SMI-32 Antibody Research Articles

Neurofilament Proteins in Y-Cells of the Cat Lateral Geniculate Nucleus: Normal Expression and Alteration with Visual Deprivation

We examined neurofilament staining in the normal and visually deprived lateral geniculate nucleus (LGN), using the SMI-32 antibody. This antibody preferentially stains LGN cells that display the morphological characteristics of Y-cells. The soma sizes of SMI-32-stained cells were consistent with those of the overall population of Y-cells, and the Golgi-like staining of their dendrites revealed a radial distribution that often crossed laminar boundaries. Labeled cells were distributed within the A laminae (primarily near laminar borders), the magnocellular portion of the C laminae, and the medial intralaminar nucleus, but they were absent in the parvocellular C laminae. Electron microscopic examination of SMI-32-stained tissue revealed that staining was confined to somata, dendrites, and large myelinated axons. Retinal synapses on SMI-32-labeled dendrites were primarily simple axodendritic contacts; few triadic arrangements were observed. In the LGN of cats reared with monocular lid suture, SMI-32 staining was decreased significantly in the A laminae that received input from the deprived eye. Dephosphorylation of the tissue did not alter the cellular SMI-32 staining patterns. Analysis of staining patterns in the C laminae and monocular zone of the A laminae suggests that changes in the cytoskeleton after lid suture reflect cell class and not binocular competition. Taken together, the results from normal and lid-sutured animals suggest that the cat LGN offers a unique model system in which the cytoskeleton of one class of cells can be manipulated by altering neuronal activity.

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

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.

Developmentally regulated markers in the postnatal cervical spinal cord of the opossum Monodelphis domestica

The aim of this study was to identify developmentally regulated immunocytochemical markers to assess development in the cervical spinal cord of Monodelphis domestica. We demonstrate that two commercially available antibodies exhibit altered patterns of distribution during early postnatal development. Although neurofilament staining was present at birth, only the phosphorylated form, recognised by monoclonal antibodies 2F11 or SMI31 could be detected. Non-phosphorylated neurofilament, recognised by monoclonal antibody SMI32, only became detectable around postnatal day 4 (P4) but was restricted to cells in the ventral horn until 5 weeks postnatum. By 7.5 weeks, SMI32 immunoreactivity (IR) was found throughout the grey matter in a pattern similar to that in the adult for both SMI32 and microtubule-associated protein 2 (MAP2). The intermediate filament proteins, glial fibrillary acidic protein (GFAP) and vimentin (VIM), were detectable at birth in radially oriented, fibrous cells, but GFAP-IR was restricted to the ventral half of the cord. This ventral to dorsal gradient of GFAP-IR diminished during the first week of postnatal life, disappearing by P8. Many astrocyte-like, GFAP-positive cells were clearly present by 38 days and, in the adult, were abundant in the white matter. A few VIM-IR cells remained in the adult cord, also within the white matter. We suggest that SMI32 and GFAP are useful, developmentally regulated markers for studies of opossum spinal cord development. We are currently using these markers to investigate the pronounced rostral to caudal gradient in the postnatal spinal cord and to assess development in the cultured spinal cord.

Multiple divisions of macaque precentral motor cortex identified with neurofilament antibody SMI-32

In brain sections stained with monoclonal antibody SMI-32, which recognizes non-phosphorylated neurofilament protein, we distinguished separate caudal, intermediate, and rostral subdivisions of gigantocellular precentral cortex (areas 4c, 4i, and 4r) in macaque monkeys. The divisions form bands extending mediolaterally across the major body-region representations of the primary motor cortex (M1). These observations provide additional evidence that primary motor cortex is not a single, structurally homogeneous cortical area.

