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Imidazole-4-acetic acid-ribotide (IAARP) is a putative neurotransmitter/modulator and an endogenous regulator of sympathetic drive, notably systemic blood pressure, through binding to imidazoline receptors. IAARP is present in neurons and processes throughout the CNS, but is particularly prevalent in regions that are involved in blood pressure control. The goal of this study was to determine whether IAARP is present in neurons in the caudal vestibular nuclei that participate in the vestibulo-sympathetic reflex (VSR) pathway. This pathway is important in modulating blood pressure upon changes in head position with regard to gravity, as occurs when humans rise from a supine position and when quadrupeds climb or rear. Sinusoidal galvanic vestibular stimulation was used to activate the VSR and cfos gene expression in VSR pathway neurons of rats. These subjects had previously received a unilateral FluoroGold tracer injection in the rostral or caudal ventrolateral medullary region. The tracer was transported retrogradely and filled vestibular neuronal somata with direct projections to the injected region. Brainstem sections through the caudal vestibular nuclei were immunostained to visualize FluoroGold, cFos protein, IAARP and glutamate immunofluorescence. The results demonstrate that IAARP is present in vestibular neurons of the VSR pathway, where it often co-localizes with intense glutamate immunofluorescence. The co-localization of IAARP and intense glutamate immunofluorescence in VSR neurons may represent an efficient chemoanatomical configuration, allowing the vestibular system to rapidly up- and down-modulate the activity of presympathetic neurons in the ventrolateral medulla, thereby altering blood pressure.

Changes in head position and posture are detected by the vestibular system and are normally followed by rapid modifications in blood pressure. These compensatory adjustments, which allow humans to stand up without fainting, are mediated by integration of vestibular system pathways with blood pressure control centers in the ventrolateral medulla. Orthostatic hypotension can reflect altered activity of this neural circuitry. Vestibular sensory input to the vestibulo-sympathetic pathway terminates on cells in the vestibular nuclear complex, which in turn project to brainstem sites involved in the regulation of cardiovascular activity, including the rostral and caudal ventrolateral medullary regions (RVLM and CVLM, respectively). In the present study, sinusoidal galvanic vestibular stimulation was used to activate this pathway, and activated neurons were identified through detection of c-Fos protein. The retrograde tracer FluoroGold was injected into the RVLM or CVLM of these animals, and immunofluorescence studies of vestibular neurons were conducted to visualize c-Fos protein and FluoroGold concomitantly. We observed activated projection neurons of the vestibulo-sympathetic reflex pathway in the caudal half of the spinal, medial and parvocellular medial vestibular nuclei. Approximately two-thirds of the cells were ipsilateral to FluoroGold injection sites in both RVLM and CVLM and the remainders were contralateral. As a group, cells projecting to RVLM were located slightly rostral to those with terminals in CVLM. Individual activated projection neurons were multipolar, globular or fusiform in shape. This study provides the first direct demonstration of the central vestibular neurons that mediate the vestibulo-sympathetic reflex.

Inflammation and compromise in structure and function of cerebral parenchymal microvasculature begins early after subarachnoid hemorrhage (SAH). We recently found greater inflammation and greater vascular compromise in male than in female rats following SAH. In this study, we investigated whether this cross-sexual difference in pathology is reflected in expression levels of genes related to vascular inflammation and structural compromise.Age-matched male and female rats underwent sham surgery or SAH by endovascular perforation. Early physiology (intracranial pressure (ICP), blood pressure (BP), heart rate, and cerebral blood flow) was monitored. Cerebral RNA was extracted at sacrifice 3 h after surgery and assayed for expression of thrombomodulin (Thbd), endothelial nitric oxide synthase (eNos;Nos3), intracellular adhesion molecule-1 (Icam1), vascular endothelial growth factor (Vegf), interleukin-1beta (Il1β) tumor necrosis factor-alpha (Tnf-α), and arginine vasopressin (Avp).Increases in ICP and BP at SAH appeared slightly greater in males but the difference did not reach statistical difference, indicating that SAH intensity did not differ significantly between the sexes. Of the seven genes studied two; Tnf-α and Vegf, did not change after injury, while the remainder showed significant responses to SAH. Response of Nos3 and Thbd was markedly different between the sexes, with expression greater in males.This study finds that sexual dimorphism is present in the response of some but not all genes to SAH. Since products of genes exhibiting sexual dimorphism have anti-inflammatory activities, our results indicate that previously found sex-based differences in vascular pathology are paralleled by sexually dimorphic changes in gene expression following SAH.

The vestibular system senses the movement and position of the head in space and uses this information to stabilize vision, control posture, perceive head orientation and self-motion in three-dimensional space, and modulate autonomic and limbic activity in response to locomotion and changes in posture. Most vestibular signals are not consciously perceived and are usually appreciated through effector pathways classically described as the vestibulo-ocular, vestibulo-spinal, vestibulo-collic and vestibulo-autonomic reflexes. The present study reviews some of the recent data concerning the connectivity and chemical anatomy of vestibular projections to autonomic sites that are important in the sympathetic control of blood pressure.

Nitric oxide is an unstable free radical that serves as a novel messenger molecule in the central nervous system (CNS). In order to understand the interplay between classic and novel chemical communication systems in vestibular pathways, the staining obtained using a monoclonal antibody directed against L-citrulline was compared with the labeling observed using more traditional markers for the presence of nitric oxide. Brainstem tissue from adult rats was processed for immunocytochemistry employing a monoclonal antibody directed against L-citrulline, a polyclonal antiserum against neuronal nitric oxide synthase, and/or NADPH-diaphorase histochemistry. Our findings demonstrate that L-citrulline can be fixed in situ by vascular perfusion, and can be visualized in fixed CNS tissue sections by immunocytochemistry. Further, the same vestibular regions and cell types are labeled by NADPH-diaphorase histochemistry, by the neuronal nitric oxide synthase antiserum, and by our anti-L-citrulline antibody. Clusters of L-citrulline-immunoreactive neurons are present in subregions of the vestibular nuclei, including the caudal portion of the inferior vestibular nucleus, the magnocellular portion of the medial vestibular nucleus, and the large cells in the ventral tier of the lateral vestibular nucleus. NADPH-diaphorase histochemical staining of these neurons clearly demonstrated their multipolar, fusiform and globular somata and long varicose dendritic processes. These results provide support for the suggestion that nitric oxide serves key roles in both vestibulo-autonomic and vestibulo-spinal pathways.

