Nonphosphorylated Neurofilament Protein Research Articles

Cyto‐ and chemoarchitecture of the cerebral cortex of an echidna (Tachyglossus aculeatus). II. Laminar organization and synaptic density

We have examined the distribution and morphology of neurons immunoreactive for nonphosphorylated neurofilament protein (SMI-32 antibody), calcium-binding proteins (parvalbumin, calbindin, calretinin), and neuropeptide Y as well as neurons reactive for NADPH diaphorase in the cerebral cortex of the Australian short-beaked echidna (Tachyglossus aculeatus). We have also studied synaptic morphology and density in S1 somatosensory cortex and assessed parameters associated with metabolic activity of the cerebral cortex (vessel volume density, mitochondrial volume density, and mitochondrial numerical density) in semi- and ultrathin sections. SMI-32 immunoreactivity was found mostly in layer V pyramidal neurons in selected cortical regions (S1, PV, V1, A). These neurons often showed atypical morphology compared with therian cortex. Neurons immunoreactive for calcium-binding proteins were broadly similar in both morphology and distribution to those seen in therian cortex, although calretinin-immunoreactive neurons were rare. Both Gray type I and Gray type II synapses could be identified in echidna S1 cortex and were similar to those seen in therian cortex. Peak synaptic density was in upper layer IV, followed by layer I, lower layer II, and upper layer III. Most synapses were of type I (72%), although types I and II were encountered with similar frequency in lower layer II and upper layer III. The capillary volume fraction values obtained for the echidna (from 1.18% in V1 to 1.34% in S1 cortex) fall within the values for rodent cortex. Similarly, values for mitochondrial volume fraction in echidna somatosensory cortex (4.68% +/- 1.76%) were comparable to those in eutherian cortex.

Topographic and laminar maturation of striate cortex in early postnatal marmoset monkeys, as revealed by neurofilament immunohistochemistry.

The maturation of pyramidal neurons in the primary visual cortex (V1) of marmoset monkeys was investigated using an antibody (SMI-32) to non-phosphorylated neurofilament protein (NNF). Analysis of animals aged between birth and postnatal day 91 (PD 91, which corresponds approximately to the peak of synaptogenesis in this species) revealed discrete changes in both the laminar and the areal distribution of NNF. At PD 0, the upper part of layer 6 contained darkly labelled neurons and associated neuropil, including axons. In this layer a centroperipheral gradient, with more labelled cells in the foveal representation, was apparent at PD 0. This topographic gradient gradually disappeared, and by PD 91 a similar density of labelled layer 6 cells was observed throughout V1. Labelled cells were not apparent in layer 3C until PD 7, and were not distributed according to a topographic gradient. Labelled cells were first observed in layer 3B(alpha) at PD 28, when they formed a centroperipheral gradient similar to that seen in layer 6. This gradient was still evident in an adult animal. These results demonstrate an inside-out profile of postnatal cortical development, with the topographic pattern of maturation of V1 mimicking the centroperipheral gradient of maturation in the retina.

Spatio-temporal changes in neurofilament proteins immunoreactivity following kainate-induced cerebellar lesion in rats.

1. Spatio-temporal changes in phosphorylated (pNFP) and nonphosphorylated (npNFP) neurofilament proteins were assessed immunocytochemicaly in adult rat cerebellum, 2-30 days following unilateral injection of kainic acid (KA) or physiological saline (s.c.). 2. Analysis of the staining intensity and pattern demonstrated that injection of both KA and physiological saline elicited significant and long-lasting increase of pNFP and npNFP immunoreactivity, at the ipsilateral, and to lesser extent at the contralateral side of lesion. 3. Kainate intoxication induced abundant expression of pNFP and npNFP in cerebellar white matter, as well as in all layers of perilesioned cortex. Higher pNFP expression was evidenced in the Purkinje cell layer, particularly at cell bodies, initial segments, and proximal dendrites, which normally do not contain pNFP. In addition, synaptophysin immunocytochemistry was used as a marker of synaptogenesis and plasticity. 4. Spatio-temporal pattern of NFP and synaptophysin expression suggests that perilesioned cortex undergoes dynamic changes following brain demage and possess a reparative capacity to abridge the consequences of brain trauma.