Stable expression and heterologous coupling of the kappa opioid receptor in cell lines of neural and nonneural origin

Signal transduction cascades initiated by the neuronal κ opioid receptor were studied following transfection of a neuronal (hippocampal) line, HN2, and the non-neural CHOs. Retinoic-acid mediated differentiation resulted in intense staining of the HN2 cells with a neurofilament protein antibody SMI 33 but not with an antibody to GFAP, thus establishing neuronal characteristics of the HN2 cell line. The k opioid receptor was stably expressed in the two cell lines by electroporation mediated transfer of a Cytomegalovirus-promoter driven construct, pCMV-κ, harboring the κ-opioid receptor cDNA. Positive clones (HN2κ24 and CHOκ18) from both lines showed high expression of the k opioid receptor, as identified by [ 3H] U-69,593 binding to membranes prepared from HN2κ24 and CHOκ 18. Scatchard analysis revealed the presence of high affinity k opioid receptors in both engineered cell lines (K D = 1.3 nM for HN2κ24 and 2.1 nM for CHOκ18). Functional coupling to adenylate cyclase was displayed by 1 μM U-69,593 mediated inhibition (55–63%) of prostaglandin eistimulated intracellular cAMP levels. A major difference between the two clones was observed in functional coupling of the expressed κ opioid receptor to phospholipases C (PL-C) and D (PL-D). U-69,593 (1 μM) treatment stimulated PL-C, but not PL-D, in HN2κ24 cells, whereas PL-D, but not PL-C, was stimulated following such treatment of CHOκ 18 cells. Our results using the model neuronal system, HN2κ24, demonstrate cell-type specific, positive coupling of the kappa opioid receptor to the major Ca 2+ mobilizing system, the PL-C cascade, which regulates neuronal firing.

Differential expression of neurofilament protein in the visual system of the vervet monkey

It has been previously reported that the monoclonal antibody SMI-32 reveals a characteristic pattern of immunostaining which may be used to delineate various cortical modules in the monkey visual system. We wished to examine staining patterns with this antibody at both the lateral geniculate nucleus (LGN) and cortical levels with regard to magno- and parvocellular processing schemes in the vervet monkey. Using standard immunohistochemical procedures, we have found that the M-layers of the LGN were intensely stained in comparison to P-layers and that there were regional variations in staining within the visual cortex that reflected this input. The transition between areas Vl and V2 was especially prominent due to differences in the laminar staining profiles. Another striking result was found within the superior temporal sulcus where heavy SMI-32 immunostaining confined to the floor of the sulcus coincided with a similar zone of intense myelin staining. We have also found a number of other areas within the intraparietal and lateral sulci that show foci of heavy SMI-32 staining. As with Cat-301 immunostaining, the regional variabilities that are observed with SMI-32 in the visual cortex reflect molecular distinctions that may provide further criteria for functional segmentation.

Immunoreactivities for calcium signaling components and neural-like properties of a Colorado potato beetle cell line.

Of the approximately 400 described insect cell lines, only three are derived from beetles (Coleoptera), and none are neural in origin. The present work was undertaken to characterize further a new cell line, designated IPLB-CPB2, derived from eggs of the Colorado potato beetle, Leptinotarsa decimlineata. Indirect immunofluorescent studies reported here indicate that these cells express neurofilament (Nf)-like immunoreactivities to antibodies directed against mammalian Nf-Medium (MW 150,000) and a heavily phosphorylated form of Nf-Heavy (MW 200,000; detected using the axonal monoclonal antibody SMI 31). This appears to be the first report of neurofilament-like immunoreactivity in an arthropod. Immunofluorescent analyses also indicate that IPLB-CPB2 cells express an antigenic epitope characteristic of the mammalian type 1 inositol trisphosphate (IP3) receptor, the ryanodine receptor (RyR) (also termed calcium-induced calcium release receptor channel, or CICR) and the sarco(endo)plasmic reticulum Ca2+ pump (SERCA). Patch-clamp recordings indicate that some IPLB-CPB2 cells are capable of producing spontaneous action potentials, while others may be photosensitive. Taken together, the findings reported here suggest that IPLB-CPB2 cells are of neural origin and that they express the major receptor channels and pumps known to be localized in the ER of the cell, which are required for receptor-mediated calcium signaling. Thus, the IPLB-CPB2 cell line may prove to be an excellent model system for studies of insect neurobiology and calcium-based signal transduction.