Object. The mechanisms underlying acute cerebral ischemia after subarachnoid hemorrhage (SAH) are not well established. Platelets aggregate within major cerebral vessels hours after SAH, but this has not been studied in the microvasculature. Platelet aggregates within the microvasculature could mechanically obstruct the lumen and initiate events that injure vessel structure. In the present study the authors examined the hypothesis that platelets aggregate within the cerebral microvasculature acutely after SAH. Methods. Subarachnoid hemorrhage was induced in the rat by using the endovascular perforation model. The animals were killed between 10 minutes and 48 hours after SAH. Immunostaining for the platelet surface receptor glycoprotein (GP)IIb/IIIa, which mediates platelet aggregation, was used to detect platelet aggregation. Sham-operated animals were used as controls. The GPIIb/IIIa immunoreactive platelet aggregates were abundant in the microvasculature of the basal and frontal cortex, striatum, and hippocampus 10 minutes after SAH. These aggregates decreased in number from 1 to 6 hours post-SAH and then increased to a peak at 24 hours. No immunoreactive aggregates were observed 48 hours after SAH. Conclusions. The data indicate that widespread platelet aggregation occurs very rapidly in response to SAH followed by a decrease within 6 hours and a subsequent increase 24 hours after SAH. Microvascular platelet aggregates may contribute to decreased cerebral blood flow and ischemic injury after SAH via a number of mechanisms.

The vestibular semicircular canals respond to angular acceleration that is integrated to angular velocity by the biofluid mechanics of the canals and is the primary origin of afferent responses encoding velocity. Surprisingly, some afferents actually report angular acceleration. Our data indicate that hair-cell/afferent synapses introduce a mathematical derivative in these afferents that partially cancels the biomechanical integration and results in discharge rates encoding angular acceleration. We examined the role of convergent synaptic inputs from hair cells to this mathematical differentiation. A significant reduction in the order of the differentiation was observed for low-frequency stimuli after γ-aminobutyric acid type B receptor antagonist administration. Results demonstrate that γ-aminobutyric acid participates in shaping the temporal dynamics of afferent responses.

OBJECTIVE: Subarachnoid hemorrhage (SAH) is associated with acute decreases and Subsequent recovery of cerebral nitric oxide (NO) levels, but the mechanisms of these alterations are not known. In this study, we measure NO synthase (NOS) protein and kinetics to determine its involvement in the alterations of cerebral NO levels after SAH. METHODS: The endovascular rat model of SAH was used. The number of NOS-1 ineuronal) and NOS-2 (inducible)-positive cells (0-96 b) was determined by counting immunoreactive cells in 8-μm cryostat sections. The tissue content of active NOS and its kinetic parameters were studied with an enzymatic t-citrulline assay. RESULTS: The number of NOS-1-positive cells increased between 1 and 3 hours after SAH, decreased to and below control values at 6 and 72 hours after SAH, and increased to control values 96 hors after SAH. The number of NOS-2-positive cells increased 1 hour after SAH, decreased to control-values at 24 hours, and increased above control values 96 hours after SAH. The Michaelis-Menten kinetic parameters (V max , K m , slope) of NOS remained unchanged at 10 and 90 minutes after SAH. CONCLUSION: NOS-1 and -2 proteins undergo a triphasic, alteration after SAH, whereas the amount of active NOS and its kinetic parameters remain unchanged during the first 90 minutes after SAH Depletion of NOS is not involved in in the acute alternations of cerebral NO levels after SAH.

Although γ-aminobutyric acid (GABA) and glutamate are known to be present in the vestibular sensory epithelia of a variety of species, the functional relationship between these two transmitters is not clear. The present study addresses the three-dimensional spatial distribution of GABA and glutamate immunoreactivity in the vestibular labyrinth of the oyster toadfish by using whole end organs labeled by immunofluorescence with monoclonal anti-GABA and/or antiglutamate antibodies and visualized as whole mounts by multiphoton confocal microscopy. We find glutamate-immunoreactive hair cells present throughout the sensory epithelium. In contrast, prominent GABA immunoreactivity is restricted to a small population of hair cells located in the central region of the crista. Double immunofluorescence reveals two distinct staining patterns in GABA-labeled hair cells. Most (∼80%) GABA-labeled cells show trace levels of glutamate, appropriate for the metabolic/synthetic role of cytoplasmic glutamate. The remainder of the GABA-stained cells contain substantial levels of both GABA and glutamate, suggesting transmitter colocalization. In the toadfish utricle, glutamatergic hair cells are present throughout the macula. GABA-immunoreactive hair cells follow the arc of the striola, and most GABA-labeled receptor cells coexpress glutamate. The localization of GABA was explored in other species as well. In the pigeon, GABAergic hair cells are present throughout the crista ampullaris. Our findings demonstrate that multiple, neurochemically distinct types of hair cells are present in vestibular sensory epithelia. These observations, together with the excitatory activity generally associated with 8th nerve afferent fibers, strongly suggest that GABA serves an important, specific, and complex role in determining primary afferent response dynamics. J. Comp. Neurol. 471:1–10, 2004. © 2004 Wiley-Liss, Inc.

Aberrant accumulation of neurofilaments is a feature of human motor neuron diseases. Experimentally motor neuron disease can be induced in transgenic mice by overexpressing the mouse neurofilament light subunit (NF-L), the human heavy subunit (NF-H), or mouse peripherin. Here we describe that mice harboring a bacterial artificial chromosome (BAC) transgene containing the human midsized neurofilament subunit (NF-M) gene develop a progressive hind limb paralysis associated with neurofilamentous accumulations in ventral horn motor neurons and axonal loss in ventral motor roots. Biochemical studies revealed that all three mouse neurofilament subunits along with the human NF-M contributed to filament formation, although filaments contained less peripherin. In addition the endogenous mouse NF-M became less phosphorylated in the presence of the human protein and accumulated in the cell bodies of affected neurons even though phosphorylated human NF-M did not. Remaining motor axons contained an increased density of neurofilaments and morphometric studies showed that principally small myelinated axons were lost in the transgenic animals. Removing half of the mouse NF-M by breeding the transgene onto the mouse NF-M heterozygous null background offered no protection against the development of disease, arguing that the effect is not simply due to elevation of total NF-M. Collectively these studies argue that the human and mouse NF-M proteins exhibit distinct biochemical properties and within mouse neurons are not interchangeable and that indeed the human protein may be toxic to some mouse neurons. These studies have implications for the use of human neurofilament transgenic mice as models of amyotrophic lateral sclerosis.

