Early postnatal development of the primary visual areas 17 and 18 of the cat cerebral cortex: An SMI-32 study.

Using anti-neurofilament H non-phosphorylated antibodies (SMI-32) as markers for the neuronal maturation level and Y channel responsible for motion processing, we investigated early postnatal development of the primary visual areas 17 and 18 in cats aged 0, 10, 14, and 34 days and in adults. Two analyzed parameters of SMI-32-immunolabeling were used: the total proportion of SMI-32-labeling and the density of labeled neurons. (i) The developmental time course of the total proportion of SMI-32-labeling shows the general increase in the accumulation of heavy-chain neurofilaments. This parameter showed a different time course for cortical layer development; the maximal increment in the total labeling in layer V occurred between the second and fifth postnatal weeks and in layers II-III and VI after the fifth postnatal week. In addition, the delay in accumulation of SMI-32-labeling was shown in layer V of the area 17 periphery representation during the first two postnatal weeks. (ii) The density of SMI-32-labeled neurons decreased in all layers of area 18, but was increased, decreased, or had a transient peak in layers II-III, V, and VI of area 17, respectively. The transient peak is in good correspondence with some transient neurochemical features previously revealed for different classes of cortical and thalamic neurons and reflects the time course of the early development of the thalamocortical circuitry. Some similarities between the time courses for the development of SMI-32-labeling in areas 17/18 and in A- and C-laminae of the LGNd allow us to propose heterochronous postnatal development of two Y sub-channels.

Inner Structure of the Lateral Geniculate Complex of Adult and Newborn Acomys cahirinus.

Acomys cahirinus is a unique Rodentia species with several distinctive physiological traits, such as precocial development and remarkable regenerative abilities. These characteristics render A. cahirinus increasingly valuable for regenerative and developmental physiology studies. Despite this, the structure and postnatal development of the central nervous system in A. cahirinus have been inadequately explored, with only sporadic data available. This study is the first in a series of papers addressing these gaps. Our first objective was to characterize the structure of the main visual thalamic region, the lateral geniculate complex, using several neuronal markers (including Ca2+-binding proteins, glutamic acid decarboxylase enzyme, and non-phosphorylated domains of heavy-chain neurofilaments) to label populations of principal neurons and interneurons in adult and newborn A. cahirinus. As typically found in other rodents, we identified three subdivisions in the geniculate complex: the dorsal and ventral lateral geniculate nuclei (LGNd and LGNv) and the intergeniculate leaflet (IGL). Additionally, we characterized internal diversity in the LGN nuclei. The "shell" and "core" regions of the LGNd were identified using calretinin in adults and newborns. In adults, the inner and outer parts of the LGNv were identified using calbindin, calretinin, parvalbumin, GAD67, and SMI-32, whereas in newborns, calretinin and SMI-32 were employed for this purpose. Our findings revealed more pronounced developmental changes in LGNd compared to LGNv and IGL, suggesting that LGNd is less mature at birth and more influenced by visual experience.

Transient expression of heavy-chain neurofilaments in the perigeniculate nucleus of cats.

The perigeniculate nucleus (PGN) is a visual part of the thalamic reticular nucleus modulating the information transfer between the lateral geniculate nucleus and the visual cortex. This study focused on the postnatal development of the PGN in cats, using the SMI-32 antibody, which recognizes non-phosphorylated heavy-chain neurofilaments responsible for neuronal structural maturation and is also used as a marker for motion processing, or Y, stream. We questioned whether transient neuronal populations exist in the PGN and can they possibly be related to the Y processing stream. We uncovered a transient, robust SMI-32 staining in the PGN of kittens aged 0-34 days with the significant decline in the cellular density of labeled cells in older animals. According to the double-labeling, in all examined age groups, perigeniculate SMI-32-immunopositive cells are part of the main parvalbumin-positive population. The maximal cellular density of the double-stained cells appeared in animals aged 10-28 days. We also revealed that the most significant growth of perigeniculate cells's soma occurred at three postnatal weeks. The possible link of our data to the development of the Y visual processing stream and to the heterogeneity of the perigeniculate neuronal population is also discussed.