403 Characterization of mAb AP422, a novel phosphorylation-dependent monoclonal antibody against Tau protein

Modified forms of tau proteins are major components of the paired helical filaments (PHFs) present in Alzheimer brains. In this study, tau from cytosolic samples obtained from normal and Alzheimer disease brains were fractionated by iron-chelated affinity chromatography (ICAC) to discriminate between isoforms phosphorylated to different extents using an stepwise pH gradient. Immunoblot analysis of the different fractions using antibody Tau-1 (recognizing art unphosphorylated epitope in tau and in PHF-tau after dephosphorylation) and antibody SMI 31 (recognizing a phosphorylated epitope in PHF-tau) have been carried out. Phosphorylated tau species (Tau 1-nonreactive and SMI 31-reactive) are only isolated from the Alzheimer samples at pH = 8.5. These tau species although having other Ser/Thr-Pro motifs susceptible of phosphorylation by proline-directed protein kinases are not further phosphorylated in vitro by MAP2 kinase whereas the fraction isolated at pH 7.0, which contains underphosphorylated tau species, is phosphorylated. Thus, soluble tau species phosphorylated both at the sites constituting the Tau-1 and the SMI 31 epitopes are present in Alzheimer but not in normal brain cytosol and can be isolated by ICAC. These modifications may be a prerequisite for PHF formation.

405 Tau in fibroblasts with and without the Swedish APP 670/671 mutation

Modified forms of tau proteins are major components of the paired helical filaments (PHFs) present in Alzheimer brains. In this study, tau from cytosolic samples obtained from normal and Alzheimer disease brains were fractionated by iron-chelated affinity chromatography (ICAC) to discriminate between isoforms phosphorylated to different extents using an stepwise pH gradient. Immunoblot analysis of the different fractions using antibody Tau-1 (recognizing art unphosphorylated epitope in tau and in PHF-tau after dephosphorylation) and antibody SMI 31 (recognizing a phosphorylated epitope in PHF-tau) have been carried out. Phosphorylated tau species (Tau 1-nonreactive and SMI 31-reactive) are only isolated from the Alzheimer samples at pH = 8.5. These tau species although having other Ser/Thr-Pro motifs susceptible of phosphorylation by proline-directed protein kinases are not further phosphorylated in vitro by MAP2 kinase whereas the fraction isolated at pH 7.0, which contains underphosphorylated tau species, is phosphorylated. Thus, soluble tau species phosphorylated both at the sites constituting the Tau-1 and the SMI 31 epitopes are present in Alzheimer but not in normal brain cytosol and can be isolated by ICAC. These modifications may be a prerequisite for PHF formation.

Neurochemical subdivisions of the inferior pulvinar in macaque monkeys.

The architecture of the pulvinar of rhesus monkeys was investigated by acetylcholinesterase (AChE) histochemistry, and by immunocytochemistry for calbindin-D28k and the SMI-32 antibody. The presence of four inferior subdivisions, comparable to those found in architectonic-connectional studies in squirrel monkeys (C.G. Cusick, J.L. Scripter, J.G. Darensbourg, and J.T. Weber, 1993, J. Comp. Neurol. 336:1-30), provided a basis for a proposed revised terminology for visual sectors of the macaque pulvinar. In the present study, the inferior pulvinar (PI) was identified as a neurochemically distinct region that included the traditional cytoarchitectonic nucleus PI and adjacent portions of the lateral and medial pulvinar nuclei, PL and PM. In calbindin-D28k stains, the lateral subdivision of the inferior pulvinar (PIL) had less intense neuropil staining than the adjacent central division, PIC. The PIL was characterized by large, intensely immunopositive neurons seldom found within PIC. PIL occupied the traditional PL and PI and exhibited a narrow shell zone, PIL-S, restricted to PL. The medial division of the inferior pulvinar (PIM) was in a location previously shown to be strongly connected with the middle temporal visual area (MT) in macaques. PIM was found in the medial one-half of the traditional PI and extended into adjacent portions of the traditional PM and PL. PIM was distinguished by less intense neuropil staining for calbindin and many cells stained with the SMI-32 antibody for neurofilament protein. In AChE stains, PIL was moderately dark, PIC appeared lighter, and PIM was characterized by small, intensely stained patches. The small posterior division (PIP) stained darkly for calbindin, lightly for AChE, and was unstained with the SMI-32 antibody. Thus, neurochemical, and perhaps connectional, subdivisions exist within PI, the region of the pulvinar that relays information to striate, "lower order" extrastriate, and inferotemporal visual cortex.