Background Brain imaging, molecular genetic, and ultrastructural evidence indicate the existence of pathologic alterations in the cortical and subcortical white matter of schizophrenic patients. Methods We performed a stereologic analysis of numbers, densities, and spatial distribution of oligodendrocytes in layer III and in the gyral white matter of Brodmann’s area 9 in the superior frontal gyrus to assess whether these cells are affected in schizophrenia. Counts were obtained on Nissl-stained materials and on sections immunolabeled for the oligodendrocyte marker 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) in seven schizophrenic and seven age-matched control cases. Results A 28% decrease in total numbers (or densities) of cortical layer III oligodendrocytes and a 27% decrease in the white matter were detected in schizophrenic compared with control cases based on CNPase immunostaining. Nissl and CNPase immunohistochemistry yielded comparable results. The spatial distribution of oligodendrocytes in area 9 white matter exhibited a less clustered arrangement in schizophrenic cases. Conclusions These results suggest a severe pathology of oligodendrocytes in schizophrenia and provide a quantitative cellular correlate of the white matter changes observed by brain imaging in vivo, showing reduced fractional anisotropy in schizophrenia. The data support recent evidence that several genes encoding myelin-related proteins consistently exhibit reduced expression in schizophrenia.

Mutations in the presenilin-1 (PS-1) gene are one cause of familial Alzheimer's disease (FAD). However, the functions of the PS-1 protein as well as how PS-1 mutations cause FAD are incompletely understood. Here we investigated if neuronal overexpression of wild-type or FAD mutant PS-1 in transgenic mice affects neurogenesis in the hippocampus of adult animals. We show that either a wild-type or an FAD mutant PS-1 transgene reduces the number of neural progenitors in the dentate gyrus. However, the wild-type, but not the FAD mutant PS-1 promoted the survival and differentiation of progenitors leading to more immature granule cell neurons being generated in PS-1 wild type expressing animals. These studies suggest that PS-1 plays a role in regulating neurogenesis in adult hippocampus and that FAD mutants may have deleterious properties independent of their effects on amyloid deposition.

The functions of the presenilin-1 (PS-1) protein remain largely unknown. In adult brain PS-1 is expressed principally in neurons. However during development PS-1 is expressed more widely including in embryonic neural progenitors. To determine if PS-1 is expressed in neural progenitors in adult hippocampus we used bromodeoxyuridine (BrdU) labeling combined with immunostaining for BrdU, PS-1 and markers of neuronal or glial differentiation. Most BrdU labeled cells also expressed PS-1 at a time when few BrdU labeled cells expressed the early neuronal markers beta-III tubulin or TOAD-64 and none expressed mature neuronal (NeuN or calbindin) or astrocytic (GFAP) markers. Cells expressing PS-1 and the neural progenitor marker nestin were also found. Thus PS-1 is expressed in neural progenitor cells in adult hippocampus implying its possible role in neurogenesis in adult brain.

The first 72 h after aneurysmal subarachnoid hemorrhage (SAH) is a critical period for the patient. Most of the deaths in the SAH patient population occur during this time, and a number of key events activate and trigger mechanisms that not only contribute to early brain injury but evolve over time and participate in the delayed complications. This review highlights the contribution of key events to the early brain injury and to overall outcome after SAH.

Here we show that presenilin-1 (PS1), a protein involved in Alzheimer's disease, binds directly to epithelial cadherin (E-cadherin). This binding is mediated by the large cytoplasmic loop of PS1 and requires the membrane-proximal cytoplasmic sequence 604–615 of mature E-cadherin. This sequence is also required for E-cadherin binding of protein p120, a known regulator of cadherin-mediated cell adhesion. Using wild-type and PS1 knockout cells, we found that increasing PS1 levels suppresses p120/E-cadherin binding, and increasing p120 levels suppresses PS1/E-cadherin binding. Thus PS1 and p120 bind to and mutually compete for cellular E-cadherin. Furthermore, PS1 stimulates E-cadherin binding to β- and γ-catenin, promotes cytoskeletal association of the cadherin/catenin complexes, and increases Ca 2+ -dependent cell–cell aggregation. Remarkably, PS1 familial Alzheimer disease mutant ΔE9 increased neither the levels of cadherin/catenin complexes nor cell aggregation, suggesting that this familial Alzheimer disease mutation interferes with cadherin-based cell–cell adhesion. These data identify PS1 as an E-cadherin-binding protein and a regulator of E-cadherin function in vivo .

The proteolipid protein (PLP) gene encodes two myelin-specific protein isoforms, DM-20 and PLP, which are members of the highly conserved lipophilin family of transmembrane proteins. While the functions of this family are poorly understood, the fact that null mutations of thePLPgene cause leukodystrophy in man is testament to the importance of DM-20 and PLP in normal CNS function. PLP differs from DM-20 by the presence of a 35 amino acid domain exposed to the cytoplasm, which is not encoded by other lipophilin genes and appears to have arisen in amphibians ∼300 million years before present. However, the lipophilin gene family can be traced back at least 550 million years and is represented inDrosophilaand silkworms. Thus, from an evolutionary perspective PLP can reasonably be anticipated to perform functions in CNS myelin that cannot be accomplished by other lipophilins. Herein we use a novel knock-in strategy to generate mice expressing wild-type levels of aPlpgene that has been modified to encode only DM-20. Although DM-20 is incorporated into functional compact myelin sheaths in young animals, our data show that the 35 amino acid PLP-specific peptide is required to engender the normal myelin period and to confer long-term stability on this multilamellar membrane.

Mammalian neurofilaments are assembled from the light (NF-L), midsized (NF-M), and heavy (NF-H) neurofilament proteins. While NF-M and NF-H cannot self-assemble into homopolymers, the data concerning NF-L has been more contradictory. In vitro bovine, porcine, and murine NF-L can homopolymerize in the absence of other subunits. However, in vivo studies suggest that neither rat nor mouse NF-L can form filaments when transfected alone into cells lacking endogenous intermediate filaments. By contrast, human NF-L forms homopolymers in similar cell lines. Recently we generated mice with null mutations in the NF-M and NF-H genes. To determine if mouse NF-L can homopolymerize in mouse axons, NF-M and NF-H null mutants were bred to create a line of double mutant animals. Here we show that axons in NF-M/H double mutant animals are largely devoid of 10-nm filaments. Instead, the axoplasm is transformed to a microtubule-based cytoskeleton-although the lack of any increase in tubulin levels per unit length of nerve or of increases in microtubule numbers relative to myelin sheath thickness argues that microtubules are not increased in response to the loss of neurofilaments. Thus in vivo rodent neurofilaments are obligate heteropolymers requiring NF-L plus either NF-M or NF-H to form a filamentous network.

