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Intraventricular hemorrhage causes spatial memory loss, but the mechanism remains unknown. Our recent studies demonstrated that traumatic brain injury activates Src family kinases, which cause spatial memory loss. To test whether the spatial memory loss was due to blood in the ventricles, which activated Src family kinases, we infused autologous whole blood or thrombin into the lateral ventricles of adult rats to model non-traumatic intraventricular hemorrhage. Hippocampal neuron loss was examined 1 day to 5 weeks later. Spatial memory function was assessed 29 to 33 days later using the Morris water maze. Five weeks after the ventricular injections of blood or thrombin, there was death of most hippocampal neurons and significant memory deficits compared with sham operated controls. These data show that intraventricular thrombin is sufficient to kill hippocampal neurons and produce spatial memory loss. In addition, systemic administration of the non-specific Src family kinase inhibitor PP2 or intraventricular injection of siRNA-Fyn, a Src family kinase family member, prevented hippocampal neuronal loss and spatial memory deficits following intraventricular hemorrhage. The data support the conclusions that thrombin mediates the hippocampal neuronal cell death and spatial memory deficits produced by intraventricular blood and that these can be blocked by non-specific inhibition of Src family kinases or by inhibiting Fyn.

Class III phosphoinositide 3-kinase (PIK3C3 or mammalian vacuolar protein sorting 34 homolog, Vps34) regulates vesicular trafficking, autophagy, and nutrient sensing. Recently, we reported that PIK3C3 is expressed in mouse cerebral cortex throughout the developmental process, especially at early embryonic stage. We thus examined the role of PIK3C3 in the development of the mouse cerebral cortex. Acute silencing of PIK3C3 with in utero electroporation method caused positional defects of excitatory neurons during corticogenesis. Time-lapse imaging revealed that the abnormal positioning was at least partially because of the reduced migration velocity. When PIK3C3 was silenced in cortical neurons in one hemisphere, axon extension to the contralateral hemisphere was also delayed. These aberrant phenotypes were rescued by RNAi-resistant PIK3C3. Notably, knockdown of PIK3C3 did not affect the cell cycle of neuronal progenitors and stem cells at the ventricular zone. Taken together, PIK3C3 was thought to play a crucial role in corticogenesis through the regulation of excitatory neuron migration and axon extension. Meanwhile, when we performed comparative genomic hybridization on a patient with specific learning disorders, a 107 Kb-deletion was identified on 18q12.3 (nt. 39554147-39661206) that encompasses exons 5-23 of PIK3C3. Notably, the above aberrant migration and axon growth phenotypes were not rescued by the disease-related truncation mutant (172 amino acids) lacking the C-terminal kinase domain. Thus, functional defects of PIK3C3 might impair corticogenesis and relate to the pathophysiology of specific learning disorders and other neurodevelopmental disorders. Acute knockdown of Class III phosphoinositide 3-kinase (PIK3C3) evokes migration defects of excitatory neurons during corticogenesis. PIK3C3-knockdown also disrupts axon outgrowth, but not progenitor proliferation in vivo. Involvement of PIK3C3 in neurodevelopmental disorders might be an interesting future subject since a deletion mutation in PIK3C3 was detected in a patient with specific learning disorders (SLD)., (© 2016 International Society for Neurochemistry.)

Context: Andrographolide, extracted from the leaves of Andrographis paniculata (Burm. f.) Nees (Acanthaceae), is a labdane diterpene lactone. It is widely reported to possess anti-inflammatory and antitumorigenic activities. Cerebral endothelial cells (CECs) play a crucial role in supporting the integrity and the function of the blood-brain barrier (BBB). However, no data are available concerning the effects of andrographolide in CECs. The aim of this study was to examine the detailed mechanisms of andrographolide on CECs., Objective: This study investigated a novel bioactivity of andrographolide on cerebral ischemia/reperfusion-induced brain injury., Materials and Methods: CECs were treated with andrographolide (20-100 µΜ) for the indicated times (0-24 h). After the reactions, cell survival rate and cytotoxicity were tested by the MTT assay and the lactate dehydrogenase (LDH) test, respectively. Western blotting was used to detect caspase-3 expression. In addition, analysis of cell cycle and apoptosis using PI staining and annexin V-FITC/PI labeling, respectively, was performed by flow cytometry. We also investigated the effect of andrographolide on middle cerebral artery occlusion (MCAO)/reperfusion-induced brain injury in a rat model., Results: In the present study, we found that andrographolide (50-100 µΜ) markedly inhibited CEC growth according to an MTT assay and caused CEC damage according to a LDH test. Our data also revealed that andrographolide (50 µM) induced CEC apoptosis and caspase-3 activation as respectively detected by PI/annexin-V double staining and western blotting. Moreover, andrographolide arrested the CEC cell cycle at the G0/G1 phase by PI staining. In addition, andrographolide (5 mg/kg) caused deterioration of MCAO/reperfusion-induced brain injury in a rat model., Conclusions: These data suggest that andrographolide may disrupt BBB integrity, thereby deteriorating MCAO/reperfusion-induced brain injury, which are, in part, associated with its capacity to arrest cell-cycle and induce CEC apoptosis.

The aim of this study was to determine the role of NADPH-cytochrome P450 reductase (CPR) and CPR-dependent enzymes in neural stem cell (NSC) genesis in the brain. A mouse model with globally suppressed Cpr gene expression (Cpr-low mouse) was studied for this purpose. Cpr-low and wild-type (WT) mice were compared immunohistochemically for the expression of markers of cell proliferation (Ki67), immature neurons (doublecortin, DCX), oligodendrocytes (oligodendrocyte transcription factor 2, OLIG2), and astrocytes (glial fibrillary acidic protein, GFAP) in the SVZ, and for the in vitro capability of their SVZ cells to form neurospheres and differentiate into astrocytes. We found that the abundance of SVZ cells that are positive for Ki67 or GFAP expression, but not the abundance of SVZ cells that are positive for DCX and OLIG2 expression, was significantly increased in Cpr-low mice, at various ages, compared with WT mice. Furthermore, extents of astrocyte differentiation and growth, but not neurosphere formation, from SVZ cells of the Cpr-low mice were significantly increased, compared with WT mice. These results suggest that CPR and CPR-dependent enzymes play a role in suppressing astrocytosis in the SVZ of adult mice., (Published by Elsevier Ireland Ltd.)

Recent studies have led to the exciting idea that adult-born neurons in the olfactory bulb (OB) may be critical for complex forms of olfactory behavior in mice. However, signaling mechanisms regulating adult OB neurogenesis are not well defined. We recently reported that extracellular signal-regulated kinase (ERK) 5, a MAP kinase, is specifically expressed in neurogenic regions within the adult brain. This pattern of expression suggests a role for ERK5 in the regulation of adult OB neurogenesis. Indeed, we previously reported that conditional deletion of erk5 in adult neurogenic regions impairs several forms of olfactory behavior in mice. Thus, it is important to understand how ERK5 regulates adult neurogenesis in the OB. Here we present evidence that shRNA suppression of ERK5 in adult neural stem/progenitor cells isolated from the subventricular zone (SVZ) reduces neurogenesis in culture. By contrast, ectopic activation of endogenous ERK5 signaling via expression of constitutive active MEK5, an upstream activating kinase for ERK5, stimulates neurogenesis. Furthermore, inducible and conditional deletion of erk5 specifically in the neurogenic regions of the adult mouse brain interferes with cell cycle exit of neuroblasts, impairs chain migration along the rostral migratory stream and radial migration into the OB. It also inhibits neuronal differentiation and survival. These data suggest that ERK5 regulates multiple aspects of adult OB neurogenesis and provide new insights concerning signaling mechanisms governing adult neurogenesis in the SVZ-OB axis.