Neurochemical phenotype of corticocortical connections in the macaque monkey: Quantitative analysis of a subset of neurofilament protein‐immunoreactive projection neurons in frontal, parietal, temporal, and cingulate cortices

The neurochemical characteristics of the neuronal subsets that furnish different types of corticocortical connections have been only partially determined. In recent years, several cytoskeletal proteins have emerged as reliable markers to distinguish subsets of pyramidal neurons in the cerebral cortex of primates. In particular, previous studies using an antibody to nonphosphorylated neurofilament protein (SMI-32) have revealed a consistent degree of regional and laminar specificity in the distribution of a subpopulation of pyramidal cells in the primate cerebral cortex. The density of neurofilament protein-immunoreactive neurons was shown to vary across corticocortical pathways in macaque monkeys. In the present study, we have used the antibody SMI-32 to examine further and to quantify the distribution of a subset of corticocortically projecting neurons in a series of long ipsilateral corticocortical pathways in comparison to short corticocortical, commissural, and limbic connections. The results demonstrate that the long association pathways interconnecting the frontal, parietal, and temporal neocortex have a high representation of neurofilament protein-enriched pyramidal neurons (45-90%), whereas short corticocortical, callosal, and limbic pathways are characterized by much lower numbers of such neurons (4-35%). These data suggest that different types of corticocortical connections have differential representation of highly specific neuronal subsets that share common neurochemical characteristics, thereby determining regional and laminar cortical patterns of morphological and molecular heterogeneity. These differences in neuronal neurochemical phenotype among corticocortical circuits may have considerable influence on cortical processing and may be directly related to the type of integrative function subserved by each cortical pathway. Finally, it is worth noting that neurofilament protein-immunoreactive neurons are dramatically affected in the course of Alzheimer's disease. The present results support the hypothesis that neurofilament protein may be crucially linked to the development of selective neuronal vulnerability and subsequent disruption of corticocortical pathways that lead to the severe impairment of cognitive function commonly observed in age-related dementing disorders.

Chemoarchitectonics and corticocortical terminations within the superior temporal sulcus of the rhesus monkey: Evidence for subdivisions of superior temporal polysensory cortex

Cortex of the upper bank of the superior temporal sulcus (STS) in macaque monkeys, termed the superior temporal polysensory (STP) region, corresponds largely to architectonic area TPO and is connectionally distinct from adjacent visual areas. To investigate whether or not the STP region contains separate subdivisions, immunostaining for parvalbumin and neurofilament protein (using the SMI-32 antibody) was compared with patterns of corticocortical terminations in the STS. Chemoarchitectonic results provided evidence for three caudal-to-rostral subdivisions: TPOc, TPOi, and TPOr. Area TPOc was characterized by patchy staining for parvalbumin and SMI-32 in cortical layers IV/III and III, respectively. Area TPOi had more uniform chemoarchitectonic staining, whereas area TPOr had a thicker layer IV than TPOi. The connectional results showed prefrontal cortex in the location of the frontal eye fields (area 8) and dorsal area 46 projected in a columnar pattern to all cortical layers of area TPOc, to layer IV of TPOi, and in a columnar fashion, with a moderate increase in density in layer IV, to TPOr. In TPOc, columns of frontal connections showed a periodicity similar to that of the SMI-32 staining. The caudal inferior parietal lobule (area 7a) and superior temporal gyrus projected to each subdivision of area TPO, displaying either panlaminar or fourth-layer terminations. In addition to STP cortex, parvalbumin and SMI-32 immunostaining allowed identification of caudal visual areas of the STS, including MT, MST, FST, and V4t. These areas received first- and sixth-layer projections from prefrontal cortex and area 7a.

