Composition and organization of the SCZ: a large germinal layer containing neural stem cells in the adult mammalian brain.

Two known germinal zones continue to generate new neurons and glia in the adult mammalian brain: the subventricular zone (SVZ), lining the lateral walls of the lateral ventricle, and the subgranular zone of the dentate gyrus. Here we describe a region we will refer to as the subcallosal zone (SCZ). The SCZ is a caudal extension of the SVZ that is no longer associated to an open ventricle. It lies between the hippocampus and the corpus callosum. Cells isolated from the SCZ and cultured as neurospheres behave as neural stem cells in vitro. Using electron and light microscopy, we describe the cell types present in this region and how their organization differs from that of the SVZ. Using retroviral labeling and homotypic-homochronic microtransplantation techniques, we show that the majority of cells born in the SCZ migrate into the corpus callosum to become oligodendrocytes in vivo. This study defines the organization and fate of cells born in a large germinal region of the adult forebrain.

The subventricular zone: source of neuronal precursors for brain repair.

The subventricular zone (SVZ) is a major germinal zone which persists in the adult brain. The SVZ contains cells that self renew and continuously produce new neurons and glia. In this chapter we discuss the development, architecture and function of the adult SVZ, as well as the fate of SVZ cells after transplantation. We focus on identification of neural stem cells, factors which regulate neurogenesis and mechanisms for neuronal migration through the adult brain. Detailed understanding of these processes is necessary to utilize the SVZ as a source of neuronal and glial precursors for genetic manipulation, transplantation or brain self repair.

Noggin antagonizes BMP signaling to create a niche for adult neurogenesis.

Large numbers of new neurons are born continuously in the adult subventricular zone (SVZ). The molecular niche of SVZ stem cells is poorly understood. Here, we show that the bone morphogenetic protein (BMP) antagonist Noggin is expressed by ependymal cells adjacent to the SVZ. SVZ cells were found to express BMPs as well as their cognate receptors. BMPs potently inhibited neurogenesis both in vitro and in vivo. BMP signaling cell-autonomously blocked the production of neurons by SVZ precursors by directing glial differentiation. Purified mouse Noggin protein promoted neurogenesis in vitro and inhibited glial cell differentiation. Ectopic Noggin promoted neuronal differentiation of SVZ cells grafted to the striatum. We thus propose that ependymal Noggin production creates a neurogenic environment in the adjacent SVZ by blocking endogenous BMP signaling.

Adult-derived neural precursors transplanted into multiple regions in the adult brain.

Neural stem cells persist in the adult brain subventricular zone (SVZ). These cells generate a large number of new neurons that migrate to the olfactory bulb, where they complete their differentiation. Here, we transplanted cells carrying beta-galactosidase under the control of neuron-specific enolase promoter (NSE::LacZ) from the SVZ of adult mice into the striatum cortex and olfactory bulb, with or without an excitotoxin lesion. Between 2 and 8 weeks after transplantation, grafted cells were present in the recipient regions, but extensive migration and differentiation into mature neurons of grafted cells were only observed in the olfactory bulb. Clusters of graft-derived neuroblasts forming chain-like structures were observed within or close to the grated sites in the cortex and striatum; electron microscopy confirmed that graft-derived cells in the olfactory bulb and a small number in the striatum were neurons. Surprisingly, most of the cells expressing NSE::LacZ outside the olfactory bulb were astrocytes. We conclude that primary precursors from the SVZ migrate and differentiate effectively only within the environment of the olfactory bulb. Only limited survival and differentiation were observed in other brain regions studied.

Young neurons from medial ganglionic eminence disperse in adult and embryonic brain.

In this study, we identified neuronal precursors that can disperse through adult mammalian brain tissue. Transplanted neuronal precursors from embryonic medial ganglionic eminence (MGE), but not from lateral ganglionic eminence (LGE) or neocortex, dispersed and differentiated into neurons in multiple adult brain regions. In contrast, only LGE cells were able to migrate efficiently from the adult subventricular zone to the olfactory bulb. In embryonic brain slices, MGE cells migrated extensively toward cortex. Our results demonstrate that cells in different germinal regions have unique migratory potentials, and that adult mammalian brain can support widespread dispersion of specific populations of neuronal precursors. These findings could be useful in repair of diffuse brain damage.

Analysis of c-Fos and glial fibrillary acidic protein (GFAP) expression following topical application of potassium chloride (KCl) to the brain surface.

Application of high K+ concentrations to a limited area of the brain surface is known to trigger spreading depression. We used this model to observe the response of cortical areas, distant to the exposed site, at the cellular level. Immunostaining of glial fibrillary acidic protein (GFAP) and of the proto-oncogene c-Fos was analyzed in brain sections at different times after K+ application. Piriform and parietal cortices, as observed in coronal sections located 3 mm rostrally from the center of the stimulated area and ipsilateral to it, showed a dramatic increase in immunostaining for both markers. However, the time course for such increments was different. c-Fos protein(s) expression was high at 1.5 h and decreased at 24 h after K+ exposure and c-fos mRNA expression correlated with the immunohistochemical results. At these initial times GFAP immunoreactivity was still low but began to rise between 2 and 7 days after treatment in exactly the same areas where c-Fos expression had been up-regulated. No significant effect, for either marker, was evident in the contralateral piriform or parietal cortices. In addition, we studied the effects of the NMDA antagonist MK-801 (4 mg/kg i.p.) on the expression of mRNA for GFAP and c-fos and demonstrated a marked reduction in the upregulation of these genes.

