Inhibition of Src family kinases improves cognitive function after intraventricular hemorrhage or intraventricular thrombin.

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.

Role of Class III phosphoinositide 3-kinase in the brain development: possible involvement in specific learning disorders.

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).

A novel bioactivity of andrographolide from Andrographis paniculata on cerebral ischemia/reperfusion-induced brain injury through induction of cerebral endothelial cell apoptosis.

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.

Suppression of cytochrome P450 reductase expression promotes astrocytosis in subventricular zone of adult mice.

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.

Targeted deletion of the ERK5 MAP kinase impairs neuronal differentiation, migration, and survival during adult neurogenesis in the olfactory bulb.

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.

[Application of immunocytochemical technique for the determination of the border between forebrain subventricular zone and striatum].

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.

An endogenous vitamin K-dependent mechanism regulates cell proliferation in the brain subventricular stem cell niche.

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.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neuroblastic apoptosis in the subventricular zone is caused by 1-methy-4-phenylpiridinium (MPP(+)) converted from MPTP through MAO-B.

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.

nNOS deficiency-induced cell proliferation depletes the neurogenic reserve.

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.

A novel preclinical rodent model of collagenase-induced germinal matrix/intraventricular hemorrhage.

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.

Alzheimer's disease modifies progenitor cell expression of monoamine oxidase B in the subventricular zone.

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.

Expression of leucine-rich-repeat-kinase 2 (LRRK2) during embryonic development.

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.

Repression of Fgf signaling by sprouty1-2 regulates cortical patterning in two distinct regions and times.

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.

Dynamic roles of FGF-2 and Anosmin-1 in the migration of neuronal precursors from the subventricular zone during pre- and postnatal development.

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.

Antagonism of the antinociceptive effect of nitrous oxide by inhibition of enzyme activity or expression of neuronal nitric oxide synthase in the mouse brain and spinal cord.

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.

Anti-allodynic effect of intracerebroventricularly administered antioxidant and free radical scavenger in a mouse model of orofacial pain.

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.

Studies of learning and memory processes in adult rats in conditions of intracerebral administration of caspase inhibitors.

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.

EphrinB3 is an anti-apoptotic ligand that inhibits the dependence receptor functions of EphA4 receptors during adult neurogenesis.

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.

[Study of learning and memory processes in adult rats under conditions of intracerebral caspases inhibitors administration].

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.

A diacylglycerol lipase-CB2 cannabinoid pathway regulates adult subventricular zone neurogenesis in an age-dependent manner.

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.