Neurofilaments are central determinants of the diameter of myelinated axons. It is less clear whether neurofilaments serve other functional roles such as maintaining the structural integrity of axons over time. Here we show that an age-dependent axonal atrophy develops in the lumbar ventral roots of mice with a null mutation in the mid-sized neurofilament subunit (NF-M) but not in animals with a null mutation in the heavy neurofilament subunit (NF-H). Mice with null mutations in both genes develop atrophy in ventral and dorsal roots as well as a hind limb paralysis with aging. The atrophic process is not accompanied by significant axonal loss or anterior horn cell pathology. In the NF-M–null mutant atrophic ventral root, axons show an age-related depletion of neurofilaments and an increased ratio of microtubules/neurofilaments. By contrast, the preserved dorsal root axons of NF-M–null mutant animals do not show a similar depletion of neurofilaments. Thus, the lack of an NF-M subunit renders some axons selectively vulnerable to an age-dependent atrophic process. These studies argue that neurofilaments are necessary for the structural maintenance of some populations of axons during aging and that the NF-M subunit is especially critical.

Neurofilaments (NFs) are prominent components of large myelinated axons. Previous studies have suggested that NF number as well as the phosphorylation state of the COOH-terminal tail of the heavy neurofilament (NF-H) subunit are major determinants of axonal caliber. We created NF-H knockout mice to assess the contribution of NF-H to the development of axon size as well as its effect on the amounts of low and mid-sized NF subunits (NF-L and NF-M respectively). Surprisingly, we found that NF-L levels were reduced only slightly whereas NF-M and tubulin proteins were unchanged in NF-H–null mice. However, the calibers of both large and small diameter myelinated axons were diminished in NF-H–null mice despite the fact that these mice showed only a slight decrease in NF density and that filaments in the mutant were most frequently spaced at the same interfilament distance found in control. Significantly, large diameter axons failed to develop in both the central and peripheral nervous systems. These results demonstrate directly that unlike losing the NF-L or NF-M subunits, loss of NF-H has only a slight effect on NF number in axons. Yet NF-H plays a major role in the development of large diameter axons.

Myelin basic proteins (MBPs) are major proteins of central nervous system (CNS) myelin, where they facilitate the apposition of cytoplasmic faces of myelin lamellae. Myelin-bearing oligodendrocytes transport MBP mRNA to myelin, where newly translated protein is directly inserted into the myelin sheath. An apparent absence of MBPs in oligodendrocyte perikarya has suggested that MBP localized to the soma is translationally inert. We now demonstrate by confocal immunofluorescence microscopy that not only are MBPs present in the majority of oligodendrocyte perikarya but oligodendrocytes are heterogeneous with respect to their localization of MBPs; MBPs are concentrated in some cells at the plasmalemma and distributed in others throughout the cytoplasm and, surprisingly, the nucleus. MBPs are present in the nuclei of over half of oligodendrocytes in the adult, but in almost all MBP+oligodendrocytes during myelinogenesis. Transport of MBPs into nuclei appears to be a regulated process since some cells exhibit robust MBP accumulation in their cytoplasm but exclude MBPs from their nuclei. We show that oligodendrocyte nuclei contain all four major MBP isoforms, but that in transgenic mice, the epitope-tagged 14 kD MBP isoform preferentially segregates to the plasmalemma. Our data demonstrate that oligodendrocytes are not required to exclude MBPs from their perikarya and suggest that MBPs have a specific function in the oligodendrocyte perikarya and nucleus.

Many individuals with familial Alzheimer disease (FAD) have mutations in a gene termed S182 or presenilin I (PS-I). Currently, the PS-I gene product has not been identified and its function remains unknown. Here we report that affinity purified antibodies against the predicted amino acid sequence of the PS-I gene product detected in homogenates of human, mouse, and rat brains a single antigen of approximately 48 kDa. This antigen was also present in immortalized human and mouse neuronal cell cultures. Brain tissue fractionation showed that all PS-I antigen was found in the membrane fraction. In stained tissue sections of mouse central nervous system (CNS), PS-I antigen was found only in neurons throughout brain and spinal cord and was located within cell bodies, axons, and dendrites. Remarkably the relative partition among these three compartments varied dramatically. A striking feature of PS-I expression was its intense concentration in some (but not all) dendrites, at levels substantially above those in the parent perikarya. In most of the cerebrum, PS-I staining in axons was very weak or undetectable. By contrast, many axons in portions of the brainstem and in the spinal cord showed marked PS-I immunoreactivity. Similarly, staining of sections from human temporal cortex showed that PS-I was present mainly in neuronal cell bodies and dendrites. These data show that in the CNS, PS-I is expressed mainly in neurons and suggests that this protein may perform a neuron specific function. The pattern of PS-I expression in the CNS would suggest that the premature neurodegeneration associated with PS-I mutations involves a primary neuronal process rather than a secondary effect of PS-I produced in non-neuronal cells. © 1996 Wiley-Liss, Inc.

Myelin sheaths develop in the central nervous system (CNS) as elaborations of the processes of oligodendrocytes. Although many details of the spiral wrapping of oligodendrocyte processes around axons and their subsequent transformation into myelin sheaths are known from thin-section electron-microscopic studies, the three-dimensional architecture of the myelin-forming cells is incompletely understood. To characterize this aspect of oligodendrocyte development, we labeled thick (100- to 300-microns) sections of developing murine CNS with oligodendrocyte marker antibodies, recorded individual cells in serial optical sections by confocal microscopy, and created whole-cell reconstructions of oligodendrocytes before and during the initiation of myelination. We distinguish three stages in the maturation of oligodendrocytes, which at all three stages are labeled by the O4, O1 and Ranscht monoclonals and by antibodies against the myelin-specific proteins CNP and myelin basic protein. Premyelinating oligodendrocytes, present before axonal ensheathment begins, emit multiple irregular processes which have predominant radial orientation. These processes, which generally terminate within 50 microns of the cell body, have a surface area 3-8 times or more that of the cell body itself and may represent a mechanism for sampling the local environment of each cell and for identifying target axons. Transitional cells have initiated one or more myelin sheaths; these cells progressively reduce the number of their radial processes as they increase the number of their myelin internodes. The radial processes of each transitional cell are most reduced in parts of the process arbor where ensheathment has begun, suggesting directional control in the elaboration or stability of the radial processes. Mature myelin-bearing oligodendrocytes entirely lack the radial processes and instead emit a few sparsely branching processes which connect cell bodies with myelin internodes. Three-dimensional analysis of the earliest stages in myelin sheath formation reveals two distinct phases. The initiating event in the formation of myelin internodes is the growth of thin unbranched processes, termed 'initiator processes', along axons. The second phase, spiral ensheathment of target axons, begins through the elaboration from each initiator process of lamellar extensions which extend circumferentially around the target axon and thereby form the first turn of its myelin sheath.