The cortical spectrum: A robust structural continuum in primate cerebral cortex revealed by histological staining and magnetic resonance imaging.

High-level characterizations of the primate cerebral cortex sit between two extremes: on one end the cortical mantle is seen as a mosaic of structurally and functionally unique areas, and on the other it is seen as a uniform six-layered structure in which functional differences are defined solely by extrinsic connections. Neither of these extremes captures the crucial neuroanatomical finding: that the cortex exhibits systematic gradations in architectonic structure. These gradations have been shown to predict cortico-cortical connectivity, which in turn suggests powerful ways to ground connectomics in anatomical structure, and by extension cortical function. A challenge to widespread use of this concept is the labor-intensive and invasive nature of histological staining, which is the primary means of recognizing anatomical gradations. Here we show that a novel computational analysis technique can provide a coarse-grained picture of cortical variation. For each of 78 cortical areas spanning the entire cortical mantle of the rhesus macaque, we created a high dimensional set of anatomical features derived from captured images of cortical tissue stained for myelin and SMI-32. The method involved semi-automated de-noising of images, and enabled comparison of brain areas without hand-labeling of features such as layer boundaries. We applied multidimensional scaling (MDS) to the dataset to visualize similarity among cortical areas. This analysis shows a systematic variation between weakly laminated (limbic) cortices and sharply laminated (eulaminate) cortices. We call this smooth continuum the "cortical spectrum". We also show that this spectrum is visible within subsystems of the cortex: the occipital, parietal, temporal, motor, prefrontal, and insular cortices. We compared the MDS-derived spectrum with a spectrum produced using T1- and T2-weighted magnetic resonance imaging (MRI) data derived from macaque, and found close agreement of the two coarse-graining methods. This suggests that T1w/T2w data, routinely obtained in human MRI studies, can serve as an effective proxy for data derived from high-resolution histological methods. More generally, this approach shows that the cortical spectrum is robust to the specific method used to compare cortical areas, and is therefore a powerful tool to understand the principles of organization of the primate cortex.

Morphological and distributional properties of SMI-32 immunoreactive ganglion cells in the rat retina.

SMI-32 is widely used to identify entire populations of alpha retinal ganglion cells (RGCs), and several SMI-32+ RGC subsets have been studied thoroughly in rodents. However, due to the thick cover of SMI-32+ neurofilaments, the morphology of SMI-32+ RGCs in the central retinal region is obscured and rarely described. Moreover, SMI-32 labels more than one morphological RGC type and the full morphological characteristics and distribution of SMI-32+ RGCs have yet to be discovered. Here, using intracellular neurobiotin injections combined with SMI-32 antibody staining, we investigated morphological and distributional properties of the entire SMI-32+ RGCs population in the rat retina. We found that SMI-32+ RGCs were evenly distributed throughout the rat retina. We compared the morphological features of SMI-32+ ON and OFF cells in the central, middle, and peripheral retinal regions. We found that SMI-32+ RGCs in different regions have distinct characteristics, such as the soma area and the dendritic field area, and Sholl analysis of ON cells and OFF cells revealed significant differences between each region. We classified SMI-32+ RGCs into five clusters based on morphological features and found that a majority of SMI-32+ RGCs belong to alpha-like cells; however, a small proportion of SMI-32+ RGCs had small soma and small dendritic fields. Together, we present a full description of the morphology and distribution of SMI-32 immunoreactive RGCs in the rat retina.

SMI-32 labeling in Cajal-Retzius cells of feline primary visual cortex.