Altered cortical glutamate neurotransmission in schizophrenia: evidence from morphological studies of pyramidal neurons.

Multiple lines of evidence from pharmacological, neuroimaging, and postmortem studies implicate disturbances in cortical glutamate neurotransmission in the pathophysiology of schizophrenia. Given that pyramidal neurons are the principal source of cortical glutamate neurotransmission, as well as the targets of the majority of cortical glutamate-containing axon terminals, understanding the nature of altered glutamate neurotransmission in schizophrenia requires an appreciation of both the types of pyramidal cell abnormalities and the specific class(es) of pyramidal cells that are affected in the illness. In this chapter, we review evidence indicating that a subpopulation of pyramidal neurons in the dorsolateral prefrontal cortex exhibits reductions in dendritic spine density, a marker of the number of excitatory inputs, and in somal volume, a measure correlated with a neuron's dendritic and axonal architecture. Specifically, pyramidal neurons located in deep layer 3 of the dorsolateral prefrontal cortex and that lack immunoreactivity for nonphosphorylated neurofilament protein may be particularly involved in the pathophysiology of schizophrenia. The presence of similar changes in pyramidal neurons located in deep layer 3 of auditory association cortex suggests that a shared property, which remains to be determined, confers cell type-specific vulnerability to a subpopulation of cortical glutamatergic neurons in schizophrenia.

Cyto- and chemoarchitecture of the hypothalamus of a wallaby ( Macropus eugenii) with special emphasis on oxytocin and vasopressinergic neurons.

We have studied the organization of the hypothalamus in an Australian diprotodontid metatherian mammal, the wallaby ( Macropus eugenii), using cytoarchitectural, histochemical and immunohistochemical techniques. Coronal sections of adult brains were processed for Nissl staining, histochemical reactivity (cytochrome oxidase, nicotinamide adenine dinucleotide phosphate diaphorase and acetylcholinesterase) and immunohistochemistry (antibodies to tyrosine hydroxylase, calbindin, calretinin, non-phosphorylated neurofilament protein, oxytocin and vasopressin). The distribution of immunoreactive neurons for these substances was mapped with the aid of a computer-linked microscope. In general, the wallaby hypothalamus showed a similar nuclear organization to that seen in rodents. The paraventricular nucleus could be divided into several subdivisions based on the different cellular parcellation, similar to that described in rodents. The ventromedial hypothalamic nucleus had cell-sparse dorsomedial and cell-dense ventrolateral subdivisions as seen in eutheria, suggesting a similar functional compartmentalization in all theria. The positions of tyrosine hydroxylase-positive neurons in the wallaby hypothalamus were also similar to those in eutheria. Oxytocin and vasopressinergic neurons were found in all the same major nuclear groups as seen in eutheria, although a nucleus circularis could not be identified. The general similarities between wallaby and eutherian hypothalamus indicate that the basic chemo- and cytoarchitectural features of the hypothalamus are common to eutheria and metatheria and validate the use of the wallaby as a mammalian model of wide applicability in investigations of hypothalamic functional development.

Neurofilament protein expression in the geniculostriate pathway of a New World monkey ( Callithrix jacchus).