Previous studies (Blank, W. F., M. B. Bunge, and R. P. Bunge. 1974. Brain Res. 67:503-518) showed that Schwann cell paranodal membranes were disrupted in calcium free medium suggesting that cadherin mediated mechanisms may operate to maintain the integrity of the paranodal membrane complex. Using antibodies against the fifth extracellular domain of E-cadherin, we now show by confocal laser and electron immunomicroscopy that E-cadherin is a major adhesive glycoprotein in peripheral nervous system Schwann cells. E-Cadherin is not found, however, in compact myelin bilayers. Rather, it is concentrated at the paranodes, in Schmidt-Lanterman incisures, and at the inner and outer loops. At these loci, E-cadherin is associated with subplasmalemmal electron densities that coordinate in register across several cytoplasmic turns of a single Schwann cell. F-Actin and beta-catenin, two proteins implicated in cellular signaling, also co-localize to E-cadherin positive sites. These complexes are autotypic adherens-type junctions that are confined to the plasma membrane synthesized by a single Schwann cell; E-cadherin was never observed between two Schwann cells, nor between Schwann cells and the axon. Our findings demonstrate that E-cadherin and its associated proteins are essential components in the architecture of the Schwann cell cytoplasmic channel network, and suggest that this network has specialized functions in addition to those required for myelinogenesis.

Most subarachnoid hemorrhage (SAH) patients exhibit clinical signs of cerebral ischemia at admission but no angiographic vasospasm. Consequently, the source of early cerebral ischemia is not understood. Parenchymal microvessels may contribute to early cerebral ischemia, but the low resolution of current imaging has prevented their analysis in SAH patients. Animal studies demonstrated that early after SAH structure and function of parenchymal vessels are compromised to the level that may very well contribute to early ischemia. We review these studies.

The vestibular system sends projections to brainstem autonomic nuclei that modulate heart rate and blood pressure in response to changes in head and body position with regard to gravity. Consistent with this, binaural sinusoidal galvanic vestibular stimulation (sGVS) in humans causes vasoconstriction in the legs, while low frequency (0.02-0.04 Hz) sGVS causes a rapid drop in heart rate and blood pressure in anesthetized rats. We have hypothesized that these responses occur through activation of vestibulo-sympathetic pathways. In the present study, c-Fos protein expression was examined in neurons of the vestibular nuclei and rostral ventrolateral medullary region (RVLM) that were activated by low frequency sGVS. We found c-Fos-labeled neurons in the spinal, medial and superior vestibular nuclei (SpVN, MVN and SVN, respectively) and the parasolitary nucleus. The highest density of c-Fos-positive vestibular nuclear neurons was observed in MVN, where immunolabeled cells were present throughout the rostro-caudal extent of the nucleus. C-Fos expression was concentrated in the parvocellular region and largely absent from magnocellular MVN. C-Fos-labeled cells were scattered throughout caudal SpVN, and the immunostained neurons in SVN were restricted to a discrete wedge-shaped area immediately lateral to the IVth ventricle. Immunofluorescence localization of c-Fos and glutamate revealed that approximately one third of the c-Fos-labeled vestibular neurons showed intense glutamate-like immunofluorescence, far in excess of the stain reflecting the metabolic pool of cytoplasmic glutamate. In the RVLM, which receives a direct projection from the vestibular nuclei and sends efferents to preganglionic sympathetic neurons in the spinal cord, we observed an approximately 3-fold increase in c-Fos labeling in the sGVS-activated rats. We conclude that localization of c-Fos protein following sGVS is a reliable marker for sGVS-activated neurons of the vestibulo-sympathetic pathway.

In ten transgenic lines, expression of a human mid-sized (M) neurofilament (NF) transgene was restricted to neurons in the central and peripheral nervous systems. However, no two lines gave identical expression patterns and none exactly matched the expression of mouse NF(M). These varied expression patterns within the neural compartment likely result from interactions of the transgene with enhancer elements located in the regions flanking the insertion site. Unexpected patterns of enhancer activity included an enhancer active in subsets of cerebellar basket cells as well as others preferentially active in subsets of motor or sensory neurons.

The primary structure of the proteolipid protein (PLP) from the central nervous system (CNS) myelin of mammals is highly conserved with only three amino acid differences between the mouse, rat, dog, bovine, and human proteins. Furthermore, within a particular species no polymorphisms in the protein have been identified. Recent interest has focused on the targeting of PLP in oligodendrocytes and the role that mutant forms of this protein play in generating dysmyelinating or hypomyelinating diseases. We previously expressed the human cDNA encoding PLP in transiently transfected Cos-7 cells and characterized the subcellular distribution of the protein in this simple heterologous system. In the current study we have used the same paradigm to examine the effect of five missense mutations in the PLP gene on processing of the encoded protein. The mutations chosen span the carboxy-terminal half of PLP and encompass that part of the protein in which most mutations have been identified. Our results show that transport of all mutations examined was arrested in the secretory pathway at an early stage, causing the mutant proteins to accumulate in the endoplasmic reticulum. Thus, a common mechanism of protein misfolding and failure of PLP to reach the cell surface of oligodendrocytes rather than the inability of the mutant protein to perform some crucial function at the cell surface may be responsible for the diseases caused by many PLP mutations. Our results, together with those of others, prompt us to speculate that the pathobiology observed in PLP mutants may result from oligodendrocyte cell death caused by the accumulation of misfolded protein in the endoplasmic reticulum. This speculation is consistent with the observations that oligodendrocytes bearing misfolded PLP, as in the jimpy mutant, proliferate but die rapidly while oligodendrocytes from PLP deletion survive and produce a myelin-like membrane which lacks PLP. © 1994 Wiley-Liss, Inc.