Cajal-Retzius cells are one of the transient elements of the developing cerebral cortex. These cells express some characteristic molecules. One of them, heavy-chain neurofilaments, participating in the construction of the mature cerebral networks, are believed to be a specific feature of the human's Cajal-Retzius cells. Using histochemical stain for SMI-32 antibody to the non-phosphorylated heavy-chain neurofilaments, large neurons having horizontally oriented soma and bipolar processes were labeled in the molecular layer of the primary visual cortex of cats aged 0-2 postnatal days. Using DiI technique, similar neurons having a well-developed system of parallel vertical branches coming from the two horizontal processes were visualized in these areas. The location and general morphology of these neurons were similar to the Cajal-Retzius cells allowing to suppose for the carnivores to share similar with primates developmental mechanisms of the corticogenesis.

What is a multisensory cortex? A laminar, connectional, and functional study of a ferret temporal cortical multisensory area.

Now that examples of multisensory neurons have been observed across the neocortex, this has led to some confusion about the features that actually designate a region as "multisensory." While the documentation of multisensory effects within many different cortical areas is clear, often little information is available about their proportions or net functional effects. To assess the compositional and functional features that contribute to the multisensory nature of a region, the present investigation used multichannel neuronal recording and tract tracing methods to examine the ferret temporal region: the lateral rostral suprasylvian sulcal area. Here, auditory-tactile multisensory neurons were predominant and constituted the majority of neurons across all cortical layers whose responses dominated the net spiking activity of the area. These results were then compared with a literature review of cortical multisensory data and were found to closely resemble multisensory features of other, higher-order sensory areas. Collectively, these observations argue that multisensory processing presents itself in hierarchical and area-specific ways, from regions that exhibit few multisensory features to those whose composition and processes are dominated by multisensory activity. It seems logical that the former exhibit some multisensory features (among many others), while the latter are legitimately designated as "multisensory."

Neurodevelopmental disorders of the prefrontal cortex in an evolutionary context.

The prefrontal cortex consists of several cytoarchitectonically defined areas that are involved in higher-order cognitive and emotional processing. The areas are highly variable in terms of organization of cortical layers and distribution of specific neuronal classes, and are affected in neurodevelopmental and psychiatric disorders. Here the focus is on microstructural anatomical characteristics of human prefrontal cortex in an evolutionary context with special emphasis on Williams syndrome. We include a pilot analysis of distribution of neurons labeled with an antibody to non-phosphorylated neurofilament protein (SMI-32) in the frontal pole of Williams syndrome to further examine microstructural characteristics of the prefrontal cortex in Williams syndrome and implications of the distribution of SMI-32 immunoreactive neurons for connectivity between the frontal pole and other cortical areas in the disorder.

Partitioning of the primate intraparietal cortex based on connectivity pattern and immunohistochemistry for Cat-301 and SMI-32.

We propose a partitioning of the primate intraparietal sulcus (IPS) using immunoarchitectural and connectivity criteria. We studied the immunoarchitecture of the IPS areas in the capuchin monkey using Cat-301 and SMI-32 immunohistochemistry. In addition, we investigated the IPS projections to areas V4, TEO, PO, and MT using retrograde tracer injections in nine hemispheres of seven animals. The pattern and distribution of Cat-301 and SMI-32 immunostaining revealed multiple areas in the IPS, in the adjoining PO cleft and in the annectant gyrus, with differential staining patterns found for areas V3d, DM, V3A, DI, PO, POd, CIP-1, CIP-2, VIPa, VIPp, LIPva, LIPvp, LIPda, LIPdp, PIPv, PIPd, MIPv, MIPd, AIPda, AIPdp, and AIPv. Areas V4, TEO, PO, MT, which belong to different cortical streams of visual information processing, receive projections from at least twenty different areas within the IPS and adjoining regions. In six animals, we analyzed the distribution of retrogradely labeled cells in tangential sections of flat-mount IPS preparations. The lateral bank of the IPS projects to regions belonging both to the ventral (V4 and TEO) and dorsal (PO and MT) streams. The region on the floor of the IPS (i.e., VIP) projects predominantly to dorsal stream areas. Finally, the medial bank of the IPS (i.e., MIP) projects solely to the dorsalmedial stream (PO). Therefore, our data suggest that ventral and dorsal streams remain segregated within the IPS, and that its projections to the dorsal stream can be further segregated based on those targeting the dorsolateral versus the dorsomedial subdivisions.

