions on in total 8589 disabled war victims in need of rehabilitation. People with severe disabilities comprised about 20% of all in need of rehabilitation. Those reported injured were 3.5 times more than those in need of physical rehabilitation. Most common types of injuries were fractures with a permanent disabling condition (3109 persons), peripheral nerve injuries (1213 persons) and amputations (956 persons). Traumatic brain injuries were registered for 594 and spinal cord injuries for 262 persons. Causes of injuries were explosive devices (such as mines, mortar shell shrapnel, etc.) in 37% of cases, bullets in 22%, accidents in 7%, other (such as fire, blast injuries, etc.) and unknown causes in 34%.

Transient fetal structure, the gangliothalamic body, connects telencephalic germinal zone with all thalamic regions in the developing human brain.

Previous studies reported that telencephalic proliferative zones contribute to the development of the pulvinar thalamic nucleus in the human brain (Rakic and Sidman [1969] Z. Anat. Entwicklungsgesch. 129:53-82). The present study examined their possible contribution to the development of other thalamic nuclei. Postmortem brain tissue from human fetuses ranging between 10.5 and 40 weeks of gestation (wg) was processed by Nissl staining, Golgi impregnation, and MAP2 (microtubule-associated protein 2) immunocytochemistry. The gangliothalamic body, suggested to serve as a conduit for cells migrating from the ganglionic eminence to the thalamus, was found in the period from 15 to 34 wg in all rostrocaudal thalamic regions, particularly at the level of the anterior nuclear complex, mediodorsal and pulvinar nucleus, and in addition, the lateral geniculate nucleus. In Nissl-stained sections, the gangliothalamic body is a thin cellular layer situated beneath the thalamic surface, near the telencephalo-diencephalic junction. In Golgi- and MAP2-stained sections, it is a stream of mostly bipolar cells extending from the ganglionic eminence to the medial thalamus. In addition, MAP2-immunoreactivity confirms the neuronal nature of its cells. The present study further supports the hypothesis that certain neurons migrate from the ganglionic eminence to the thalamus through the transient gangliothalamic body during fetal development. Moreover, our data indicate that both the association (mediodorsal and pulvinar), as well as the anterior (limbic) and specific relay nuclei are potential recipients of the telencephalic neurons.

Volume and number of neurons of the human hippocampal formation in normal aging and Alzheimer's disease.

In order to observe changes owing to aging and Alzheimer's disease (AD) in the volumes of subdivisions of the hippocampus and the number of neurons of the hippocampal formation, 18 normal brains from subjects who died of nonneurological causes and had no history of long-term illness or dementia (ten of these brains comprised the aged control group) and 13 AD brains were analyzed. An optimized design for sampling, measuring volume by using the Cavalieri principle, and counting the number of neurons by using the optical disector was implemented on 50 microns-thick cresyl-violet sections. The mean total volume of the principal subdivisions of the hippocampal formation (fascia dentata, hilus, CA3-2, CA1, and subiculum) showed a negative correlation with age in normal subjects (r = -0.56, 2P < 0.05), and a 32% mean reduction in the AD group compared with controls (P < 0.001). This finding supports the measurement of the coronal cross-sectional area and the volume of the hippocampal formation in the clinical diagnosis of AD. There was an inverse relationship between the age of normal subjects and the number of neurons in CA1 (r = -0.84, 2P < 0.0001) and subiculum (r = -0.49, 2P < 0.05) but not in other subdivisions. Pronounced AD-related reductions in neuron number were found only in the subiculum and the fascia dentata. Compared with controls, both losses represented 23% of neurons (P < 0.05). These results 1) confirm that AD is a qualitatively different process from normal aging and 2) reveal the regional selectivity of neuron loss within the hippocampal formation in aging and AD, which may be relevant to understanding the mechanisms involved in the neuron loss associated with the two processes.

Laminar distribution of neuropeptide Y-immunoreactive neurons in human prefrontal cortex during development.