Intraperitoneal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration induces apoptosis of subventricular zone (SVZ) doublecortin (Dcx)-positive neural progenitor cells (migrating neuroblasts, A cells). Actually, a metabolite of MPTP, 1-methy-4-phenylpiridinium (MPP(+)), is responsible for neural progenitor cell toxicity. In the present study, to examine whether the MPTP-induced SVZ cell apoptosis is caused directly by MPP(+) metabolized through monoamine oxidase B (MAO-B), MPTP or MPP(+) was intracerebroventricularly (icv) injected into C57BL/6 mice. At Day 1 postinjection, many terminal deoxynucleotidyl transferase-mediated dUTP endlabeling (TUNEL)-positive cells were observed in the SVZ of both low (36 μg) and high (162 μg) dose MPTP- and MPP(+)-injected mice. The number of Dcx-positive A cells showed a significant decrease following high dose of MPTP- or MPP(+)-injection on Days 1 and 3, respectively, whereas that of EGFR-positive C cells showed no change in mice with any treatment. In addition, prior icv injection of a MAO-B inhibitor, R(-)-deprenyl (deprenyl), inhibited MPTP-induced apoptosis, but not MPP(+)-induced apoptosis. MAO-B- and GFAP-double positive cells were detected in the ependyma and SVZ in all mice. It is revealed from these results that icv injection of MPTP induces apoptosis of neural progenitor cells (A cells) in the SVZ via MPP(+) toxicity. In addition, it is suggested that the conversion from MPTP to MPP(+) is caused mainly by MAO-B located in ependymal cells and GFAP-positive cells in the SVZ., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Neural stem cells (NSC) persist in the adult mammalian brain, within the subventricular zone (SVZ). The endogenous mechanisms underpinning SVZ stem and progenitor cell proliferation are not fully elucidated. Vitamin K-dependent proteins (VKDPs) are mainly secreted factors that were initially discovered as major regulators of blood coagulation. Warfarin ((S(-)-3-acetonylbenzyl)-4-hydroxycoumarin)), a widespread anticoagulant, is a vitamin K antagonist that inhibits the production of functional VKDP. We demonstrate that the suppression of functional VKDPs production, in vitro, by exposure of SVZ cell cultures to warfarin or, in vivo, by its intracerebroventricular injection to mice, leads to a substantial increase in SVZ cell proliferation. We identify the anticoagulant factors, protein S and its structural homolog Gas6, as the two only VKDPs produced by SVZ cells and describe the expression and activation pattern of their Tyro3, Axl, and Mer tyrosine kinase receptors. Both in vitro and in vivo loss of function studies consisting in either Gas6 gene invalidation or in endogenous protein S neutralization, provided evidence for an important novel regulatory role of these two VKDPs in the SVZ neurogenic niche. Specifically, we show that while a loss of Gas6 leads to a reduction in the numbers of stem-like cells and in olfactory bulb neurogenesis, endogenous protein S inhibits SVZ cell proliferation. Our study opens up new perspectives for investigating further the role of vitamin K, VKDPs, and anticoagulants in NSC biology in health and disease., (Copyright © 2012 AlphaMed Press.)

Forebrain subventricular zone (SVZ)--the putative major source of neural stem cells in the brain of adult mammals--can hardly be visualized using routine histological staining. The present study was focused on the possibility of application of immunocytochemical approach for accurate delineation of the border between SVZ and striatum. It was shown that immunocytochemical reactions demonstrating tyrosine hydroxylase or synaptophysin were optimal for the determination of the border between SVZ and striatum in different mammals.

The consequences of nitric oxide synthase (NOS) gene knockout on proliferation, survival and differentiation of neuronal precursors in the subgranular (SGZ) and subventricular (SVZ) zones were analyzed. Comparative studies were performed in neonatal, adult and old (18-month) wild-type (WT), nNOS, eNOS, and iNOS knockout (KO) mice. Effects on brain cell proliferation were studied by sacrificing animals at 24h after injecting BrdU, while effects on survival and differentiation of dividing brain cells were studied by sacrificing other animals at three weeks after injections and double immunostaining with cell phenotype-specific antibodies. In the neonatal SGZ, cell proliferation was higher than at any other age, with a significantly decreased level in eNOS-KO mice. In the neonatal SVZ, cell proliferation in each of the three NOS-KO strains was significantly lower than in WT. In the adult, in both the SGZ and SVZ, all strains showed lower levels of cell proliferation than in neonates. Thereby, the significant highest cell proliferation was found in the SGZ and SVZ of nNOS-KO mice. In the SGZ and SVZ of old mice, in each strain, BrdU-positive cell counts were further reduced from adult levels, whereby cell proliferation of nNOS-KO mice attained the most massive reduction (in the SGZ almost to zero). In adult animals sacrificed 21 days after BrdU injections, values of BrdU-/NeuN-positive cells in all knockout animals were the same as WT, indicating that the initial cell proliferation changes were not sustained or translated into neuronal differentiation. The effect of nNOS-KO, inducing cell proliferation only temporarily, consists with the concept that neuronal stem cells have a finite proliferation capacity., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Germinal matrix/intraventricular hemorrhage (GMH/IVH) is a complication that arises in premature infants associated with neurological sequelae. Greater understanding of GMH/IVH is needed to develop therapies, a goal that depends on the existence of appropriate animal models. Towards this goal, we aimed to develop a rodent model of GMH/IVH based on collagenase-induced hemorrhage that exhibits histological and neurological consequences similar to that seen in patients. Male 6-day-old rats were placed on a warming pad and anesthetized with halothane/nitrous oxide delivered by face mask. Uni- or bilateral periventricular injections of 2-μl collagenase (2.0 U) were performed freehand with a needle inserted percutaneously. Sham rats were infused with saline. Early neonatal development, long-term motor and cognitive performances and alterations in brain volume were assessed. Collagenase-based GMH/IVH negatively affected ambulation, surface righting and negative geotaxis outcomes more evidently in bilaterally infused rats, which also presented an early decrease in brain volume, as assessed by the Cavalieri method. In adult animals, a unilateral collagenase infusion produced no significant alteration on forepaw preference. Only bilaterally infused rats presented an impairment of object recognition memory and locomotor deficit. Nevertheless, histological evaluation also demonstrated a persistent brain volume reduction in bilaterally infused rats. Our study provides a pioneering animal model of collagenase-based GMH/IVH, which can be used to evaluate preventive strategies and potential therapeutic interventions for this disorder., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In the adult brain, progenitor cells remaining in the subventricular zone (SVZ) are frequently identified as glial fibrillary acidic protein (GFAP)-positive cells that retain attributes reminiscent of radial glia. Because the very high expression of monoamine oxidase B (MAO-B) in the subventricular area has been related to epithelial and astroglial expression, we sought to ascertain whether it was also expressed by progenitor cells of human control and Alzheimer's disease (AD) patients. In the SVZ, epithelial cells and astrocyte-like cells presented rich MAO-B activity and immunolabeling. Nestin-positive cells were found in the same area, showing a radial glia-like morphology. When coimmunostaining and confocal microscopy were performed, most nestin-positive cells showed MAO-B activity and labeling. The increased progenitor activity in SVZ proposed for AD patients was confirmed by the positive correlation between the SVZ nestin/MAO-B ratio and the progression of the disease. Nestin/GFAP-positive cells, devoid of MAO-B, can represent a distinct subpopulation of an earlier phase of maturation. This would indicate that MAO-B expression takes place in a further step of nestin/GFAP-positive cell differentiation. In the early AD stages, the discrete MAO-B reduction, different from the severe GFAP decrease, would reflect the capacity of this population of MAO-B-positive progenitor cells to adapt to the neurodegenerative process.