Neuroplastic change of cytoskeleton in inferior colliculus after auditory deafferentation.

Neural plasticity is a characteristic of the brain that helps it adapt to changes in sensory input. We hypothesize that auditory deafferentation may induce plastic changes in the cytoskeleton of the neurons in the inferior colliculus (IC). In this study, we evaluated the dynamic status of neurofilament (NF) phosphorylation in the IC after hearing loss. We induced auditory deafferentation via unilateral or bilateral cochlear ablation in rats, aged 4 weeks. To evaluate cytoskeletal changes in neurons, we evaluated mRNA fold changes in NF heavy chain expression, non-phosphorylated NF protein fold changes using SMI-32 antibody, and the ratio of SMI-32 immunoreactive (SMI-32-ir) neurons to the total neuronal population in the IC at 4 and 12 weeks after deafness. In the bilateral deafness (BD) group, the ratios of SMI-32-ir neurons significantly increased at 4 weeks after ablation in the right and left IC (6.1 ±â€¯4.4%, 5.0 ±â€¯3.4%, respectively), compared with age-matched controls (P < 0.01, P < 0.01). At 12 weeks after ablation, the ratio of SMI-32 positive neurons was higher (right, 3.4 ±â€¯2.0%; left, 3.2 ±â€¯2.3%) than that in the age-matched control group, albeit not significant in the right and left side (P = 0.38, P = 0.24, respectively). Consistent with the results of the ratio of SMI-32-ir neurons, SMI-32-ir protein expression was increased at 4 weeks after BD, and the changes at 12 weeks after bilateral ablation were not significant in the right or left IC. The age-matched control fold changes of NF mRNA expression after bilateral deafness were not significant at 4 and 12 weeks after deafness in right and left IC. Unilateral deafness did not induce significant change of NF mRNA expression, SMI-32-ir protein expression, and the ratio of SMI-32-ir neurons in the IC at 4 and 12 weeks after hearing loss. Bilateral auditory deafferentation induces structural changes in the neuronal cytoskeleton within the IC, which is prominent at 4 weeks after BD. The structural remodeling of neurons stabilized at 12 weeks after BD. Unlike BD, unilateral auditory deafferentation did not affect the dynamic status of NFs in the IC.

Morphological Identification of Melanopsin-Expressing Retinal Ganglion Cell Subtypes in Mice.

Melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) are a relatively recently discovered class of photoreceptor. ipRGCs can be subdivided into at least five subtypes (M1-M5), each of which has a distinct complement of morphological and physiological properties. ipRGC subtypes can be identified morphologically based on a combination of dendritic morphology and immunostaining for a cell-type specific marker. In this chapter, we describe methods for conclusively identifying each of the five ipRGC subtypes through a combination of patch clamp electrophysiology, Neurobiotin filling, visualization of ipRGC dendrites, and immunostaining for the marker SMI-32.

Metabolomics and neuroanatomical evaluation of post-mortem changes in the hippocampus.

Understanding the human brain is the ultimate goal in neuroscience, but this is extremely challenging in part due to the fact that brain tissue obtained from autopsy is practically the only source of normal brain tissue and also since changes at different levels of biological organization (genetic, molecular, biochemical, anatomical) occur after death due to multiple mechanisms. Here we used metabolomic and anatomical techniques to study the possible relationship between post-mortem time (PT)-induced changes that may occur at both the metabolomics and anatomical levels in the same brains. Our experiments have mainly focused on the hippocampus of the mouse. We found significant metabolomic changes at 2 h PT, whereas the integrity of neurons and glia, at the anatomical/ neurochemical level, was not significantly altered during the first 5 h PT for the majority of histological markers.