Neuropeptide Y (NPY) is present in neurons of the adult human cerebral cortex. In view of the reported roles of NPY in the central nervous system in health and during certain disease conditions, we have studied normal development of NPY immunoreactivity (-ir) in the human prefrontal cortex (PFC), Brodmann areas 9 and 46. Twenty-six specimens ranging from the ages of 14 postovulatory weeks to 34 years exhibited patterns that revealed six periods in the development of the laminar distribution and density of NPY-ir neurons. Changes during prenatal and perinatal periods reflect the onset, development, and resolution of the transient fetal telencephalic compartments, including the subplate zone, in which NPY-ir neurons are especially abundant. Before the age of 1 year, the majority of NPY-ir neurons were found in the subplate zone, whereas, after 1 year, the majority were seen in the cortical layers. This is in contrast with the human visual cortex, where the majority of NPY-ir neurons were still located in the white matter. The density of cortical NPY-ir neurons increased in the fifth developmental period (ages 4-7 years), coinciding with the increase of cortical volume and marked progression of cognitive functions. The adult pattern of a relatively low density of cortical NPY-ir neurons was reached in period 6 (from about 8 years), when individual variation also became apparent. Our data point to a protracted maturation of NPY-ir in the human PFC and to different distribution patterns of NPY-ir neurons in different cortical areas.

Transient patterns of calbindin-D28k expression in the developing striatum of man.

In fetal and adult human brains, calbindin immunoreactivity (CB-ir) of neostriatal neuropil showed inhomogeneous pattern. A mosaic of CB-ir neuropil patches matching the acetylcholinesterase (AChE)-reactive patches and most of the encapsulated cell-dense islands was distributed in a lighter stained background matrix. During infancy, the pattern of CB expression changed from one of CB-rich patches to one of CB-poor striosomes and rich matrix. Furthermore, we observed a steady development of population of medium-sized neurons in the striatal matrix; in addition, a transient CB-expression was found in cells of the ganglionic eminence and presumably in a subset of striatal interneurons.

Transient neuronal population of the internal capsule in the developing human cerebrum.

The present study reveals the presence of a distinct group of cells, resembling reticular thalamic neurones, in the internal capsule during fetal development. This cell population rapidly decreases in size during early infancy and few cells are apparent in the 1-year-old infant. Internal capsule cells are well differentiated, multipolar or polymorphous, AChE (acetylcholinesterase)-reactive neurones. The following specific molecular markers were demonstrated in the neurones of the internal capsule: MAP2 (microtubule-associated protein 2), somatostatin, calbindin-D28K and p75 low-affinity NGF (nerve growth factor) receptor. A group of neurones described here corresponds to the perireticular thalamic nucleus found in certain mammalian species, hitherto unidentified in the primate brain, which may play an important role during development.

Ontogenesis of goal-directed behavior: anatomo-functional considerations.

Recent neuroanatomical and neurophysiological studies in man have revealed ontogenetic events which coincide with broadly defined phases of behavioral and cognitive development. During the early fetal period, early produced neurons make initial synapses which form the basis for the earliest electrical activity of the human brain. The overall immaturity of neuronal connections, in particularly in cortical areas, correlates with the absence of any behavioral pattern or goal-directed movements. In the late fetus and preterm infant, transient accumulation of major afferent pathways, the presence of transient layers (subplate zone) and transient pattern of transmitter-related organization form the neurological basis of cortical electric responses as well as transient behavioral states and sleep patterns. Parallel to the profound structural and chemical reorganization of the human cerebrum during the first 6 postnatal months there is a disappearance of transient behavioral and motor patterns. The previously close spatio-temporal correlation between these events becomes progressively looser. The overproduction of circuitry elements during the subsequent period peaks in associative cortex between 1 and 2 years of age, corresponding to the emergence of skilled actions and cognitive functions. After the elimination of some circuitry elements after the second year of life, the prolonged maturation of goal-directed behavior and the protracted emergence of different cognitive functions correlates with the development plateau of synapse production which can be seen up to 16 years of age. Parallel to the prolonged maturation of postsynaptic elements, there are well defined maturational changes in the chemical properties of associative pyramidal neurons of cortical layer III. These findings correspond to the prolonged maturation of movement-related brain macropotentials as well as other cognition-related potentials, where the last prominent changes were seen after 10 years of age. Although the coincidence of the developmental events does not necessarily mean a causal relationship, the combination of structural and physiological data opens new vistas for the further investigation of the neurobiological basis of goal-directed movement and cognitive behavior.

Identification of war victims in Croatia.

The intention of this paper is to describe the organizational principles and indicate the results already achieved in the identification of war victims in Croatia. By 25 February 1993, 6,493 victims had been identified. A model is proposed that could be used in the course of identification processes, examining the methods and principles of identification which have been complicated by the time interval of more than a year from the time of death, for a presumed number of several thousand (up to 14,000) unidentified victims, possibly in mass graves. Identification is further complicated by the lack of ante-mortem medical and dental records and the incapacity to utilize more expensive methods of identification. Attention is drawn to a group of more complex cases examined at the Institute of Forensic Medicine and Criminology.