Isolation of a phosphorylated soluble Tau fraction from Alzheimer's disease brain

Modified forms of tau proteins are major components of the paired helical filaments (PHFs) present in Alzheimer brains. In this study, tau from cytosolic samples obtained from normal and Alzheimer disease brains were fractionated by iron-chelated affinity chromatography (ICAC) to discriminate between isoforms phosphorylated to different extents using an stepwise pH gradient. Immunoblot analysis of the different fractions using antibody Tau-1 (recognizing art unphosphorylated epitope in tau and in PHF-tau after dephosphorylation) and antibody SMI 31 (recognizing a phosphorylated epitope in PHF-tau) have been carried out. Phosphorylated tau species (Tau 1-nonreactive and SMI 31-reactive) are only isolated from the Alzheimer samples at pH = 8.5. These tau species although having other Ser/Thr-Pro motifs susceptible of phosphorylation by proline-directed protein kinases are not further phosphorylated in vitro by MAP2 kinase whereas the fraction isolated at pH 7.0, which contains underphosphorylated tau species, is phosphorylated. Thus, soluble tau species phosphorylated both at the sites constituting the Tau-1 and the SMI 31 epitopes are present in Alzheimer but not in normal brain cytosol and can be isolated by ICAC. These modifications may be a prerequisite for PHF formation.

SMI-32 antibody against non-phosphorylated neurofilaments identifies a subpopulation of cultured cortical neurons hypersensitive to kainate toxicity

SMI-32, an antibody against a non-phosphorylated neurofilament epitope identifies a subpopulation of human cortical neurons preferentially lost in Alzheimer's or Huntington's disease. In murine cortical cultures SMI-32 labeled a small subset of neurons exhibiting enhanced vulnerability to kainate toxicity. Most SMI-32(+) neurons were GABAergic and exhibited kainate-activated C0 2+ uptake. Thus expression of Ca 2+ permeable AMPA or kainate receptor-gated channels likely underlies the heightened vulnerability of SMI-32(+) cortical neurons to kainate.

Information processing within the motor cortex. II. Intracortical connections between neurons receiving somatosensory cortical input and motor output neurons of the cortex

Connections between motor cortical neurons receiving somatosensory inputs from area 2 and large pyramidal cells in layer V were examined in the cat via intracellular injection of biocytin and immunohistochemistry of nonphosphorylated neurofilament proteins (npNFP). Biocytin was injected into pyramidal cells in layers II/III of the motor cortex that responded monosynaptically and polysynaptically to microstimulation of the somatosensory cortex and subsequently stained black by the avidin-biotinylated peroxidase complex method with diaminobenzidine (DAB) and nickel. By using a monoclonal antibody SMI-32 and a modified peroxidase-antiperoxidase method with Tris-aminophenyl-methane (TAPM) and p-cresol as a chromogen, pyramidal cells in layers III and V of the motor cortex were stained red for npNFP. In particular, all the large pyramidal cells in layer V, Betz cells, displayed intense npNFP immunoreactivity not only in the perikarya but also in the dendrites. Double staining with DAB/nickel and TAPM/p-cresol showed that biocytin-filled axon varicosities of the pyramidal cells, which were thought to receive monosynaptic inputs from area 2, made contacts with npNFP-positive dendrites in layers I-III around the biocytin-injected cell and in layers V-VI beneath the cell. The present results suggest that the corticocortical input from area 2 to pyramidal cells in layers II/III of the motor cortex is transferred to layer V pyramidal cells, including Betz cells, as well as to neighboring layer II/III pyramidal cells. Since tetanic stimulation of the somatosensory cortex reportedly produces long-term potentiation in layer II/III cells of the motor cortex, it seems reasonable to assume that a given area of the somatosensory cortex can produce a long-lasting change in the activity of a given group of output cells in the motor cortex.