We examined the expression profile of non-phosphorylated neurofilament protein in the dorsal lateral geniculate nucleus (LGN) and striate cortex (V1) of a New World simian, the marmoset monkey, using the monoclonal antibody SMI-32. The overall distribution of neurofilament protein in the marmoset resembled that previously described in Old World monkeys. While immunostained neurones were observed throughout the LGN, there were clear laminar differences in terms of both cellular and neuropil labelling. Neurones in the magnocellular layer cells stained more densely than those in the parvocellular layers. The marmoset's well-defined koniocellular layers showed an overall light stain of both neurones and neuropil. In V1, densely stained pyramidal cells and heavy neuropil label were observed in the two sublayers that send projections to the middle temporal area (MT): a supragranular band located in layer 3C (Brodmann's layer 4B) and an infragranular band located near the top of layer 6. More lightly stained, small pyramidal cells were also found in layer 3Balpha. Accordingly, in both New World and Old World monkeys the expression of neurofilament protein is correlated with specific functional subdivisions of the geniculocortical pathway. In particular, projection neurones associated with fast-conducting pathways to the extrastriate 'dorsal stream' appear to contain higher levels of this protein.

Neurofilament-Immunoreactive Neurons in Alzheimer's Disease and Dementia with Lewy Bodies

The cortical neurons thought to be selectively affected in dementia with Lewy bodies (DLB) are those containing nonphosphorylated 200-kDa neurofilament (NF) protein. As these neurons are largely spared in Alzheimer's disease (AD), DLB and AD may impact on different cortical neuronal populations. The present study quantifies the NF-containing neurons in frontal and temporal cortex of 8 AD, 8 DLB, and 8 control cases. Formalin-fixed paraffin-embedded tissue was immunohistochemically stained with antibodies against nonphosphorylated and phosphorylated NF. Immunoreactive neurons were quantified by areal fraction analysis and corrected for cortical volume. As expected, nonphosphorylated and phosphorylated NF accumulated in the pathological hallmarks of AD and DLB. However, rather than a decrease in NF-containing neurons, a doubling of this population was observed in DLB, compared with AD and controls. This increased number of cortical NF-containing neurons reveal novel widespread cortical changes, beyond those explained by Lewy body formation, that are specific for DLB.

Task-dependent and cell-type-specific Fos enhancement in rat sensory cortices during audio-visual discrimination.

Attention modulates neural activities in sensory cortices. Because cortical neurons are composed of many types of neurons, the activities of these different types of cells can exhibit different modifications depending on whether an animal pays attention to a particular sensory stimulus or not. In the present study, we examined which types of cortical neurons change their activities in rats during one of two types of audio-visual discrimination (AVD) tasks by using Fos immunohistochemistry. In the tasks, both auditory and visual stimuli were simultaneously presented but only one of the two modalities was task-relevant. Once the rats had learned one of the AVD tasks, presenting only relevant sensory stimuli was sufficient for them to perform the task correctly. These results suggest that the rats indeed attended to the relevant stimuli during the performance of the tasks. We found that Fos expression in the primary auditory and visual cortices was enhanced in a task-dependent manner during the performance of the AVD tasks. The enhancement of Fos expression depended on the behavioural significance of the stimulus in the tasks. Moreover, using double immunohistochemistry of Fos and a cell type-specific marker protein (phosphate-activated glutaminase, nonphosphorylated neurofilament protein, parvalbumin, calretinin or somatostatin), the task-dependent Fos expression was observed preferentially in excitatory neurons but not in inhibitory interneurons. These results suggest that modulation in cortical excitatory neurons might have critical roles in selecting and processing behaviourally relevant sensory stimuli.

Neurochemical organization of the human basal ganglia: anatomofunctional territories defined by the distributions of calcium-binding proteins and SMI-32.