The LRRK2 gene was recently found to have multiple mutations that are causative for the most common inherited form of late onset Parkinson's disease. In the adult brain, LRRK2 mRNA is broadly expressed, also in regions other than the nigrostriatal system. In order to establish a basis for assessing more detailed functional implications of LRRK2 in development, we provide here an in-depth analysis of its mRNA expression patterns in neural and extra-neural tissues with a focus on murine embryonic development. LRRK2 mRNA is detectable at E8.5 in non-neural and at E10.5 in neural tissues. From E12.5 to E16.5, LRRK2 mRNA is prominently expressed throughout the neocortex and subsequently highly concentrated in ventricular and subventricular zones and cortical plate. In addition, developing cerebellar granule and Purkinje neurons and spinal cord neurons display robust LRRK2 expression. In non-neural tissues LRRK2 was highly expressed in limb interdigital zones, developing kidney glomeruli, and spermatogenetic cells. Together, our results suggest roles for LRRK2 in controlling proliferation, migration, and differentiation of neural cells as well as in morphogenesis of extra-neural tissues., (Copyright 2010 ISDN. Published by Elsevier Ltd. All rights reserved.)

FGF-2 and Anosmin-1 are diffusible proteins which act in cell proliferation and/or migration during CNS development. We describe their developmental expression patterns in the subventricular zone (SVZ) of the forebrain and the neuronal precursors (NPs) that migrate from this neurogenic site towards the olfactory bulb, forming the rostral migratory stream (RMS). The analysis is carried out before (E14), during (E17, P5) and after (P15) the peaks of migration along the RMS and before this acquires its mature conformation. At all these stages, FGF-2 exerts a FGFR1-mediated motogenic effect on NPs and induces the proliferation of SVZ astrocytes (putatively type B cells from triads), and Anosmin-1 works as a typical chemotropic agent for the NPs (mediated by FGFR1 at P5-P15). Altogether, our results are consistent with the notion that FGF-2 increases cell proliferation in the SVZ and would be the motogenic cue which feeds the migration of the newly produced NPs once generated, from early development (E14) and at least until P15, while Anosmin-1 cooperates in this migration attracting the NPs. In this sense, both cues should be considered as two of the first to be chronologically identified as actors in the formation of the RMS., (Copyright 2010 Elsevier Inc. All rights reserved.)

A fundamental question in developmental biology is how signaling pathways establish a transcription factor code that controls cell proliferation, regional fate and cell fate. Morphogenesis of the rostral telencephalon is controlled in part by Fgf signaling from the rostral patterning center. How Fgf signaling is regulated in the telencephalon is critical for understanding cerebral cortex formation. Here we show that mouse Sprouty1 and Sprouty2 (Spry1-2), which encode negative feedback regulators of Fgf signaling, are affecting cortical proliferation, differentiation, and the expression of genes regulating progenitor identity in the ventricular zone. In addition, Spry2 has a later function in regulating the MAPK pathway, proliferation, and gene expression in the cortex at mid-neurogenesis. Finally, we provide evidence that Coup-TFI, a transcription factor that promotes caudal fate, does so through repressing Fgf signaling, in part by promoting Spry expression.

Previous studies have implicated nitric oxide (NO) in the antinociceptive response to the anesthetic gas nitrous oxide (N(2)O). The present study was conducted to confirm this NO involvement using pharmacological and gene knockdown and knockout strategies to inhibit the supraspinal and spinal production of NO. Antinociceptive responsiveness to 70% N(2)O was assessed using the acetic acid (0.6%) abdominal constriction test in NIH Swiss mice following intracerebroventricular (i.c.v.) or intrathecal (i.t.) pretreatment with the NOS-inhibitor l-N(G)-nitro arginine methyl ester (L-NAME) or an antisense oligodeoxynucleotide (AS-ODN) directed against neuronal NOS (nNOS). Experiments were also conducted in mice homozygous for a defective nNOS gene (nNOS(-/-)). Mice that were pretreated i.c.v. or i.t. with L-NAME (1.0 microg) both exhibited 80-90% reduction in the magnitude of the N(2)O-induced antinociceptive response. Mice that were pretreated i.c.v. or i.t. with nNOS AS-ODN (3 x 25microg) exhibited a 60-80% antagonism of the antinociceptive response. Compared to wild-type mice, nNOS knockout mice showed a 60% reduction in N(2)O-induced antinociception. These findings consistently demonstrate that transient or developmental suppression of nNOS expression significantly reduces antinociceptive responsiveness to N(2)O. NO of both supraspinal and spinal origin, therefore, plays an important role in the antinociceptive response to N(2)O.

The effects of caspase inhibitors on different types of learning and memory were studied in adult rats on administration into the cerebral ventricles and application to the vermis of the cerebellum. The wide-spectrum caspase inhibitor z-VAD-fmk, given into the lateral ventricles of adult rats, facilitated the formation of long-term spatial memory in a water maze and increased the ability to rearrange the habit at the early stages of acquisition of this skill. Application of the specific caspase 3 inhibitor z-DEVD-CHO to the cerebellar vermis stimulated the extinction of an acoustic startle reaction but had no effect on its retention or reproduction. These results indicate that caspases may be involved in the mechanisms of learning and memory both via indirect influences on the linked processes of neurogenesis and apoptosis in the adult brain and by regulating synaptic efficiency.

Aims: To evaluate possible effects of the intracerebroventricular (icv) injection of either O-Tricyclo [5.2.1.0(2,6)] dec-9-yl dithiocarbonate potassium salt (D609), a potent antioxidant and inhibitor of phosphatidylcholine specific phospholipase C (PtdCho-PLC) and acid sphingomyelinase (ASMase), or the spin trap/free radical scavenger N-tert-Butyl-alpha-phenylnitrone (PBN), on mechanical allodynia induced by facial carrageenan injection in mice., Methods: Balb/c mice received icy injection of D609/PBN plus facial carrageenan injection, and the number of face wash strokes to von Frey hair mechanical stimulation of the maxillary skin was quantified. PtdCho-PLC and ASMase activities were also assayed in the brainstem, thalamus, and somatosensory cortex., Results: Mice that received the icy injection of 10 nmol D609 plus facial carrageenan injection showed significantly fewer face wash strokes evoked by von Frey hair stimulation (indicating reduced mechanical allodynia) at 1 and 3 days post-injection, compared to mice that received icy injection of isotonic saline plus facial carrageenan injection. Mice that received icy injection of 1.13 micromol PBN plus facial carrageenan injection likewise showed significantly fewer face wash strokes after facial carrageenan injection, compared to isotonic saline-injected plus carrageenan-injected controls. D609 injection also resulted in significantly reduced ASMase activity in the brainstem, thalamus, and somatosensory cortex 3 days after injection, compared to controls., Conclusion: The icv injections of D609 and PBN were effective in reducing mechanical allodynia after facial carrageenan injection-induced pain. Together, the results point to a possible role of central nervous system sphingolipids and/or free radicals in orofacial pain.