Adult-onset Krabbe's disease in siblings with novel mutations in the galactocerebrosidase gene.

Krabbe's disease or globoid cell leukodystrophy is a rare demyelinating disorder of the central and peripheral nervous systems, the diagnosis of which is based on clinical findings and the determination of low to absent functional activity of the enzyme beta-galactocerebrosidase. We report the presentation of late-onset Krabbe's disease in 2 siblings, a 17-year-old boy and his 16-year-old sister, both with marked deficiency of the enzyme beta-galactocerebrosidase. Only the older sibling manifested clinical signs and symptoms of the disease, while the younger sister remained asymptomatic to date. Molecular analyses disclosed the presence in this family of two novel single point mutations within the gene for galactocerebrosidase.

Activation of c-fos in the brain.

Activation of the proto-oncogene c-fos in the brain was described initially almost a decade ago and represents one of the most studied immediate early genes in the brain. Transient c-fos expression in the central nervous system was first observed after seizure activity and following noxious stimulation in the spinal cord. Since then, multiple studies have shown that different stimuli can induce c-fos expression. Seizure activity induces rapid and transient expression of c-fos in hippocampal structures. Similarly, transient activation of c-fos follows cortical brain injury in a pattern that resembles that of spreading depression. Many other stimuli have been shown to induce the expression of this proto-oncogene in the brain and c-fos immunostaining and in situ hybridization are now used to map brain metabolism under different physiological and non-physiological conditions. Here we review the variety of inducible patterns of c-fos expression in the brain.

Differential regulation of c-fos expression after cortical brain injury during development.

Unilateral cortical brain injury is accompanied by widespread expression of c-fos protein(s) throughout the wounded cortex, including areas far from the lesion site. Here we report that this phenomenon is differentially regulated during development. At postnatal day (PD) 10 or 15, when rats were sacrificed 1.5 h after a mechanical cortical injury, they did not show an increase in c-fos immunoreactivity far from the wound, despite the fact that some of these animals (PD 15) displayed a positive response close to the lesion. At PD 22, the same injury induced an increase in c-fos-immunoreactive nuclei in the piriform cortex ipsilateral, but not contralateral, to the lesion. This pattern was maintained up to at least PD 360. Similarly, the presence of c-fos-immunoreactive cells was observed in the ipsilateral cingulate cortex in animals 22 days old and older. The pattern of c-fos expression in adult animals after mechanical damage was compared with other models of focal brain injury: application of potassium to the cortical surface and devascularization. Though all models generated c-fos expression far from the lesion site, potassium application resulted in higher numbers of c-fos-positive cells, particularly in the cingulate cortex. This study shows that c-fos expression after cortical brain injury is regulated differently during development, and that dissimilar models of cortical injury induce qualitatively similar responses although c-fos-like protein expression differs quantitatively.

Spreading depression induces c-fos-like immunoreactivity and NGF mRNA in the rat cerebral cortex.

Application of potassium chloride (KCl) to the brain surface elicits spreading depression which leads to a marked induction of the proto-oncogene c-fos in the treated cerebral cortex at the earliest time examined (90 min). High levels of c-fos immunoreactivity are observed up to 6 h after KCl treatment. The areas affected include the cingulate, entorhinal and frontoparietal cortex throughout the treated hemisphere. The c-fos expression preceded an increase in both NGFmRNA and NGF-like protein(s). A maximal increase in c-fos was detected within 3 h, whereas NGFmRNA peaked at 12 h and NGF-like protein(s) reached their maximum level 24 h after KCl application. The most prominent increase in NGFmRNA was measured in the entorhinal cortex (50-fold), but other cortical areas also showed a moderate increase of 2-3-fold. In conclusion, our results provide evidence that increases in c-fos and NGF expression are early adaptive responses following brain injury.

Induction of c-FOS immunoreactivity in the hippocampus following potassium stimulation.

In microdialysis procedures high potassium ion concentrations are generally used to induce neurotransmitter release. However, the widespread effects, if any, of such a treatment have not been described. In order to establish a possible link between c-fos expression and stimulating conditions for neurotransmitter release in microdialysis procedures we administered KCl (100 mM) into the hippocampus. Proto-oncogene c-FOS-like immunoreactivity is upregulated in granule cells of the dentate gyrus, pyramidal cells of the hippocampus, cingulate, piriform and frontoparietal cortices at 2 h, but not 24 h after K+ administration. Neither implantation of the probes nor perfusion with artificial cerebrospinal fluid resulted in similar patterns of c-FOS immunoreactivity. In addition, we investigated whether the impairment of the cholinergic septohippocampal pathway would modify the K(+)-induced expression of the immediate early gene c-fos in the hippocampus. The expression of c-fos induced by KCl was not altered in the animals with fimbria-fornix lesion despite the marked decrease in acetylcholine release in the hippocampus. Glutamate concentrations measured in the same superfusates showed that a significantly greater glutamate release occurs in denervated hippocampi. Furthermore, abolishment of seizure-like activity (induced by KCl) in anesthetized animals did not alter expression of c-FOS immunoreactivity in the K(+)-stimulated hippocampi. The results from these studies confirm that most of the releasable acetylcholine of the hippocampus is linked to the fimbrial input and may suggest that c-FOS upregulation in this model does not respond to any cholinergic input from the medial septum via the fimbria-fornix.