Intrinsic Connections of the Core Auditory Cortical Regions and Rostral Supratemporal Plane in the Macaque Monkey.

In the ventral stream of the primate auditory cortex, cortico-cortical projections emanate from the primary auditory cortex (AI) along 2 principal axes: one mediolateral, the other caudorostral. Connections in the mediolateral direction from core, to belt, to parabelt, have been well described, but less is known about the flow of information along the supratemporal plane (STP) in the caudorostral dimension. Neuroanatomical tracers were injected throughout the caudorostral extent of the auditory core and rostral STP by direct visualization of the cortical surface. Auditory cortical areas were distinguished by SMI-32 immunostaining for neurofilament, in addition to established cytoarchitectonic criteria. The results describe a pathway comprising step-wise projections from AI through the rostral and rostrotemporal fields of the core (R and RT), continuing to the recently identified rostrotemporal polar field (RTp) and the dorsal temporal pole. Each area was strongly and reciprocally connected with the areas immediately caudal and rostral to it, though deviations from strictly serial connectivity were observed. In RTp, inputs converged from core, belt, parabelt, and the auditory thalamus, as well as higher order cortical regions. The results support a rostrally directed flow of auditory information with complex and recurrent connections, similar to the ventral stream of macaque visual cortex.

Neurofilament heavy chain expression and neuroplasticity in rat auditory cortex after unilateral and bilateral deafness.

Deafness induces many plastic changes in the auditory neural system. For instance, dendritic changes cause synaptic changes in neural cells. SMI-32, a monoclonal antibody reveals auditory areas and recognizes non-phosphorylated epitopes on medium- and high-molecular-weight subunits of neurofilament proteins in cortical pyramidal neuron dendrites. We investigated SMI-32-immunoreactive (-ir) protein levels in the auditory cortices of rats with induced unilateral and bilateral deafness. Adult male Sprague-Dawley rats were divided into unilateral deafness (UD), bilateral deafness (BD), and control groups. Deafness was induced by cochlear ablation. All rats were sacrificed, and the auditory cortices were harvested for real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analyses at 2, 4, 6, and 12 weeks after deafness was induced. Immunohistochemical staining was performed to evaluate the location of SMI-32-ir neurons. Neurofilament heavy chain (NEFH) mRNA expression and SMI-32-ir protein levels were increased in the BD group. In particular, SMI-32-ir protein levels increased significantly 6 and 12 weeks after deafness was induced. In contrast, no significant changes in protein level were detected in the right or left auditory cortices at any time point in the UD group. NEFH mRNA level decreased at 4 weeks after deafness was induced in the UD group, but recovered thereafter. Taken together, BD induced plastic changes in the auditory cortex, whereas UD did not affect the auditory neural system sufficiently to show plastic changes, as measured by neurofilament protein level.

The influence of aging on the number of neurons and levels of non-phosporylated neurofilament proteins in the central auditory system of rats.

In the present study, an unbiased stereological method was used to determine the number of all neurons in Nissl stained sections of the inferior colliculus (IC), medial geniculate body (MGB), and auditory cortex (AC) in rats (strains Long Evans and Fischer 344) and their changes with aging. In addition, using the optical fractionator and western blot technique, we also evaluated the number of SMI-32-immunoreactive (-ir) neurons and levels of non-phosphorylated neurofilament proteins in the IC, MGB, AC, and visual cortex of young and old rats of the two strains. The SMI-32 positive neuronal population comprises about 10% of all neurons in the rat IC, MGB, and AC and represents a prevalent population of large neurons with highly myelinated and projecting processes. In both Long Evans and Fischer 344 rats, the total number of neurons in the IC was roughly similar to that in the AC. With aging, we found a rather mild and statistically non-significant decline in the total number of neurons in all three analyzed auditory regions in both rat strains. In contrast to this, the absolute number of SMI-32-ir neurons in both Long Evans and Fischer 344 rats significantly decreased with aging in all the examined structures. The western blot technique also revealed a significant age-related decline in the levels of non-phosphorylated neurofilaments in the auditory brain structures, 30-35%. Our results demonstrate that presbycusis in rats is not likely to be primarily associated with changes in the total number of neurons. On the other hand, the pronounced age-related decline in the number of neurons containing non-phosphorylated neurofilaments as well as their protein levels in the central auditory system may contribute to age-related deterioration of hearing function.