Eph receptors have been implicated in regulating a diverse array of cellular functions in the developing nervous system. Recently, Eph receptors have been shown to promote cell death in adult germinal zones; however, their mechanisms of action remain ill-defined. In this study, we demonstrate that EphA4 is a new member of the dependence receptors family, which can initiate cell death in the absence of its ligand ephrinB3. Upon removal of its ligand, EphA4 triggers cell death that is dependent on caspase activation as caspase inhibitors prevent cell death. EphA4 itself is cleaved by caspase-3-like caspase in the intracellular domain at position D773/774, which is necessary for cell death initiation as mutation of the cleavage site abolishes apoptosis. In the adult subventricular zone, abolishing ephrinB3 results in increased cell death, while the absence of EphA4 results in excessive numbers of neuroblasts. Furthermore, infusion of soluble ephrinB3 into the lateral ventricle reduced cell death, and together these results support a dependence role for EphA4 in adult neurogenesis.

The subventricular zone (SVZ) is a major site of neurogenesis in the adult. We now show that ependymal and proliferating cells in the adult mouse SVZ express diacylglycerol lipases (DAGLs), enzymes that synthesise a CB1/CB2 cannabinoid receptor ligand. DAGL and CB2 antagonists inhibit the proliferation of cultured neural stem cells, and the proliferation of progenitor cells in young animals. Furthermore, CB2 agonists stimulate progenitor cell proliferation in vivo, with this effect being more pronounced in older animals. A similar response was seen with a fatty acid amide hydrolase (FAAH) inhibitor that limits degradation of endocannabinoids. The effects on proliferation were mirrored in changes in the number of neuroblasts migrating from the SVZ to the olfactory bulb (OB). In this context, CB2 antagonists reduced the number of newborn neurons appearing in the OB in the young adult animals while CB2 agonists stimulated this in older animals. These data identify CB2 receptor agonists and FAAH inhibitors as agents that can counteract the naturally observed decline in adult neurogenesis that is associated with ageing.

Effects of caspase inhibitors injected intracerebroventricularly or applicated to cerebellar vermis of adult rats on different types of learning and memory were studied. Pancaspase inhibitor z-VAD-fmk introduced into lateral cerebral ventriculi enhanced elaboration of long-term spatial memory in Morris water maze and stimulated habit alteration at the early stage of its formation. Caspase-3 inhibitor z-DEVD-CHO applied to cerebellar vermis enhanced the elaboration of acoustic startle habituation but had no effect on its storage and retrieval. These results indicate caspase participation in mechanisms of learning and memory both through influence on coupled processes of neurogenesis-apoptosis and through modulation of synaptic efficiency.

During anuran metamorphosis, the tadpole brain is transformed producing the sensorial and motor systems required for the frog's predatory lifestyle. Nervous system remodeling simultaneously implicates apoptosis, cell division, and differentiation. The molecular mechanisms underlying this remodeling have yet to be characterized. Starting from the observation that active caspase-9 and the Bcl-X(L) homologue, XR11 are highly expressed in tadpole brain during metamorphosis, we determined their implication in regulating the balance of apoptosis and proliferation in the developing tadpole brain. In situ hybridization showed caspase-9 mRNA to be expressed mainly in the ventricular area, a site of neuroblast proliferation. To test the functional role of caspase-9 in equilibrating neuroblast production and elimination, we overexpressed a dominant-negative caspase-9 protein, DN9, in the tadpole brain using somatic gene transfer and germinal transgenesis. In both cases, abrogating caspase-9 activity significantly decreased brain apoptosis and increased numbers of actively proliferating cells in the ventricular zone. Moreover, overexpression of XR11 with or without DN9 was also effective in decreasing apoptosis and increasing cell division in the tadpole brain. We conclude that XR11 and caspase-9, two key members of the mitochondrial death pathway, are implicated in controlling the proliferative status of neuroblasts in the metamorphosing Xenopus brain. Modification of their expression during the critical period of metamorphosis alters the outcome of metamorphic neurogenesis, resulting in a modified brain phenotype in juvenile Xenopus.

After brain injury, neuroblast cells from the subventricular zone (SVZ) expand and migrate toward damaged tissue. The mechanisms that mediate these neurogenic and migratory responses remain to be fully dissected. Here, we show that bromodeoxyuridine-labeled and doublecortin-positive cells from the SVZ colocalize with the extracellular protease matrix metalloproteinase-9 (MMP-9) during the 2 week recovery period after transient focal cerebral ischemia in mice. Treatment with the broad spectrum MMP inhibitor GM6001 significantly decreases the migration of doublecortin-positive cells that extend from the SVZ into the striatum. These data suggest that MMPs are involved in endogenous mechanisms of neurogenic migration as the brain seeks to heal itself after injury.

Fatty acid amides and fatty acid ethanolamides are novel signalling molecules exemplified by the sleep-inducing lipid oleamide and the endocannabinoid anandamide, respectively. These substances are inactivated by fatty acid amide hydrolase (FAAH), an enzyme that is expressed by neurons and non-neuronal cells in the brain. In the rat, FAAH-immunoreactivity has been detected in epithelial cells of the choroid plexus and, in accordance with this finding, here we report FAAH mRNA expression in rat choroid plexus epithelium using in situ hybridisation methods. Surprisingly, a comparative analysis of mouse brain did not reveal FAAH mRNA expression or FAAH-immunoreactivity in the choroid plexus of this species. FAAH-immunoreactivity was, however, detected in non-choroidal ventricular ependymal cells in the mouse brain and the specificity of this immunostaining was confirmed by analysis of FAAH-knockout mice. FAAH-immunoreactivity was detected in ependymal cells throughout the ventricles of the mouse brain but with regional variation in the intensity of immunostaining. Intriguingly, in rat brain, although FAAH expression is observed in choroid plexus epithelial cells, little or no FAAH-immunoreactivity is present in the ventricular ependyma. Thus, there are mutually exclusive patterns of FAAH expression in the ventricular epithelium of rat and mouse brain. Our observations provide the basis for an experimental analysis that exploits differences in FAAH expression in rat and mouse to investigate FAAH function in ventricular epithelial cells and, in particular, the role of FAAH in regulating the sleep-inducing agent oleamide in cerebrospinal fluid.