The distribution of the calcium-binding proteins calbindin-D28K (CB), parvalbumin (PV) and calretinin (CR), and of the nonphosphorylated neurofilament protein (with SMI-32) was investigated in the human basal ganglia to identify anatomofunctional territories. In the striatum, gradients of neuropil immunostaining define four major territories: The first (T1) includes all but the rostroventral half of the putamen and is characterized by enhanced matriceal PV and SMI-32 immunoreactivity (-ir). The second territory (T2) encompasses most part of the caudate nucleus (Cd) and rostral putamen (PuT), which show enhanced matriceal CB-ir. The third and fourth territories (T3 and T4) comprise rostroventral parts of Cd and PuT characterized by complementary patch/matrix distributions of CB- and CR-ir, and the accumbens nucleus (Acb), respectively. The latter is separated into lateral (prominently enhanced in CB-ir) and medial (prominently enhanced in CR-ir) subdivisions. In the pallidum, parallel gradients also delimit four territories, T1 in the caudal half of external (GPe) and internal (GPi) divisions, characterized by enhanced PV- and SMI-32-ir; T2 in their rostral half, characterized by enhanced CB-ir; and T3 and T4 in their rostroventral pole and in the subpallidal area, respectively, both expressing CB- and CR-ir but with different intensities. The subthalamic nucleus (STh) shows contrasting patterns of dense PV-ir (sparing only the most medial part) and low CB-ir. Expression of CR-ir is relatively low, except in the medial, low PV-ir, part of the nucleus, whereas SMI-32-ir is moderate across the whole nucleus. The substantia nigra is characterized by complementary patterns of high neuropil CB- and SMI-32-ir in pars reticulata (SNr) and high CR-ir in pars compacta (SNc) and in the ventral tegmental area (VTA). The compartmentalization of calcium-binding proteins and SMI-32 in the human basal ganglia, in particular in the striatum and pallidum, delimits anatomofunctional territories that are of significance for functional imaging studies and target selection in stereotactic neurosurgery.

Cytology of human caudomedial cingulate, retrosplenial, and caudal parahippocampal cortices.

Brodmann showed areas 26, 29, 30, 23, and 31 on the human posterior cingulate gyrus without marking sulcal areas. Histologic studies of retrosplenial areas 29 and 30 identify them on the ventral bank of the cingulate gyrus (CGv), whereas standardized atlases show area 30 on the surface of the caudomedial region. This study evaluates all areas on the CGv and caudomedial region with rigorous cytologic criteria in coronal and oblique sections Nissl stained or immunoreacted for neuron-specific nuclear binding protein and nonphosphorylated neurofilament proteins (NFP-ir). Ectosplenial area 26 has a granular layer with few large pyramidal neurons below. Lateral area 29 (29l) has a dense granular layer II-IV and undifferentiated layers V and VI. Medial area 29 (29m) has a layer III of medium and NFP-ir pyramids and a layer IV with some large, NFP-ir pyramidal neurons that distinguish it from areas 29l, 30, and 27. Although area 29m is primarily on the CGv, a terminal branch can extend onto the caudomedial lobule. Area 30 is dysgranular with a variable thickness layer IV that is interrupted by large NFP-ir neurons in layers IIIc and Va. Although area 30 does not appear on the surface of the caudomedial lobule, a terminal branch can form less that 1% of this gyrus. Area 23a is isocortex with a clear layer IV and large, NFP-ir neurons in layers IIIc and Va. Area 23b is similar to area 23a but with a thicker layer IV, more large neurons in layer Va, and a higher density of NFP-ir neurons in layer III. The caudomedial gyral surface is composed of areas 23a and 23b and a caudal extension of area 31. Although posterior area 27 and the parasubiculum are similar to rostral levels, posterior area 36' differs from rostral area 36. Subregional flat maps show that retrosplenial cortex is on the CGv, most of the surface of caudomedial cortex is areas 23a, 23b, and 31, and the retrosplenial/parahippocampal border is at the ventral edge of the splenium. Thus, Brodmann's map understates the rostral extent of retrosplenial cortex, overstates its caudoventral extent, and abridges the caudomedial extent of area 23.