MK-801 affects the potassium-induced increase of glial fibrillary acidic protein immunoreactivity in rat brain.

Exposure of a limited brain surface to a high potassium (K+) concentration produces an injury limited to the underlying cortex, without apparently affecting other brain areas. Such a treatment produces an increased expression of glial fibrillary acidic protein (GFAP) in astrocytes, as assessed by immunohistochemical techniques, throughout the cortex ipsilateral to K+ exposure. This effect is evident 2 days after treatment and persists up to, at least, day 7. Thirty days after K+ exposure GFAP immunostaining is similar in both hemispheres. Administration of the non-competitive NMDA antagonist MK-801 (4 mg/kg i.p.) prior to the injury prevented the rise in GFAP immunoreactivity (IR) at 2 but not 7 days after the treatment. Administration of MK-801 after the injury appeared to have no effect on GFAP expression. This work confirms that brain injury, associated with spreading depression, can induce a glial response far from the lesion site. Furthermore, the fact that this phenomenon can be modified by an NMDA receptor antagonist suggests that glutamate may play a role, in vivo, in the regulation of astrocytic response to injury and introduces the possibility that brain injury-induced gliosis may be pharmacologically manipulated.

Glial fibrillary acidic protein immunoreactivity following cortical devascularizing lesion.

Disruption of a restricted area of the pia-arachnoid compromises vascular irrigation of the underlying cortex, leading to infarction of the tissue. This study was undertaken to examine the effects of such brain injury on glial cells. Adult male Wistar rats were processed for glial fibrillary acidic protein immunoreactivity at 1, 4, 7, 15 and 30 days after undergoing cortical devascularization. One day post-lesion, glial fibrillary acidic protein-positive cells were observed only at the lesion site. Glial fibrillary acidic protein-positive cells were present in the ipsilateral remaining cortex, distant from the wound, between days 4 and 15, and were also evident in the thalamus beginning 4 days post-lesion. These diverse temporal patterns of GFAP immunoreactivity in different brain structures suggest that various mechanisms can mediate increased GFAP immunostaining following injury.

Application of potassium chloride to the brain surface induces the c-fos proto-oncogene: reversal by MK-801.

The proto-oncogene c-fos is activated in the brain by a variety of stimuli including brain injury. In unilateral brain injury, c-fos immunoreactivity is confined to the damaged hemisphere, an effect reminiscent of spreading depression. Here we show that topical application of KCl (3 M) to the brain surface (which induces spreading depression) is accompanied by ipsilateral increase in c-fos immunolabeling. The activation of c-fos, like spreading depression, is markedly reduced by the non-competitive NMDA antagonist MK-801 (3 mg/kg i.p.).

N-methyl-D-aspartate receptors mediate activation of the c-fos proto-oncogene in a model of brain injury.

The proto-oncogene c-fos is rapidly and transiently induced in the CNS by a variety of stimuli. Brain injury, disruption of pia-arachnoid in a limited area, is one of the situations that leads to a dramatic increase in c-fos immunoreactivity. This increase is limited to the lesioned hemisphere. Injections of atropine (25 mg/kg, i.p.), naltrexone (5 mg/kg, i.p.), nifedipine (5 mg/kg, i.p.), and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (20 mg/kg, i.p.), prior to the injury, did not affect the activation of c-fos as assessed by immunohistochemistry in adult Sprague-Dawley rats perfused 2 h after the lesion. The non-competitive N-methyl-D-aspartate antagonists ketamine (100 mg/kg, i.p.) and MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate] (1 and 3 mg/kg, i.p.) markedly reduced c-fos activation. Phencyclidine (10 mg/kg, i.p.) produced a slight reduction in damage-induced fos activation. This study suggests that c-fos activation in this particular model is N-methyl-D-aspartate receptor-mediated and supports the idea that the fos proto-oncogene might play a role in plasticity and/or neurotoxic changes following brain damage.

Unilateral induction of c-fos protein in cortex following cortical devascularization.

The proto-oncogene c-fos is rapidly induced by a variety of stimuli including wounding of the cortex. In an attempt to define the conditions necessary for c-fos activation, we have studied the effects of cerebral devascularization (CD; unilateral pial disruption), a procedure which results in ischaemia without producing direct physical damage to the tissues. Cerebral devascularization produces c-fos activation throughout the ipsilateral cortex; this effect lasts for about 4 h and is not confined to the area subjacent to pial disruption. The activation of c-fos is not prevented by even high doses of benzodiazepines but is effectively reduced by the N-methyl-D-aspartate (glutamate) receptor antagonist ketamine.