Disabled-1 (Dab1) forms part of the Reelin-Dab1 signalling pathway that controls neuronal positioning during brain development; Dab1 deficiency gives rise to a reeler-like inversion of cortical layers. To establish a timetable of Dab1 expression in developing human brain, Dab1 mRNA and protein expression were studied in prenatal human cortex. The earliest Dab1 signal was detected at 7 gestational weeks (GW), the stage of transition from preplate to cortical plate, suggesting a role of the Reelin-Dab1 signalling pathway in preplate partition. From 12 to 20 GW, the period of maximum cortical migration, Dab1 expression was prominent in the upper tiers of the cortical plate, to decline after midgestation. Radially orientated apical dendrites of Dab1-expressing neurons indicated a predominant pyramidal phenotype. Pyramidal cells in hippocampus and entorhinal cortex displayed a more protracted time of Dab1 expression compared to neocortex. In addition, at later stages (18-25 GW), Dab1 was also expressed in large neurons scattered throughout intermediate zone and subplate. From 14 to 22 GW, particularly high levels of Dab1 mRNA and protein were observed in cells of the ventricular/subventricular zone displaying the morphology of radial glia. The partial colocalization of vimentin and Dab1 in cells of the ventricular zone supported a radial glia phenotype. The concentration of Dab1 protein in ventricular endfeet and initial portions of radial processes of ventricular-zone cells points to a possible involvement of Dab1 in neurogenesis. Furthermore, a subset of Cajal-Retzius cells in the marginal zone colocalized Dab1 and Reelin, and may thus represent a novel target of the Reelin-Dab1 signalling pathway.

Objectives: This study establishes reference data for human lumbar CSF butyrylcholinesterase (E.C.3.1.1.8.) activity and investigates the enzyme activity in ventricular CSF. We comment on the relationship between CSF butyrylcholinesterase activity and other laboratory parameters., Subjects and Methods: We investigated 64 lumbar CSF samples obtained from a clinically healthy population and 169 ventricular CSF samples collected from 90 neurosurgical patients., Results: The reference range we recommend for lumbar CSF butyrylcholinesterase activity is 5.4 to 17.0 nmol/min x ml. The majority of ventricular butyrylcholinesterase activities in our patient subset ranged up to 5 nmol/min x ml., Conclusions: We established the relative influence of serum and CNS components on total CSF butyrylcholinesterase activity. The CNS fraction predominates the total butyrylcholinesterase activity in normal lumbar CSF. In ventricular CSF enzyme influx from serum outweighs the CNS component.

Background: Soluble acetylcholinesterase (AChE, E.C. 3.1.1.7.) is released by neurons, glial and meningeal cells into the CSF. AChE activity in cerebrospinal fluid (CSF) is altered in various disorders of the nervous system. The objects of this study are to define a reference range for CSF AChE activity in human lumbar CSF, to prove that the enzyme activity does not depend on the blood/CSF barrier function, and to provide information about AChE in ventricular CSF. In addition, drugs used in neurosurgical care have been examined for their in vitro effects on CSF AChE activity to exclude interference with the test system., Methods: We tested the AChE activity in 64 lumbar CSF samples collected from a clinically healthy population and in 169 ventricular CSF samples obtained from 90 neurosurgical patients. AChE activity was assayed with our inhibitor-free test procedure., Results: The reference range determined for lumbar CSF AChE activity is 9.2-24.4 nmol/min per ml. Lumbar CSF AChE activity does not correlate with parameters characterising the status of the blood/CSF barrier. Ventricular puncture is only justified for underlying pathology making it impossible to provide reference data for ventricular CSF. Most measurements reveal ventricular enzyme activity below 4 nmol/min per ml., Conclusion: The results of this study suggest the utility of lumbar CSF AChE activity as a measure of specific secretory function in enzyme releasing cells of the nervous system.

The present study showed the expression of induced nitric oxide synthase (iNOS) immunoreactivity in amoeboid microglia following an exposure to transient hypoxia in postnatal rats. iNOS immunoreactivity was expressed mainly in the amoeboid microglia in corpus callosum and subependymal regions of the ventricles within 3 h after hypoxia. The expression declined after 5 h, and became undetectable after 15 h and in longer surviving rats. The immunoreactivity of these cells with OX-42, which is a marker for microglia cells and detects complement type three receptors (CR3), was comparable in the rats exposed to hypoxia and the control rats. Immunoglobulin G (IgG) immunoreactivity was observed in the amoeboid microglia up to 3 h after hypoxia but it was undetectable in longer surviving rats and in the control rats. The iNOS expression in the amoeboid mircoglial cells may be related to the host defense and maintenance of structural integrity of the highly vulnerable periventricular white matter after hypoxia. The immunostaining of amoeboid microglial cells with IgG following hypoxia indicates leakage of plasma immunoglobulin from the blood vessels and its removal by the amoeboid microglial cells.

In turtles, crocodilians, lizards and snakes, the dorsal ventricular ridge (DVR) is a nuclear cell mass that contains distinct visual and auditory thalamorecipient cell groups. In the tuatara (Sphenodon punctatus), the DVR is not organized into diverse cell groups but instead possesses a trilaminar cytoarchitecture resembling that characteristic of the telencephalic cortex in reptiles. To determine if visual and auditory fields might also be present in the DVR of Sphenodon punctatus, we used succinic dehydrogenase (SDH) histochemistry, which has been shown to delineate the visual and auditory fields of the DVR in turtles, crocodilians and lizards. We also used acetylcholinesterase (AChE) histochemistry to determine the boundary between the DVR and the basal ganglia in Sphenodon. We found an SDH-rich region in the neuropil ventral to the cell plate of the rostrolateral DVR and a slightly less intense SDH-rich zone in the neuropil deep to the cell plate of the ventromedial DVR. These SDH-rich zones appear to be located at the apical dendrites of the neurons of the adjacent cell plate. These SDH-rich zones were clearly located within the DVR and were distinct from the AChE-rich striatal part of the basal ganglia, which occupied the ventrolateral wall of the telencephalon. Based on findings in other reptiles, it seems likely that the SDH-rich zone in rostrolateral DVR represents the zone of termination of nucleus rotundus visual input to the DVR, whereas the zone in ventromedial DVR represents the zone of termination of nucleus reuniens auditory input. Because a trilaminar DVR such as that in Sphenodon might be the primitive DVR condition for reptiles, our results suggest that the cytoarchitecture of the DVR and the synaptic organization of its thalamic sensory input in the common ancestor of living reptiles might have been much like of the dorsal cortex., (Copyright (R) 2000 S. Karger AG, Basel)

Thyroid hormone is an important epigenetic factor in brain development, acting by modulating rates of gene expression. The active form of thyroid hormone, 3,5,3'-triiodothyronine (T3) is produced in part by the thyroid gland but also after 5'-deiodination of thyroxine (T4) in target tissues. In brain, approximately 80% of T3 is formed locally from T4 through the activity of the 5'-deiodinase type 2 (D2), an enzyme that is expressed mostly by glial cells, tanycytes in the third ventricle, and astrocytes throughout the brain. D2 activity is an important point of control of thyroid hormone action because it increases in situations of low T4, thus preserving brain T3 concentrations. In this work, we have studied the expression of D2 by quantitative in situ hybridization in hypothyroid animals during postnatal development. Our hypothesis was that those regions that are most dependent on thyroid hormone should present selective increases of D2 as a protection against hypothyroidism. D2 mRNA concentration was increased severalfold over normal levels in relay nuclei and cortical targets of the primary somatosensory and auditory pathways. The results suggest that these pathways are specifically protected against thyroid failure and that T3 has a role in the development of these structures. At the cellular level, expression was observed mainly in glial cells, although some interneurons of the cerebral cortex were also labeled. Therefore, the T3 target cells, mostly neurons, are dependent on local astrocytes for T3 supply.