Neurofilament-Immunoreactive Neurons Are Not Selectively Vulnerable in Alzheimer's Disease

Abnormal neurofilament protein distribution and phosphorylation contributes to the cytoskeletal pathology of Alzheimer's disease. Anatomical studies suggest that cortical neurons immunoreactive for nonphosphorylated 200-kDa neurofilament are most vulnerable. We repeated these studies in formalin-fixed temporal lobe tissue from five Alzheimer's disease cases with tissue volume loss compared to five controls without tissue loss. Immunohistochemistry for nonphosphorylated and phosphorylated forms of the neurofilament protein was counterstained for Nissl substance and immuno-positive and -negative pyramidal neurons quantified using areal fraction counts. Compared with controls, cases with Alzheimer's disease had similar numbers of neurons expressing the nonphosphorylated neurofilament protein, suggesting these neurons are largely spared by the disease process. In Alzheimer's disease there was a significant increase in neurons containing phosphorylated neurofilament and tau proteins and a decrease in neurons devoid of neurofilament protein. Our results challenge the theory that neurons containing 200 kDa neurofilament are selectively vulnerable in Alzheimer's disease.

Organization of the human paraventricular hypothalamic nucleus

The cyto- and chemoarchitecture of the human paraventricular hypothalamic nucleus (Pa) was studied with the aid of three-dimensional computer reconstruction. The adult human Pa is a vertically elongated structure that abuts the wall of the third ventricle (3V) medially and is indented dorsolaterally by the descending fornix. Chemoarchitecture revealed the following five subnuclei in the human Pa. The most prominent of these is the magnocellular subnucleus (PaM) occupying the ventrolateral quadrant of the Pa and comprised of a concentration of large arginin-vasopressin (AVP)- and acetylcholinesterase (AChE)-positive cells, and small calbindin (Cb)-positive neurons. Rostrally, the PaM is succeeded by the small anterior parvicellular subnucleus (PaAP), which contains small AChE-, AVP- and tyrosin hydroxylase (TH)-positive cells. Dorsal to the PaM is found the dorsal subnucleus (PaD), containing large spindle-shaped TH-, oxytocin (OXY)-, and AChE-positive cells, as well as a population of small Cb-positive neurons. Abutting the wall of the 3V and medial to PaM and PaD is the parvicellular subnucleus (PaP). The PaP contains small cells immunoreactive for corticotropin-releasing factor (CRF), neuromedin K receptor (NK3), and nonphosphorylated neurofilament protein (SMI32). The posterior subnucleus (PaPo) is situated posterior to the descending column of the fornix; it replaces all above-mentioned subdivisions caudally, and is a chemoarchitectonic amalgam that includes dispersed large AChE-, OXY-, AVP- and TH-positive cells, as well as small NK3-, CRF-, SMI32- and Cb-immunoreactive neurons. The present findings suggest that the human PaM and PaD are homologues to the magnocellular subnuclei of the rat Pa, whereas the human PaP and PaPo correspond to the rat medial parvicellular and posterior subnuclei, respectively. J. Comp. Neurol. 423:299–318, 2000. © 2000 Wiley-Liss, Inc.

NMDAR-1 staining in the lateral geniculate nucleus of normal and visually deprived cats.

In normal adult cats, a monoclonal antibody directed toward the NR-1 subunit of the N-methyl-D-aspartate (NMDA) receptor (Pharmingen, clone 54.1) produced dense cellular and neuropil labeling throughout all layers of the lateral geniculate nucleus (LGN) and adjacent thalamic nuclei, including the thalamic reticular, perigeniculate, medial intralaminar, and ventral lateral geniculate nuclei. Cellular staining revealed well-defined somata, and in some cases proximal dendrites. NMDAR-1 cell labeling was also evident in the LGN of early postnatal kittens, suggesting that developing LGN cells possess this receptor subunit at or before eye opening. Within the A-layers of the adult LGN, staining encompassed a wide range of soma sizes. Soma size comparisons of NMDAR-1 stained cells with those stained with an antibody directed toward a nonphosphorylated neurofilament protein (SMI-32), which selectively stains Y-relay cells (Bickford et al., 1998), or an antibody to glutamic acid decarboxylase (GAD), which stains for GABAergic interneurons, suggested that NMDA receptors are utilized by relay cells and interneurons. NMDAR-1 staining was also observed in the LGN of cats with early monocular lid suture. Although labeling was apparent in both deprived and nondeprived A-layers of LGN, the distribution of soma sizes was significantly different. In the deprived A-layers of LGN, staining was limited to small- and medium-sized cells. Cells with relatively large soma were lacking. However, cell density measurements as well as soma size comparisons with cells stained for Nissl substance suggested these differences were due to deprivation-induced cell shrinkage and not to a loss of NMDAR-1 staining in Y-cells. Taken together, these results suggest that NMDA receptors are utilized by both relay cells and interneurons in LGN and that alterations in early visual experience do not necessarily affect the expression of NMDA receptors in the LGN.