Background Subarachnoid haemorrhage (SAH) elicits rapid pathological changes in the structure and function of parenchymal vessels (≤ 100 μm). The role of neutrophils in these changes has not been determined. This study investigates the role of neutrophils in early microvascular changes after SAH Method Rats were either untreated, treated with vinblastine or anti-polymorphonuclear (PMN) serum, which depletes neutrophils, or treated with pyrrolidine dithiocarbamate (PDTC), which limits neutrophil activity. SAH was induced by endovascular perforation. Neutrophil infiltration and the integrity of vascular endothelium and basement membrane were assessed immunohistochemically. Vascular collagenase activity was assessed by in situ zymography. Results Vinblastine and anti-PMN serum reduced post-SAH accumulation of neutrophils in cerebral vessels and in brain parenchyma. PDTC increased the neutrophil accumulation in cerebral vessels and decreased accumulation in brain parenchyma. In addition, each of the three agents decreased vascular collagenase activity and post-SAH loss of vascular endothelial and basement membrane immunostaining. Conclusions Our results implicate neutrophils in early microvascular injury after SAH and indicate that treatments which reduce neutrophil activity can be beneficial in limiting microvascular injury and increasing survival after SAH.

During the last decade much effort has been invested in understanding the events that occur early after SAH. It is now widely accepted that these early events not only participate in the early ischemic injury but also set the stage for the pathogenesis of delayed vasospasm. That early cerebral ischemia occurs after SAH is documented in both experimental SAH and in human autopsy studies; however, angiographic evidence for vasoconstriction early after SAH is lacking and the source of early ischemic injury is therefore unclear. Recently, the cerebral microvasculature has been identified as an early target of SAH. Changes in the anatomical structure of cerebral microvessels, sufficient to cause functional deficits, are found early after experimental SAH. These changes may explain cerebral ischemia in human in the absence of angiographic evidence of large vessel vasoconstriction. This paper summarizes known alterations in cerebral microvasculature during the first 48h after SAH.

Our previous studies established that targeted axonal outgrowth into the host median eminence (ME) from grafted gonadotropin-releasing hormone (GnRH) neurons is essential for stimulation of reproductive function in hypogonadal (HPG) mice homozygous for a deletion in the GnRH gene. In the current experiments transgenic mice expressing the human midsized neurofilament NF(M) were used as sources of grafts to clarify the extent of transplant-derived innervation of the host that accompanies this dramatic recovery process. Preoptic area (POA) tissue from 1- or 2-day-old transgenic pups was implanted in the third ventricle of adult male HPG mice. Polymerase chain reaction (PCR) analysis verified that each donor bore the transgene. Immunohistochemistry using a monoclonal antibody specific for human NF(M) revealed intensely immunoreactive parvicellular soma and proximal dendrites in the grafts. Interestingly, human NF(M)-positive neurons also migrated out of the graft into the host hypothalamus. In the animal with the most dramatic increase in testicular and seminal vesicle weight, human NF(M)-positive cells were observed within the host ME. In some cases, human NF(M)-positive axonal bundles were present within the wall of the third ventricle of the host as well as in the host ME. These were GnRH negative. More extensive anatomical studies will delineate the degree and patterning of axonal outgrowth. In these early studies, however, it is apparent that neuronal fiber outgrowth into the host brain is not exclusive to GnRH fibers from the preoptic area grafts. This is important information in regard to our studies of the specificity of the signals derived from the ME that may be involved in GnRH fiber targeting.

Transgenic mice of line MβP3 express bacterial β-galactosidase in oligodendrocytes but not other cells of the CNS. The marker enzyme, demonstrated histochemically or by immunostaining in oligodendrocyte cell bodies and along myelin internodes, appears at the time of myelination and persists thereafter; in transplantation experiments, the marker may serve to indicate both the source of particular cells and their state of differentiation. The subventricular zone of the lateral ventricle, grafted from transgenic to wild-type perinatal recipient mice, yields histochemically labeled oligodendrocytes in surrounding host tissue. When grafts are placed in cerebral cortex near callosal radiations, graft-derived oligodendrocytes are found in cerebral cortex and subcortical white matter as far as 1.5 mm from the site of implant but not in nearby caudoputamen. This study is the first to document differentiation of transplant-derived oligodendrocytes in normal developing CNS. Our results are consistent with the well- established notion that oligodendrocyte progenitors migrate during normal development and suggest that such migration might be guided or restricted by mechanisms yet to be identified.

Deletion of the humanSHANK3gene near the terminus of chromosome 22q is associated with Phelan–McDermid syndrome and autism spectrum disorders. Nearly all such deletions also span the tightly linkedIB2gene. We show here that IB2 protein is broadly expressed in the brain and is highly enriched within postsynaptic densities. Experimental disruption of theIB2gene in mice reduces AMPA and enhances NMDA receptor-mediated glutamatergic transmission in cerebellum, changes the morphology of Purkinje cell dendritic arbors, and induces motor and cognitive deficits suggesting an autism phenotype. These findings support a role for humanIB2mutation as a contributing genetic factor in Chr22qter-associated cognitive disorders.

The pathophysiology of early ischemic injury after aneurysmal subarachnoid hemorrhage (SAH) is not understood. This study examined the acute effect of endovascular puncture-induced SAH on parenchymal vessel function in rat, using intravascular fluorescent tracers to assess flow and vascular permeability and immunostaining to assess structural integrity and to visualize platelet aggregates. In sham-operated animals, vessels were well filled with tracer administered 10 seconds before sacrifice, and parenchymal escape of tracer was rare. At ten minutes and 3 hours after hemorrhage, patches of poor vascular filling were distributed throughout the forebrain. Close examination of these regions revealed short segments of narrowed diameter along many profiles. Most vascular profiles with reduced perfusion contained platelet aggregates and in addition showed focal loss of collagen IV, a principal component of basal lamina. In contrast, vessels were well filled at 24 hours post-hemorrhage, indicating that vascular perfusion had recovered. Parenchymal escape of intravascular tracer was detected at 10 minutes post-hemorrhage and later as plumes of fluorescence emanating into parenchyma from restricted microvascular foci. These data demonstrate that parenchymal microvessels are compromised in function by 10 minutes after SAH and identify focal microvascular constriction and local accumulation of luminal platelet aggregates as potential initiators of that compromise.