Objective: To investigate the levels of glutathione transferase (GST), a protective enzyme against aldehydes, and especially 4-hydroxynonenal (HNE) in the brain and ventricular CSF of autopsied AD and normal control subjects., Background: Studies have implicated increased levels of oxidative stress in the brain in the pathogenesis of AD. Decreased levels of polyunsaturated fatty acids and increased levels of markers of lipid peroxidation have been reported in the brain in AD, particularly in areas severely affected in the disease. HNE, one marker of lipid peroxidation, is neurotoxic in neuronal culture and in vivo and is elevated in AD brain and CSF., Methods: We measured levels of GST activity and protein in multiple brain regions and ventricular CSF in short-postmortem-interval AD patients and age-matched prospectively evaluated control subjects., Results: A decrease in GST activity in all brain areas was observed in AD compared with controls with significant decreases in the amygdala, hippocampus and parahippocampal gyrus, inferior parietal lobule, and nucleus basalis of Meynert. Levels of GST protein also were depleted in most brain regions in AD. A significant decrease in GST activity and protein levels was also found in ventricular CSF in AD., Conclusion: Reduced levels of GST, a protective mechanism against HNE, may have a role in the pathogenesis of neuron degeneration in AD.

Type 2 transglutaminase (Tg), which catalyzes the covalent cross-linking of cytoplasmic proteins during apoptosis, also functions as the alpha subunit of a heterodimeric G-protein (Gh) which can activate phospholipase C-delta1 during the signal transduction pathway linked to alpha1-adrenoreceptors. Continued stimulation of rat forebrain ventricular zone (VZ) germinal cells with the alpha1-agonist phenylephrine during development in vitro suppresses apoptosis and promotes DNA synthesis [Pabbathi et al., Brain Res., 760, 1997, 22-33]. Immunocytochemistry with a monoclonal antibody to Galphah/Tg reveals that alpha1-agonist deprivation during culture of VZ cells in the presence of a protein synthesis inhibitor results after 20 h in a loss of peripheral distribution of the protein and an increase in the reaction product of Tg in the cytoplasm of cells undergoing apoptosis. Using photoaffinity labelling, we observed reduced GTP binding to Galphah/Tg in phenylephrine-deprived cultures. Formation of inositol triphosphate (IP3) and intracellular Ca2+ transients occurred in the presence of phenylephrine. In cultures grown in phenylephrine-deprived conditions in the presence of protein synthesis inhibitor, both the IP3 response and the amplitude and duration of Ca2+ transients were reduced. These results show that loss of signal transduction coincides with the onset of transglutaminase activity in VZ cells during a period when cell survival is reduced following withdrawal of alpha1-agonist, and support the hypothesis that Tg/Galphah could be implicated in both signal transduction and programmed cell death., (Copyright 1998 Elsevier Science B.V.)

The distribution of immunoreactivity to neuronal nitric oxide synthase (nNOS) and vasopressin (AVP) was studied in the circumventricular organs of the female rat. The occurrence of NOS immunoreactivity showed correspondence to nicotinamide dinucleotide phosphate diaphorase reactivity, a previously used but less specific marker for neuronal NOS. nNOS immunolabeling was detected in the two most rostrally located circumventricular organs - the organum vasculosum of the lamina terminalis and the subfornical organ. In the latter, AVP immunoreactivity was observed in some cell bodies, which also were nNOS-immunoreactive. In the median eminence and the neurohypophysis there were large amounts of nNOS- and AVP-immunoreactive nerve fibers, which often displayed similarities in distribution and morphology. Within the pineal gland, only very few nNOS-immunoreactive varicose terminals were observed, which ran along blood vessels. nNOS immunoreactivity was also seen in the epithelium of the choroid plexus, whereas no nNOS immunoreactivity could be found in the subcommissural organ or in the area postrema. The present demonstration of nNOS and AVP immunoreactivity in the subfornical organ, median eminence, and neurohypophysis, and the occurrence of nNOS immunoreactivity also in the choroid plexus and organum vasculosum of the lamina terminalis, provides a morphological background for a functional role for nitric oxide in water homeostatic mechanisms, both as executed through the hypothalamohypophyseal system and via the production of cerebrospinal fluid.

The distribution of neuronal nitric oxide synthase (nNOS) has been studied in the more rostral portion of the lateral ventricle, subfornical organ, area postrema and blood vessels of the rat central nervous system. nNOS was located by means of a specific polyclonal antibody, by using light and electron microscopy. Light microscopy showed immunoreactive varicose nerve fibers and terminal boutons-like structures in the lateral ventricle, positioned in supra- and subependimal areas. The spatial relationships between immunoreactive neuronal processes and the wall of the intracerebral blood vessels were studied. Electron microscopy showed numerous nerve fibers in the wall of the lateral ventricle; many were nNos-immunoreactive and established very close contact with ependymal cells. Immunoreactive neurons and processes were found in the subependymal plate of the ventricular wall, the subfornical organ, the area postrema, and the circularis nucleus of the hypothalamus. In these last three areas, the immunoreactive neurons were found close to the perivascular space of fenestrated and nonfenestrated blood vessels. The nNOS immunoreactivity was localized to the endoplasmic reticulum, cisterns, ribosomes, neurotubules, and in the inner part of the external membrane. In the terminal boutons, the reaction product was found surrounding the vesicle membranes. This distribution showed nNOS as a predominantly membrane-bound protein. The nitrergic nerve fibers present in the wall of the ventricular system might regulate metabolic functions as well as neurotransmission in the subfornical organ, area postrema and circularis nucleus of the hypothalamus.

Immunocytochemical staining for the presence of catecholamine synthesizing enzymes, tyrosine hydroxylase and dopamine beta-hydroxylase, was used to characterize the regional distribution of catecholaminergic neurons in the hypothalamus and adjacent areas of domestic cattle, Bos taurus. In steers, heifers and cows, tyrosine hydroxylase-immunoreactive perikarya was located throughout periventricular regions of the third cerebral ventricle, in both anterior and retrochiasmatic divisions of the supraoptic nucleus, suprachiasmatic nucleus, and ventral and dorsolateral regions of the paraventricular nucleus, dorsal hypothalamus, ventrolateral aspects of the arcuate nucleus, along the ventral hypothalamic surface between the median eminence and optic tract, and in the posterior hypothalamus. Immunostained perikarya ranged from small (10-20 microns, parvicellular) to large (30-50 microns, magnocellular) and were of multiple shapes: round, triangular, fusiform or multipolar, often with 2-5 processes of branched arborization. There were no dopamine-beta-hydroxylase immunoreactive perikarya observed within the hypothalamus and adjacent structures. However, both tyrosine hydroxylase and dopamine-beta-hydroxylase immunoreactive fibers and punctate varicosities were observed throughout regions of tyrosine hydroxylase immunoreactivity perikarya. Generally, the location and pattern of hypothalamic tyrosine hydroxylase immunoreactivity and dopamine-beta-hydroxylase immunoreactive were similar to those reported for most other large brain mammalian species, however, there were several differences with commonly used small laboratory animals. These included intense tyrosine hydroxylase immunoreactivity of perikarya within the retrochiasmatic division of the supraoptic nucleus (ventral A15 region), the absence of tyrosine hydroxylase immunoreactive perikarya below the anterior commissure or within the bed nucleus of stria terminalis (absence of the dorsal A15 region), an abundance of tyrosine hydroxylase immunoreactive perikarya within the ependymal layer of the median eminence, heavy innervation of the arcuate nucleus with dopamine-beta-hydroxylase immunoreactive fibers and varicosities, and the paucity of dopamine-beta-hydroxylase immunoreactive throughout the median eminence.