Moniliform deformation of retinal ganglion cells by formaldehyde-based fixatives.

Protocols for characterizing cellular phenotypes commonly use chemical fixatives to preserve anatomical features, mechanically stabilize tissue, and stop physiological responses. Formaldehyde, diluted in either phosphate-buffered saline or phosphate buffer, has been widely used in studies of neurons, especially in conjunction with dyes and antibodies. However, previous studies have found that these fixatives induce the formation of bead-like varicosities in the dendrites and axons of brain and spinal cord neurons. We report here that these formaldehyde formulations can induce bead formation in the dendrites and axons of adult rat and rabbit retinal ganglion cells, and that retinal ganglion cells differ from hippocampal, cortical, cerebellar, and spinal cord neurons in that bead formation is not blocked by glutamate receptor antagonists, a voltage-gated Na(+) channel toxin, extracellular Ca(2+) ion exclusion, or temperature shifts. Moreover, we describe a modification of formaldehyde-based fixatives that prevents bead formation in retinal ganglion cells visualized by green fluorescent protein expression and by immunohistochemistry.

Modified areal cartography in auditory cortex following early- and late-onset deafness.

Cross-modal plasticity following peripheral sensory loss enables deprived cortex to provide enhanced abilities in remaining sensory systems. These functional adaptations have been demonstrated in cat auditory cortex following early-onset deafness in electrophysiological and psychophysical studies. However, little information is available concerning any accompanying structural compensations. To examine the influence of sound experience on areal cartography, auditory cytoarchitecture was examined in hearing cats, early-deaf cats, and cats with late-onset deafness. Cats were deafened shortly after hearing onset or in adulthood. Cerebral cytoarchitecture was revealed immunohistochemically using SMI-32, a monoclonal antibody used to distinguish auditory areas in many species. Auditory areas were delineated in coronal sections and their volumes measured. Staining profiles observed in hearing cats were conserved in early- and late-deaf cats. In all deaf cats, dorsal auditory areas were the most mutable. Early-deaf cats showed further modifications, with significant expansions in second auditory cortex and ventral auditory field. Borders between dorsal auditory areas and adjacent visual and somatosensory areas were shifted ventrally, suggesting expanded visual and somatosensory cortical representation. Overall, this study shows the influence of acoustic experience in cortical development, and suggests that the age of auditory deprivation may significantly affect auditory areal cartography.

The RNA binding protein RBPMS is a selective marker of ganglion cells in the mammalian retina.