By a combination of DNase I footprinting, methylation interference, and gel shift analyses we have identified multiple binding sites for nuclear proteins within the promoter region of the human neurofilament H gene. Two sites likely bind the transcription factor Sp1 while two others may be targets for previously unrecognized DNA binding proteins. One site, PAL, occurs within the 10 bp sequence GGGGAGGAGG. Two copies of the PAL sequence form an interrupted palindrome around one of the Sp1 sites. A second site, PROX, is found within the sequence GGTTGGACC. Nuclear extracts prepared from both neural and non-neural cell lines, mouse brain, and mouse liver contain proteins that recognize and bind to the PROX and PAL sequences indicating that proteins which bind to these target sequences are widespread. The appearance of these target sequences in the 5′ upstream region of several neuron specific genes suggests that they play key roles in the transcription of neuron specific genes. The functional activity of these target DNA sequences was demonstrated by transfection assays using a reporter gene fused to nested deletions of the NF(H) promoter region. Interestingly, these assays revealed that maximal transient expression was obtained with DNA fusion genes containing the PAL, PROX and TATA sequences. Inclusion of the Sp1 sites into the fusion genes failed to enhance the expression of the reporter gene. To determine if the NF(H) promoter can be activated in a tissue specific manner during development transgenic mice containing the promoter region linked to a β-galactosidase reporter gene were generated. In one line sporadic expression of the transgene occurred in the CNS and testis while in four other lines no expression occurred. Collectively these results suggest that the NF(H) gene promoter is active in a tissue specific manner only by interactions with regulatory elements that lie further upstream or downstream of the start site of initiation.

Object The role of adenosine A2A receptors in the early vascular response after subarachnoid hemorrhage (SAH) is unknown. In other forms of cerebral ischemia both activation and inhibition of A2A receptors is reported to be beneficial. However, these studies mainly used pharmacological receptor modulation, and most of the agents available exhibit low specificity. The authors used adenosine A2A receptor knockout mice to study the role of A2A receptors in the early vascular response to SAH. Methods Subarachnoid hemorrhage was induced in wild-type mice (C57BL/6) and A2A receptor knockout mice by endovascular puncture. Cerebral blood flow, intracranial pressure, and blood pressure were recorded, and cerebral perfusion pressure was deduced. Animals were sacrificed at 1, 3, or 6 hours after SAH or sham surgery. Coronal brain sections were immunostained for Type IV collagen, the major protein of the basal lamina. The internal diameter of major cerebral arteries and the area fraction of Type IV collagen–positive microvessels (< 100 μm) were determined. Results The initial increase in intracranial pressure and decrease in cerebral perfusion pressure at SAH induction was similar in both types of mice, but cerebral blood flow decline was significantly smaller in A2A receptor knockout mice as compared with wild-type cohorts. The internal diameter of major cerebral vessels decreased progressively after SAH. The extent of diameter reduction was significantly less in A2A receptor knockout mice than in wild-type mice. Type IV collagen immunostaining decreased progressively after SAH. This decrease was significantly less in A2A receptor knockout mice than in wild-type mice. Conclusions These results demonstrate that global inactivation of A2A receptors decreases the intensity of the early vascular response to SAH. Early inhibition of A2A receptors after SAH might reduce cerebral injury.

Platelet aggregates are present in parenchymal vessels as early as 10 minutes after experimental subarachnoid hemorrhage (SAH). Structural injury to parenchymal vessel walls and depletion of collagen-IV (the major protein of basal lamina) occur in a similar time frame. Since platelets upon activation release enzymes which can digest collagen-IV, we investigated the topographical relationship between platelet aggregates, endothelium, and basal lamina after SAH produced by endovascular perforation, using triple immunofluorescence and confocal microscopy with deconvolution. The location of platelet aggregates in relation to zymography-detected active collagenase was also examined. As reported previously, most cerebral vessels profiles contained platelets aggregates at 10 minutes after SAH. High-resolution three-dimensional image analysis placed many platelets at the ab-luminal (basal) side of endothelium at 10 minutes, and others either within the vascular basal lamina or in nearby parenchyma. By 24 hours post-hemorrhage, large numbers of platelets had entered the brain parenchyma. The vascular sites of platelet movement were devoid of endothelium and collagen IV. Collagenase activity colocalized with vascular platelet aggregates. Our data demonstrate that parenchymal entry of platelets into brain parenchyma begins within minutes after hemorrhage. Three-dimensional analysis suggests that platelet aggregates initiate or stimulate local disruption of endothelium and destruction of adjacent basal lamina after SAH.

gamma-H2AX is emerging as an important marker of ionizing radiation-induced double-strand breaks. Development of a significantly automated method to quantify gamma-H2AX would have broad application in assessing physiological responses to radiation exposure. PC-3 and DU145 prostate cancer cells grown on glass cover slips and 96-well plates were irradiated and assessed for gamma-H2AX focus formation by immunofluorescence analysis. The gamma-H2AX immunofluorescence staining was performed either manually or by using a preprogrammed automated robotic liquid handling system. A computer-controlled charge-coupled device camera acquired images serially throughout the thickness of each cell. Image analysis was performed manually and/or with automated image segmentation software. A robust relationship between radiation dose and gamma-H2AX focus numbers was demonstrated with both manual and automated image analysis methods, with excellent agreement observed between the two techniques. The r(2) correlation coefficients and Z factors exceeded 0.9 and 0.5, respectively, when gamma-H2AX focus formation was correlated with radiation dose using the automated technique. Inhibition of gamma-H2AX foci by drugs readily detected with this assay. Robotic specimen preparation with automated image acquisition and analysis can be used to quantify gamma-H2AX foci in irradiated cells, and the results agree well those obtained by manual counts. These data suggest that this assay has an excellent signal-to-noise ratio and is suitable for high-throughput applications.

The production of beta-amyloid precursor protein (beta APP) by primary mixed glial cell cultures from cerebral cortex of 2-3 day postnatal rats was examined by Northern and Western blotting and by immunocytochemistry. A single 3.5 kb beta APP RNA transcript was detected using probes recognizing all forms of beta APP messenger RNA. No signal was detected with a probe specific for the alternately spliced Kunitz protease inhibitor (KPI) region. In Western blot analysis of protein extracts, antisera specific for either the cytoplasmic or extracellular parts of beta APP detected several proteins ranging from 105 to 140 kDa. None of these were recognized by an antiserum specific to the KPI insert of the beta APP. Multicolor immunofluorescence showed beta APP immunoreactivity in type I (GFAP+ A2B5-) astrocytes, distributed in a fibrillar pattern like that of glial fibrillary acidic protein (GFAP). No beta APP immunoreactivity was detected in oligodendrocytes (GC+) or in A2B5+ progenitor cells. Moreover, no cultured cells showed immunostaining with an antiserum specific for the KPI sequence of beta APP. We conclude that type I astrocytes in primary culture produce amyloid precursor protein, but that oligodendrocytes and their precursors do not. Moreover, type I astrocytes produce predominantly the beta APP subtypes which lacks the KPI sequence.