Hypothalamic dopaminergic periventricular and arcuate nuclei are known to project to the pituitary gland and contain serotonin in their terminals. In order to elucidate the potential of these neurons to synthesize serotonin, we studied immunohistochemically the possible tryptophan hydroxylase content of periventriculo-hypophyseal neurons, identified by retrograde tracing from the pituitary gland. These neurons were found to contain tryptophan hydroxylase-immunoreactivity (TpOH-IR), which was enhanced after colchicine treatment. All of the TpOH-IR neurons contained tyrosine hydroxylase-immunoreactivity as well. However, none of them were immunoreactive for serotonin in either intact animals or in animals pretreated with serotonin precursor L-tryptophan and MAO inhibitor pargyline. Thus, neurons of the dopaminergic periventriculo-hypophyseal pathway express tryptophan hydroxylase, but are unable to synthesize serotonin. These findings (i) raise the possibility that, in these nerves, serotonin might serve a function other than regular synaptic transmission, and (ii) suggest that expression of an enzyme synthesizing certain transmitter does not necessarily confirm the corresponding transmitter phenotype of that neuron.

A primary culture system of cells derived from two circumventricular organs (CVO) of the rat brain was established. The subfornical organ (SFO) and the organum vasculosum of the lamina terminalis (OVLT) were dissected from the rostral wall of the third ventricle and its cells taken into culture after mechanical dissociation. The cells were cultured in a modified microculture chamber system ensuring relatively high cell density despite their low absolute number. When animals were injected with Evans blue prior to cell preparation, the macroscopically visible penetration of the dye into the parenchyma of the CVOs could be used as guidance during tissue isolation and labelled cells could be identified in culture. Cultured CVO neurones and astrocytes were identified using antibodies against cell type specific marker proteins. The histochemical NADPH-diaphorase staining was used for the detection of nitric oxide synthase in tissue sections of both CVOs and in their cultured neurones. In addition, angiotensin II (ANG II)-evoked elevations of the intracellular Ca2+ concentration ([Ca2+]i) in single cultured OVLT neurones were measured. The described methods will be useful for further characterization of CVO neurones and astrocytes.

Brain aromatase has been considered to be an important clue in elucidating the actions of androgen on brain sexual differentiation. Using highly specific anti-P450arom antiserum, the regional and subcellular distributions were immunohistochemically evaluated in the preoptic, strial, and amygdaloid regions of developing rat brains. Aromatase-immunoreactive (AROM-I) neurons were classified into three groups. The first, in which immunostaining occurs only during certain pre- or neonatal days (E16-P2), included the anterior medial preoptic nucleus, the periventricular preoptic nucleus, neurons associated with the strial part of the preoptic area, and the rostral portion of the medial preoptic nucleus. The second is a striking AROM-I cell group in the "medial preopticoamygdaloid neuronal arc," which extends from the medial preoptic nucleus to the principal nucleus of the bed nucleus of the stria terminalis and the posterodorsal part of the medial amygdaloid nucleus. The AROM-I neurons appeared by E16, reaching a peak in staining intensity between E18 and P2 and diminishing after the perinatal stage. After P14, a third group of AROM-I neurons emerged in the lateral septal nucleus, the oval nucleus of the bed nucleus of the stria terminalis, and the central amygdaloid nucleus. The second group was thought to be the major aromatization center in developing rat brains, while the center might partly shift to the third group of neurons after the late infantile stage. The distribution and developmental patterns were basically similar in males and females, suggesting that the neonatally prominent aromatase is not induced by male-specific androgen surges occurring around birth. On immunoelectron microscopy, subneuronal aromatase was predominantly localized on the nuclear membrane and endoplasmic reticulum, which appeared to be appropriate for the efficient conversion of androgen into estrogen just prior to binding to the nuclear receptors.

Peripheral axotomy of the spinal nerve and avulsion of the ventral roots have been found to induce increase in expression of nitric oxide synthase (NOS) in the spinal motor neurones and the dorsal root ganglion. The present study investigated changes of NOS, using NADPH-diaphorase (NADPH-d) reactivity as the marker, in vagal complex after axotomy in the rat. Eight days after left cervical vagotomy the NADPH-d reactivity was found to be markedly enhanced in the dorsal motor nucleus of the vagus nerve, the ambiguus nucleus, the solitary tract and the nucleus of the tractus solitarius, and the nodose ganglion. This study offers the first evidence of changes in NOS expression in cranial visceral components following axotomy.

The effects of active oxygen species in the development of congenital hydrocephalus have been investigated. Superoxide dismutase (SOD) is one of the scavengers of active oxygen species and there have been many recent reports on the relationship between neurological disorders by active oxygen species following reperfusion for ischemic brain and SOD. In this study, the localization of Cu-SOD and Zn-SOD in WIC-Hyd congenitally hydrocephalic rat brains was identified by the enzyme unlabeled antibody method. We examined the localization of SOD in the choroid plexus, hippocampus, and ependymal cells of the lateral ventricle and aqueduct of WIC-Hyd rats. SOD was hardly observed in the choroid plexus and faintly localized in the hippocampus and ependymal cells of the congenitally hydrocephalic brain, but was observed equally in the cytoplasm of the choroid plexus, hippocampus, and ependymal cells in control animals. In the hippocampus, less SOD was found in hydrocephalic rats than in controls. The SOD was slightly observed in the CA1 pyramidal cells in hydrocephalic rats. In the lateral ventricle and aqueductal ependyma, less SOD was found in hydrocephalic than in controls rats. The amount of Cu, Zn-SOD in the congenitally hydrocephalic rat brain was less than in the control, especially in the choroid plexus. Therefore, we suspect that the production of SOD is congenitally reduced in the congenitally hydrocephalic rat brain, and this may promote the impairment of the function of choroid plexus and cilia due to increased active oxygen species. The reduction of SOD in the choroid plexus, hippocampus and ependymal cells of ventricles or aqueduct may promote the development of hydrocephalus in the congenitally hydrocephalic rat.

Nitric oxide, a free radical, has recently been shown to exert major influences on CNS functions in mammals. It is synthesized by NO-synthase. For the first time, this study reveals this enzyme's existence in the CNS of a teleost fish and describes its distribution in the diencephalon, where the paraventricular organ displays an extraordinarily high activity. The study contributes to an evolutionary perspective of the biological role played by nitric oxide in the vertebrate CNS, and raises questions regarding the significance of this gas in cerebrospinal fluid-contacting neurons.

To evaluate the potential for adenovirus-mediated central nervous system (CNS) gene transfer, the replication deficient recombinant adenovirus vectors Ad.RSV beta gal (coding for beta-galactosidase) and Ad-alpha 1AT (coding for human alpha 1-antitrypsin) were administered to the lateral ventricle of rats. Ad.RSV beta gal transferred beta-galactosidase to ependymal cells lining the ventricles whereas Ad-alpha 1AT mediated alpha 1-antitrypsin secretion into the cerebral spinal fluid for 1 week. These observations, together with beta-galactosidase activity in the globus pallidus and substantia nigra following stereotactic administration of Ad.RSV beta gal to the globus pallidus, suggest that adenovirus vectors will be useful for CNS gene therapy.