There are few neurochemical markers that reliably identify retinal ganglion cells (RGCs), which are a heterogeneous population of cells that integrate and transmit the visual signal from the retina to the central visual nuclei. We have developed and characterized a new set of affinity-purified guinea pig and rabbit antibodies against RNA-binding protein with multiple splicing (RBPMS). On western blots these antibodies recognize a single band at 〜24 kDa, corresponding to RBPMS, and they strongly label RGC and displaced RGC (dRGC) somata in mouse, rat, guinea pig, rabbit, and monkey retina. RBPMS-immunoreactive cells and RGCs identified by other techniques have a similar range of somal diameters and areas. The density of RBPMS cells in mouse and rat retina is comparable to earlier semiquantitative estimates of RGCs. RBPMS is mainly expressed in medium and large DAPI-, DRAQ5-, NeuroTrace- and NeuN-stained cells in the ganglion cell layer (GCL), and RBPMS is not expressed in syntaxin (HPC-1)-immunoreactive cells in the inner nuclear layer (INL) and GCL, consistent with their identity as RGCs, and not displaced amacrine cells. In mouse and rat retina, most RBPMS cells are lost following optic nerve crush or transection at 3 weeks, and all Brn3a-, SMI-32-, and melanopsin-immunoreactive RGCs also express RBPMS immunoreactivity. RBPMS immunoreactivity is localized to cyan fluorescent protein (CFP)-fluorescent RGCs in the B6.Cg-Tg(Thy1-CFP)23Jrs/J mouse line. These findings show that antibodies against RBPMS are robust reagents that exclusively identify RGCs and dRGCs in multiple mammalian species, and they will be especially useful for quantification of RGCs.

Carbon nanotubes in neuroregeneration and repair.

In the last decade, we have experienced an increasing interest and an improved understanding of the application of nanotechnology to the nervous system. The aim of such studies is that of developing future strategies for tissue repair to promote functional recovery after brain damage. In this framework, carbon nanotube based technologies are emerging as particularly innovative tools due to the outstanding physical properties of these nanomaterials together with their recently documented ability to interface neuronal circuits, synapses and membranes. This review will discuss the state of the art in carbon nanotube technology applied to the development of devices able to drive nerve tissue repair; we will highlight the most exciting findings addressing the impact of carbon nanotubes in nerve tissue engineering, focusing in particular on neuronal differentiation, growth and network reconstruction.

Immunochemical changes of calbindin, calretinin and SMI32 in ischemic retinas induced by increase of intraocular pressure and by middle cerebral artery occlusion.

The reaction of neuroactive substances to ischemic conditions in the rat retina evoked by different methods was immunochemically evaluated in adult Sprague-Dawley rats. Ocular ischemic conditions were unilaterally produced by elevating intraocular pressure (EIOP) or by middle cerebral artery occlusion (MCAO). Two EF-hand calcium binding proteins, calbindin D28K (CB) and calretinin (CR), in the normal retina showed similar immunolocalization, such as the amacrine and displaced amacrine cells, the ganglion cells, and their processes, particularly CB in horizontal cells. CB immunoreactive neurons in the ganglion cell layer in both types of ischemic retinas were more reduced in number than CR neurons compared to those in a normal retina. The CB protein level in both ischemic retinas was reduced to 60-80% of normal. The CR protein level in MCAO retinas was reduced to about 80% of normal but increased gradually to the normal value, whereas that in the EIOP showed a gradual reduction and a slight recovery. SMI32 immunoreactivity, which detects a dephosphorylated epitope of neurofilaments-M and -H, appeared in the axon bundles of ganglion cells in the innermost nerve fiber layer of normal retinas. The reactivity in the nerve fiber bundles appeared to only increase slightly in EIOP retinas, whereas a moderate increase occurred in MCAO retinas. The SMI32 protein level in MCAO retinas showed a gradual increasing tendency, whereas that in the EIOP showed a slight fluctuation. Interestingly, the MCAO retinas showed additional SMI32 immunoreactivity in the cell soma of presumed ganglion cells, whereas that of EIOP appeared in the Müller proximal radial fibers. Glial fibrillary acidic protein (GFAP) immunoreactivity appeared in the astrocytes located in the nerve fiber layer of normal retinas. Additional GFAP immunoreactivity appeared in the Müller glial fibers deep in EIOP retinas and at the proximal end in MCAO retinas. These findings suggest that the neurons in the ganglion cell layer undergo degenerative changes in response to ischemia, although EIOP retinas represented a remarkable Müller glial reaction, whereas MCAO retinas had only a small-scaled axonal transport disturbance.