Transient immunohistochemical labelling of rat retinal axons during Wallerian degeneration by a monoclonal antibody to neurofilaments

Immunohistochemical labelling with the monoclonal antibody SMI32 to non-phosphorylated epitopes on neurofilament proteins of high molecular weight class was low in rat central optic fibers of controls. After unilateral transection of optic nerve, a strong, transient increase of labelling with SMI32 occurred in degenerating fibers of optic tract at 2 and 4 days, which then declined at 8 and remained low at 21 days. Consequently, immunostaining with SMI32 may serve as a positive marker for degenerating fibers in rat optic system.

A study of SMI 32-stained pyramidal cells, parvalbumin-immunoreactive chandelier cells, and presumptive thalamocortical axons in the human temporal neocortex.

Immunocytochemical studies in the primate neocortex have shown that particular populations of pyramidal cells can be identified by antibody SMI 32 that recognizes a nonphosphorylated epitope of neurofilament protein, while chandelier cells (a powerful type of cortical inhibitory interneuron) and presumptive thalamocortical axons can be identified by antibodies directed against the calcium-binding protein parvalbumin (PV). We used these antibodies in correlative light and electron microscopic immunocytochemical studies to analyze certain aspects of the synaptic circuitry of human temporal neocortex. In sections cut in the tangential plane, many PV-immunoreactive chandelier cell axon terminals and apical dendrites of SMI 32-stained pyramidal cells were distributed in small clusters. Combination of immunocytochemistry for PV and SMI 32 revealed four subpopulations of pyramidal cells with regard to the immunocytochemical staining by SMI 32 and the innervation of their axon initial segments by PV-positive or -negative chandelier cell axon terminals, but there were differences in the concentration and proportion of these subpopulations by layers. Furthermore, we present electron microscopic evidence suggesting that the characteristic layer III dense band of PV-immunoreactive puncta is made up mainly of presumptive thalamocortical axon terminals. Besides, coincidence was found between the dense PV-immunoreactive band and the dendritic plexus formed by the SMI 32-stained pyramidal cells in the lower half of layer III, which leads us to think that they are probably a major target of PV-immunoreactive thalamic terminations.

Ultrastructural studies of diffuse axonal injury in humans.

Diffuse axonal injury (DAI) is observed commonly in traumatically brain injured humans. However, traditional histologic methods have proven of limited use in identifying reactive axonal change early (< 12 h) in the posttraumatic course. Recently, we have reported, in both humans and animals, that antibodies targeting neurofilament subunits are useful in the light microscopic recognition of early reactive change. In the present study, we extend our previous efforts in humans by analyzing the progression of traumatic brain injury (TBI)-induced axonal change at the ultrastructural level. This effort was initiated to follow the subcellular progression of reactive axonal change in humans and to determine whether this progression parallels that described in animals. Two commercially prepared antibodies were used to recognize reactive axonal change in patients surviving from 6 to 88 h. The NR4 antibody was used to target the light neurofilament subunit (NF-L), and the SMI32 antibody was used to target the heavy neurofilament subunit (NF-H). Plastic-embedded tissue sections were screened for evidence of reactive axonal change, and once identified, this reactive change was analyzed at the ultrastructural level. At 6 h survival, focally enlarged, immunoreactive axons with axolemmal infolding or disordered neurofilaments were seen within fields of axons exhibiting no apparent abnormality. By 12 h, some axons exhibited continued neurofilamentous misalignment, pronounced immunoreactivity, vacuolization, and, occasionally, disconnection. At later stages, specifically 30 and 60 h survival, further accumulation of neurofilaments and organelles had led to the further expansion of the axis cylinder, and clearly disconnected reactive swellings were recognized. These contained a dense core of disordered immunoreactive neurofilaments partially encompassed by a cap of less densely aggregated organelles. At 88 h, the reactive axons were larger and elongated, consistent with the continued delivery of organelles by axoplasmic transport. At the later time points, considerable heterogeneity was observed, with focally enlarged disconnected axons being observed in relation to axons showing less advanced reactive change. Our findings suggest that neurofilamentous disruption is a pivotal event in axonal injury.