Changes of glutaminase immunoreactivity in rat brain were examined after intracranial injection of 6-diazo-5-oxo-L-norleucine (DON), an irreversible inhibitor of glutaminase. When 1 M DON was injected into the lateral ventricle, a half-lethal dose was 7.5-10 mumol. After intraventricular injection of 2-7.5 mumol DON, glutaminase immunoreactivity was dose dependently enhanced with the maximum enhancement 3-5 days after the injection. The enhanced glutaminase immunoreactivity was recognized by enlarged granular immunodeposits in both perikarya and neuropil in many regions, such as the hippocampus, thalamus, hypothalamus, periaqueductal gray, and some brain stem, cerebellar, and spinal cord regions. Intrathalamic injection of 0.2 mumol DON enhanced glutaminase immunoreactivity in many neuronal perikarya in the thalamus and in some perikarya in layer VI of the cerebral cortex. Intrastriatal injection of the same dose of DON enhanced glutaminase immunoreactivity in neuropil of the caudoputamen and in many neuronal perikarya of the intralaminar thalamic nuclei. These results suggested that DON induced a new massive synthesis of glutaminase in the affected neurons.

The acetylcholinesterase activity (AChE) in the human subfornical organ (SFO) was detected by the method of Koelle and Friedenwald in 16 human brains collected between 6 and 12 hrs postmortem. The only AchE-positive structures found were neuronal cell bodies and processes, morphologically classified as stellate and fusiform neurons of large, medium and small size. Large ones prevailed in the dorsal zone. The neurons were homogeneously distributed in the rostral area of the SFO. The penetration and ramification of large blood vessels produced a decreasing neuronal density in the medium-caudal area. The architecture of the SFO in sagittal sections comprised a central zone with neurons juxtaposed to the walls of the vascular plexus, whose dendritic and axonal processes showed an intricate pattern without a special arrangement. This neuronal zone of the SFO was surrounded by a peripheral layer of neurons with axonal projections to the rostral area. This layer was thicker in the dorsal zone of the SFO, where axonal fibers "climbing-up" from the central perivascular neurons could be demonstrated. In coronal sections some neurons with prolongations of arcuate distribution connected the dorsal and ventral zones.

The glutathione S-transferases are a complex group of multifunctional enzymes which may detoxify a wide range of toxic substances including drugs and carcinogens. Different isoenzymes vary in substrate specificity, tissue distribution and level of expression during development. Following reports of cell-specific and age-dependent expression in rat brain we have studied, immunohistochemically, expression of the Pi and Alpha class isoenzymes in 10 adult and 21 human fetal brains. Whilst Alpha isoenzyme is expressed only in adult brain, and then only focally, Pi isoenzyme is strongly expressed from as early as 12 weeks gestation. In the adult, expression is localized to choroid plexus, vascular endothelium, ventricular lining cells, pia-arachnoid and astrocytes. In fetal brain, expression is also strong in cells with the morphology of tanycytes and in the cell bodies of radial glia. Neurons are consistently negative. Pi isoenzyme thus localizes to the sites of the blood-CSF barrier, blood-brain barrier, CSF-brain barrier and pia-arachnoid-brain barrier. It is ideally placed to regulate neuronal exposure to potentially toxic substances derived from blood or cerebrospinal fluid. Expression so early in gestation is of significance and may imply a role in protection of the developing human brain.

The authors describe the localization of monoaminooxidases oxidizing the protoxin MPTP to the active neurotoxic MPP+ ion, which induces parkinsonism by destroying the dopaminergic neurons of the nigrostriatum. Apart from some types of magnocellular neurons of the hypothalamus, enzymatic activity is localized in the endothelium of segments of the circumventricular vascular bed communicating with the enzymatically equally positive processes of the tanycytes of the third brain ventricle. The findings are briefly discussed from the aspect of the pathogenesis of experimental parkinsonism.

The presence of monoamine oxidase (MAO) in the cerebrospinal fluid (CSF) and MAO positive tracts bridging the CSF and the subependyma strongly suggest the involvement of CSF in the neuroendocrine control of hypophysial function.

The catecholaminergic and peptidergic neurons in the area postrema and adjacent portion of the medial nucleus tractus solitarii (mNTS) were characterized by the immunocytochemical localization of the catecholamine synthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) and two neuropeptides, substance P and (Leu5)-enkephalin. The catecholamine synthesizing enzymes TH and DBH, found jointly only in noradrenergic and adrenergic neurons, were localized in cells having a similar morphology and topographical distribution. These cells were located throughout the rostrocaudal and dorsoventral extent of the area postrema, as well as in neurons within the mNTS. The processes showing TH and DBH immunoreactivity appear to form reciprocal connections between the area postrema and mNTS. Phenylethanolamine-N-methyltransferase, the enzymatic marker found only in adrenergic neurons, was detected immunocytochemically in terminals distributed throughout the area postrema and in neuronal perikarya and varicosities within the adjacent mNTS. Like the catecholamine synthesizing enzymes TH and DBH, enkephalin-like immunoreactivity was localized to perikarya, proximal processes and varicose axon terminals within the area postrema and the adjacent mNTS. However, in contrast to the widespread distribution of the enzymes, the enkephalin-like immunoreactivity was localized predominantly along the dorsal and ventrolateral margins of the area postrema. The distribution of substance P immunoreactivity, which was detected only in varicose processes, paralleled the distribution of enkephalin-like immunoreactivity, being predominantly located along the dorsal and ventrolateral margins of the area postrema. Within the mNTS adjacent to the area postrema, substance P immunoreactivity was localized to neuronal perikarya, proximal processes and varicose axon terminals. Based upon the presence of appropriate biosynthetic enzyme markers and neuropeptide localization, these findings suggest that neurons within the area postrema contain noradrenalin and enkephalin and that the afferent axons contain substance P, adrenalin and, probably, noradrenalin.

We performed an ultracytochemical study of Mg++-ATPase as a marker of maturation in circumventricular capillaries at the developmental stage of rats, from 19 gestational days to 18 postnatal day, comparing with hippocampal capillaries. During the course of perinatal development, the predominant site of the Mg++-ATPase activity in circumventricular capillaries was shifted from the luminal cell membrane to the antiluminal cell membrane and the total enzyme activity was markedly increased. In all hippocampal capillaries observed, the predominant site of the Mg++-ATPase activity was the antiluminal cell membrane. It was suggested that immaturity of circumventricular capillaries may be one of the causative factors leading to intraventricular and subependymal hemorrhages in neonates.

Catechol-O-methyltransferase (COMT) was localized in the organum vasculosum of the lamina terminals, subfornical organ, subcommissural organ and area postrema of rat brain using an indirect immunofluorescence technique. COMT immunofluorescence was apparent in neuroglia within the organum vasculosum and was most intense in the ependyma between this structure and the optic recess of the third ventricle. In both the subfornical organ and the area postrema, COMT was localized in a neuroglial network, but was noticeably absent in the ependymal layer. COMT immunofluorescence in the ependyma of the subcommissural organ was continuous with the more intense immunofluorescence of the cuboidal ependyma of the third ventricle. Each of the circumventricular organs studied, with the exception of the subcommissural organ, lies outside the blood-brain barrier. However, the unique pattern of COMT immunofluorescence in the area postrema and the subfornical organ suggests that these two structures, of all circumventricular organs, are most likely to permit the entry of peripherally circulating catechols to the cerebrospinal fluid.