Organization of the human paraventricular hypothalamic nucleus

The cyto- and chemoarchitecture of the human paraventricular hypothalamic nucleus (Pa) was studied with the aid of three-dimensional computer reconstruction. The adult human Pa is a vertically elongated structure that abuts the wall of the third ventricle (3V) medially and is indented dorsolaterally by the descending fornix. Chemoarchitecture revealed the following five subnuclei in the human Pa. The most prominent of these is the magnocellular subnucleus (PaM) occupying the ventrolateral quadrant of the Pa and comprised of a concentration of large arginin-vasopressin (AVP)- and acetylcholinesterase (AChE)-positive cells, and small calbindin (Cb)-positive neurons. Rostrally, the PaM is succeeded by the small anterior parvicellular subnucleus (PaAP), which contains small AChE-, AVP- and tyrosin hydroxylase (TH)-positive cells. Dorsal to the PaM is found the dorsal subnucleus (PaD), containing large spindle-shaped TH-, oxytocin (OXY)-, and AChE-positive cells, as well as a population of small Cb-positive neurons. Abutting the wall of the 3V and medial to PaM and PaD is the parvicellular subnucleus (PaP). The PaP contains small cells immunoreactive for corticotropin-releasing factor (CRF), neuromedin K receptor (NK3), and nonphosphorylated neurofilament protein (SMI32). The posterior subnucleus (PaPo) is situated posterior to the descending column of the fornix; it replaces all above-mentioned subdivisions caudally, and is a chemoarchitectonic amalgam that includes dispersed large AChE-, OXY-, AVP- and TH-positive cells, as well as small NK3-, CRF-, SMI32- and Cb-immunoreactive neurons. The present findings suggest that the human PaM and PaD are homologues to the magnocellular subnuclei of the rat Pa, whereas the human PaP and PaPo correspond to the rat medial parvicellular and posterior subnuclei, respectively.

Macaque monkey retrosplenial cortex: I. three-dimensional and cytoarchitectonic organization.

This is the first in a series of reports on the neuroanatomic organization and connectivity of the macaque monkey retrosplenial cortex, i.e., areas 29 and 30. To elucidate the topographic configuration of the retrosplenial cortex and adjacent structures, we have made three-dimensional computer reconstructions of the posterior cingulate region that includes the retrosplenial cortex. The largest portion of the posterior cingulate gyrus is located dorsal to the corpus callosum. At the caudal limit of the corpus callosum, the gyrus curves around the splenium, turns laterally and forms a region called the isthmus that links the cingulate and parahippocampal gyri. The isthmus contains the caudomedial lobule, which is a rostrally oriented bulge that is made up, in part, of portions of the retrosplenial cortex. To delineate the subdivisions of the retrosplenial and adjacent cortices, we conducted a cytoarchitectonic analysis by using cerebral hemispheres that were cut at oblique angles and stained with a variety of techniques, including immunohistochemistry for nonphosphorylated neurofilament protein. The dorsal bank of the callosal sulcus and the rostral surface of the isthmus are covered by the retrosplenial cortical areas 29l, 29m, and 30, whereas most of the medial surface of the posterior cingulate gyrus and the ventral bank of the posterior cingulate sulcus consist of areas 23i and 23e. The most caudoventral portion of the cingulate gyrus is composed of an area (area 23v) that resembles the retrosplenial and posterior cingulate cortices but has a much more prominent layer IV. On the dorsal bank of the calcarine sulcus, we also defined a transitional zone, area 30v, located between the retrosplenial cortex and the prestriate visual cortex.