Immunohistochemical patterns of selective cellular vulnerability in human cerebral ischemia

Although specific patterns of cellular vulnerability have been identified in experimental models of cerebral ischemia, there is little data on the occurrence of similar abnormalities in human ischemia. We therefore used a variety of histochemical methods to define changes affecting specific classes of cells in post-mortem specimens from seven patients with hippocampal and neocortical ischemic lesions. In acute lesions, staining with SMI-32, an antibody directed against nonphosphorylated neurofilaments that labels pyramidal projection neurons, was prominently depleted even when conventional Nissl staining revealed only mild pyknosis. In contrast, staining for other markers such as microtubule-associated protein 2 (MAP-2), another cytoskeletal protein, or parvalbumin, a calcium-binding protein found in γ-aminobutyric acid (GABA)-ergic interneurons, were relatively preserved. SMI-32 antibody also labeled dystrophic axons and axonal retraction balls in and around acute ischemic lesions. The pattern of differential changes in immunoreactivity was essentially the same in all acute ischemic injuries, including both diffuse lesions in the CA1 field (Sommer's sector) and discrete infarcts in CA1 and neocortex. In addition, immunoreactivity for the immediate early gene product c- fos was enhanced in and around the acute ischemic lesions that we studied. In some very acute lesions, immunoreactivity for glial fibrillary acidic protein (GFAP) was depleted in areas of severe ischemia and necrosis, but, as expected, GFAP immunoreactivity was increased in lesions more than a few days old. In contrast, the loss of SMI-32 immunoreactivity persisted in chronic lesions. These findings are consistent with those of experimental ischemia in animals and confirm the relevance of these studies for human cerebral ischemia. The pattern of selective changes also resembles that of injuries induced directly by excitatory amino acids, which may play a significant role in the pathogenesis of ischemic damage.

Quantitative analysis of a vulnerable subset of pyramidal neurons in Alzheimer's disease: II. Primary and secondary visual cortex

In this study we investigated the primary and secondary visual areas of normal and Alzheimer's disease brains by using the SMI32 antibody. It is known that in Alzheimer's disease primary sensory areas are usually less devastated than association cortices, although visual symptomatology has been documented early in the course of the disease. In area 17, the SMI32 antibody primarily labeled the perikarya and dentritic tree of the large Meynert cells and cells in layer IVB. Smaller neurons in layers III, V, and VI were also immunoreactive (ir). In area 18, very large SMI32-ir pyramidal neurons in layers III and V were observed. In both areas, staining intensity was correlated with cell size, the largest neurons being the most intensely stained. Only a few changes were observed in the Alzheimer's disease cases. The only statistically significant differences in SMI32-ir neuron counts between control and Alzheimer's disease brains occurred in layer IVB cells and Meynert cells in area 17, and in layer III cells in area 18. In contrast with association cortices, there were no changes in staining intensity in the visual areas. There were fewer neurofibrillary tangles and neuritic plaques in these areas than in prefrontal and inferior temporal cortex, and a correlation between neurofibrillary tangle counts and SMI32-ir neuron loss was only observed in layer III of area 18. These observations show that in the primary and secondary visual cortex, SMI32 also labeled a distinct subset of pyramidal cells that are known from data obtained in the monkey brain to furnish long corticocortical as well as subcortical projections. Interestingly, although there is much less cell and/or neurofibrillary tangle formation in these occipital regions than in prefrontal and temporal association areas, there is significant loss within key subsets of pyramidal cells. The selective loss of this particular subpopulation of pyramidal neurons will disrupt association pathways linking primary visual cortex with areas involved in higher level visual processing. The partial disconnection of such pathways may be relevant to the visual symptomatology frequently observed in Alzheimer's disease patients. These data further support the hypothesis that subtypes of pyramidal neurons with specific anatomical and molecular profiles may display a differential vulnerability in Alzheimer's disease.