1. In order to characterize some of the molecular events leading to repair of myelin in the adult central nervous system (CNS), we examined the expression of transcripts for myelin basic protein (MBP) during remyelination in the mouse. C57B1/6 mice develop a demyelinating disease when glial cells are selectively infected by the A59 strain of mouse coronavrius. The virus is spontaneously cleared from the mice by 4 weeks postinfection (WPI), a time when remyelination is starting. 2. At 3 WPI total MBP transcripts are decreased by 75% in demyelinating lesions compared to control white matter. Using RNase protection assays andin situ hybridization with probes for particular MBP exons, we detected an increase in MBP transcripts containing exon 2 information, coincident with the earliest histological signs of remyelination. 3. The expression of MBP transcripts containing exon 2 information was first seen clustered in the perinuclear cytoplasm of oligodendrocytes scattered within the lesions. This is reminiscent of the increased levels and perinuclear clustering of MBP transcripts containing exon 2 seen during early developmental myelination. The peak abundance of exon 2-containing transcripts in the lesions was 13-fold that seen in control white matter. At later stages of remyelination, additional forms of MBP transcripts (without exon 2) increased and their distribution was more diffuse. 4. Thus, during remyelination, preforms of MBP transcripts, which are normally present at low levels in the adult CNS, are abundantly expressed and regulated in a manner similar to that observed in developmental myelination.

A demyelinating disease induced in C57B1/6N mice by intracranial injection of a coronavirus (murine hepatitis virus strain A59) is followed by functional recovery and efficient CNS myelin repair. To study the biological properties of the cells involved in this repair process, glial cells were isolated and cultured from spinal cords of these young adult mice during demyelination and remyelination. Using three-color immunofluorescence combined with [3H]thymidine autoradiography, we have analyzed the antigenic phenotype and mitotic potential of individual glial cells. We identified oligodendrocytes with an antibody to galactocerebroside, astrocytes with an antibody to glial fibrillary acidic protein, and oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells with the O4 antibody. Cultures from demyelinated tissue differed in several ways from those of age-matched controls: first, the total number of O-2A lineage cells was strikingly increased; second, the O-2A population consisted of a higher proportion of O4-positive astrocytes and cells of mixed oligodendrocyte-astrocyte phenotype; and third, all the cell types within the O-2A lineage showed enhanced proliferation. This proliferation was not further enhanced by adding PDGF, basic fibroblast growth factor (bFGF), or insulin-like growth factor I (IGF-I) to the defined medium. However, bFGF and IGF-I seemed to influence the fate of O-2A lineage cells in cultures of demyelinated tissue. Basic FGF decreased the percentage of cells expressing galactocerebroside. In contrast, IGF-I increased the relative proportion of oligodendrocytes. Thus, O-2A lineage cells from adult mice display greater phenotypic plasticity and enhanced mitotic potential in response to an episode of demyelination. These properties may be linked to the efficient remyelination achieved in this demyelinating disease.

Autophagy is a regulated lysosomal degradation process that involves autophagosome formation and transport. Although recent evidence indicates that basal levels of autophagy protect against neurodegeneration, the exact mechanism whereby this occurs is not known. By using conditional knockout mutant mice, we report that neuronal autophagy is particularly important for the maintenance of local homeostasis of axon terminals and protection against axonal degeneration. We show that specific ablation of an essential autophagy gene, Atg7 , in Purkinje cells initially causes cell-autonomous, progressive dystrophy (manifested by axonal swellings) and degeneration of the axon terminals. Consistent with suppression of autophagy, no autophagosomes are observed in these dystrophic swellings, which is in contrast to accumulation of autophagosomes in the axonal dystrophic swellings under pathological conditions. Axonal dystrophy of mutant Purkinje cells proceeds with little sign of dendritic or spine atrophy, indicating that axon terminals are much more vulnerable to autophagy impairment than dendrites. This early pathological event in the axons is followed by cell-autonomous Purkinje cell death and mouse behavioral deficits. Furthermore, ultrastructural analyses of mutant Purkinje cells reveal an accumulation of aberrant membrane structures in the axonal dystrophic swellings. Finally, we observe double-membrane vacuole-like structures in wild-type Purkinje cell axons, whereas these structures are abolished in mutant Purkinje cell axons. Thus, we conclude that the autophagy protein Atg7 is required for membrane trafficking and turnover in the axons. Our study implicates impairment of axonal autophagy as a possible mechanism for axonopathy associated with neurodegeneration.

Object Structural changes in brain parenchymal vessels occur within minutes after subarachnoid hemorrhage (SAH). These changes include platelet aggregation, activation of vascular collagenases, and destruction of perivascular collagen IV. Because collagen IV is an important component of the basal lamina, the authors attempted to further define changes in vascular structure (length and luminal diameter) and their relationship to vascular permeability immediately after SAH. In addition, the authors explored whether such alterations were attenuated by administration of a nitric oxide (NO) donor. Methods Endovascular perforation was used to induce SAH in rats. Two sets of experiments were performed. The first established changes in vascular structure and permeability (collagen IV and endothelial barrier antigen [EBA] dual immunofluorescence) during the first 24 hours after SAH. In the second, the investigators examined the effects of an NO donor on vascular structure, permeability, and collagenase activity (in situ zymography). In this second study, animals received intravenous infusion of the NO donor S-nitrosoglutathione (GSNO, 1 μM/8 μl/min) 15 minutes after induction of SAH and were killed 3 hours after SAH onset. Controls were naive unoperated animals for the first study and saline-infused SAH animals for the second. The authors found a time-dependent decrease in area fraction, length, and luminal diameter of collagen IV– and EBA-immunofluorescent vessels after SAH. The greatest change occurred at 3 hours after onset of SAH. Administration of GSNO was associated with striking preservation of collagen IV and EBA immunofluorescence compared with saline treatment. Zymography indicated decreased collagenase activity in GSNO-treated SAH animals compared with saline-treated SAH animals. Conclusions These results demonstrate changes in the structure and permeability of brain parenchymal microvessels after SAH and their reversal by treatment with an NO donor.