Neurochemical gradients along monkey sensory cortical pathways: calbindin‐immunoreactive pyramidal neurons in layers II and III

We examined the distribution of neurons containing immunoreactivity for three calcium-binding proteins, calbindin, parvalbumin and calretinin, as well as nonphosphorylated neurofilament protein, in cortical areas along the ventral and dorsal cortical visual pathways, and in ventrally-directed somatosensory and auditory cortical pathways. Calbindin-immunoreactive pyramidal neurons showed the most prominent regional differences. They were largely restricted to layers II and III and their number monotonically increased from the primary sensory areas to the anteroventral areas along the ventral visual pathway and along the ventrally-directed somatosensory and auditory pathways. The number of calbindin-immunoreactive pyramidal neurons in layers II and III also increased along the dorsal visual pathway, but the number in the last recognized stage of the dorsal visual pathway (area 7a) was significantly smaller than that at the corresponding stage in the ventral visual pathway (TE). The number of calbindin-immunoreactive pyramidal neurons was highest in layers II and III of areas 35/36, TG, and TF/TH, which represent terminal cortical regions of the pathways. These results show neurochemical differences between cortical areas located at early and late stages along serial corticocortical pathways, as well as confirming differences between pyramidal neurons in the supragranular and infragranular layers.

Parcellation of the lateral premotor cortex of the macaque monkey based on staining with the neurofilament antibody SMI-32.

In macaque monkey, frontal and parasagittal brain sections were stained with SMI-32, an antibody directed against a nonphosphorylated neurofilament protein that labels pyramidal cells. The goal of this investigation was to find reliable criteria with which to draw the border between the motor (M1) and premotor (PM) cortex and delineate subdivisions within the lateral PM. Two-dimensional reconstruction of the staining patterns was also performed by flattening the series of frontal sections. The distribution of SMI-32 immunoreactivity in layers III and V of the cortex revealed the existence of three subregions in the ventral rostral PM and a clear mediolateral boundary within the dorsal PM defined by clusters of SMI-32-positive pyramidal cells in layer V. The border between M1 and PM was easily distinguished at the level of the dorsal PM by a strong loss of immunoreactive pyramidal cells in layers III and V. At the level of the ventral PM there was no clear disruption of layer V pattern, and the border was set using the pattern of layer III immunoreactivity.

Carbon filaments direct the growth of postlesional plastic axons after spinal cord injury

The effect of implantation of carbon filaments and fetal tissues on the axonal regeneration following contusion injury in a rat model was investigated by in situ immunofluorescence. Female Sprague-Dawley rats were subjected to severe contusion injury to the spinal cord at T9–T10. All animals were divided into 5 groups (N=5/group): normal controls, surgical controls, with carbon filament implants, with fetal tissue implants and with implants consisting of fetal tissue cocultured with carbon filaments. After a 10-week survival period, the astroglial response was assessed by immunoreactive glial fibrillary acidic protein and the neuro-axonal profile by immunoreactive phosphorylated and nonphosphorylated neurofilament proteins. The contusion injury resulted in: (a) dramatically increased immunoreactivity of glial fibrillary acidic protein indicating injury-associated reactive astrogliosis, (b) increase in immunoreactive phosphorylated neurofilament protein indicating upregulated phosphorylation of neurofilament protein, (c) with no change in the highly differentiated nonphosphorylated neurofilament protein which normally occur in the nonregenerating mature neurons. Implantation of fetal tissues alone following contusion injury did not show any appreciable change with regard to the immunoreactivities for the glial and neuronal markers studied, compared to the injury controls. However, the implantation of carbon filaments alone or together with fetal tissues directed the growth of glial fibrillary acidic protein-positive astroglia and phosphoneurofilament-positive neurites along the carbon fibers, with no effect on nonphosphoneurofilament protein. In conclusion, implantation of carbon filaments appears to be critical for facilitating the attachment of astroglia forming a substrate and scaffolding that can further support and direct the growth of postlesional plastic axons across the lesion. In addition, carbon filament prostheses in combination with fetal tissue implants provides an improved combinational approach to promote regrowth of injured neurons following injury.

Expression of neuronal markers in oligodendrogliomas: an immunohistochemical study.

The oligodendroglioma has been considered to be a tumour showing oligodendrocyte differentiation, but studies of the expression of oligodendrocyte markers have not conclusively demonstrated this and the pattern of differentiation of this tumour remains uncertain. Recent studies have suggested that some oligodendrogliomas may show neuronal differentiation. The aim of this study was to determine whether there was evidence of neuronal differentiation in a series of oligodendrogliomas, and, if so, to determine whether this identified a biologically or clinically distinct group. Immunohistochemistry was carried out on paraffin sections using antibodies to synaptophysin, phosphorylated and non-phosphorylated neurofilament proteins. An archival series of 32 oligodendrogliomas had been previously characterized for histological features, histological grade, Ki-67 labelling index, apoptosis index and prognosis. Six per cent of tumours showed expression of synaptophysin. Thirty-one per cent of cases showed expression of neurofilament proteins with an antibody to non-phosphorylated epitopes, but no cases were positive with antibodies to phosphorylated neurofilament epitopes. Tumours showing expression of neuronal markers did not show a difference in the distribution of histological grade or GFAP expression from those which did not express these markers, and there was no difference in labelling indices or prognosis between the two groups. In conclusion, a subset of oligodendrogliomas showed expression of neuronal lineage markers; this is discussed in relation to histogenesis and differential diagnosis. The expression of such markers did not identify a biologically or clinically distinct subgroup.

Developmental changes in human cerebellum: Expression of intracellular calcium receptors, calcium-binding proteins, and phosphorylated and nonphosphorylated neurofilament protein

Few recent data are available on the development of the precise projection maps of the cerebellar cortex in humans. To address this topic, we studied temporal and spatial distribution of several antigens involved in calcium (Ca)-dependent processes: the intracellular Ca receptors, inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) and ryanodine receptor (RyR); the Ca-binding proteins, calbindin D-28k (CB), parvalbumin (PV), and synaptophysin; and phosphorylated (SMI 31) and nonphosphorylated (SMI 32) forms of neurofilament protein. All antigens were studied in the human cerebellum during intrauterine development. The results of this study show that immunocytochemical markers appeared in the following sequence: CB and both forms ofneurofilament protein were observed at 4-5 gestational weeks (g.w.), PV appeared in the external granular layer and in a few Purkinje cells at 11 g.w., a diffuse immunostaining for IP3R1 and synaptophysin were observed at 13 g.w., whereas RyR was observed at 17-18 g.w. From 24 g.w. on, Purkinje cells expressed all four examined markers of intracellular Ca signaling as well as two forms of neurofilament protein. At the same time, compartmentation of the Purkinje cell layer was detected with three intracellular Ca-signaling molecules (IP3R1, CB, and PV) and with SMI 32. These results indicate that the developmentally regulated expression of antigens studied here may play a role in establishing a highly regular organization of terminal fields in the human cerebellar cortex. Moreover, the initial expression of these antigens is correlated temporally with other developmental processes in the cerebellum, such as cellular maturation, revealed by the immunoreaction to cytoskeletal protein, and synaptogenesis, revealed by immunoreaction to synaptophysin.