Neural progenitor cells persist throughout the adult forebrain subependyma, and neurons generated from them respond to brain-derived neurotrophic factor (BDNF) with enhanced maturation and survival. To induce neurogenesis from endogenous progenitors, we overexpressed BDNF in the adult ventricular zone by transducing the forebrain ependyma to constitutively express BDNF. We constructed a bicistronic adenovirus bearing BDNF under cytomegalovirus (CMV) control, and humanized green fluorescent protein (hGFP) under internal ribosomal entry site (IRES) control. This AdCMV:BDNF:IRES:hGFP (AdBDNF) was injected into the lateral ventricles of adult rats, who were treated for 18 d thereafter with the mitotic marker bromodeoxyuridine (BrdU). Three weeks after injection, BDNF averaged 1 microg/gm in the CSF of AdBDNF-injected animals but was undetectable in control CSF. In situ hybridization demonstrated BDNF and GFP mRNA expression restricted to the ventricular wall. In AdBDNF-injected rats, the olfactory bulb exhibited a >2.4-fold increase in the number of BrdU(+)-betaIII-tubulin(+) neurons, confirmed by confocal imaging, relative to AdNull (AdCMV:hGFP) controls. Importantly, AdBDNF-associated neuronal recruitment to the neostriatum was also noted, with the treatment-induced addition of BrdU(+)-NeuN(+)-betaIII-tubulin(+) neurons to the caudate putamen. Many of these cells also expressed glutamic acid decarboxylase, cabindin-D28, and DARPP-32 (dopamine and cAMP-regulated phosphoprotein of 32 kDa), markers of medium spiny neurons of the neostriatum. These newly generated neurons survived at least 5-8 weeks after viral induction. Thus, a single injection of adenoviral BDNF substantially augmented the recruitment of new neurons into both neurogenic and non-neurogenic sites in the adult rat brain. The intraventricular delivery of, and ependymal infection by, viral vectors encoding neurotrophic agents may be a feasible strategy for inducing neurogenesis from resident progenitor cells in the adult brain.

Neural progenitor cells are widespread throughout the adult central nervous system but only give rise to neurons in specific loci. Negative regulators of neurogenesis have therefore been postulated, but none have yet been identified as subserving a significant role in the adult brain. Here we report that nitric oxide (NO) acts as an important negative regulator of cell proliferation in the adult mammalian brain. We used two independent approaches to examine the function of NO in adult neurogenesis. In a pharmacological approach, we suppressed NO production in the rat brain by intraventricular infusion of an NO synthase inhibitor. In a genetic approach, we generated a null mutant neuronal NO synthase knockout mouse line by targeting the exon encoding active center of the enzyme. In both models, the number of new cells generated in neurogenic areas of the adult brain, the olfactory subependyma and the dentate gyrus, was strongly augmented, which indicates that division of neural stem cells in the adult brain is controlled by NO and suggests a strategy for enhancing neurogenesis in the adult central nervous system. T he vast majority of neurons in the mammalian brain are produced during embryonic development. However, remnants of the germinal zones of the developing brain continue to proliferate into adulthood, generating large numbers of neurons in the adult brain (1-3). The subventricular zone (SVZ) of the lateral ventricles (LVs), its anterior extension, the rostral migratory stream (RMS), and the subgranular cell layer (S-GCL) of the dentate gyrus (DG) of the hippocampus are the major sites of adult neurogenesis, although other regions of the adult brain retain the potential to generate new neurons (4-6). Many of the newly generated neurons undergo physiological cell death (7), but it is becoming clear that some of these new neurons become integrated into existing neuronal circuits, thus potentially contributing to a previously unanticipated form of neuroplasticity (8). Several protein growth factors have been shown to affect adult neurogenesis in vivo (5, 6, 9-11). However, the signaling systems involved in regulating cell division in the adult brain are only beginning to be understood.Increasingly diverse functions of NO, a transcellular signaling molecule (12), are continuing to be demonstrated, and there is growing evidence that NO may be involved in controlling proliferation of neuronal cells. Neuronal NO synthase (nNOS), the major NOS isoform in the mammalian brain, is transiently expressed in the developing brain in a pattern suggesting its involvement in neural development (13). Furthermore, NO has been shown to effectively and reversibly suppress cell division (14, 15); this property of NO, coupled to its ability to regulate gene expression, is exploited in a number of developmental contexts (16). Materials and MethodsA full description of the methods used in this work can be found in Supporting Materials and Methods, which is published as supporting information on the PNAS web site, www.pnas.org.All a...

Neurogenesis persists in the adult mammalian hippocampus. To identify and isolate neuronal progenitor cells of the adult human hippocampus, we transfected ventricular zone-free dissociates of surgically-excised dentate gyrus with DNA encoding humanized green fluorescent protein (hGFP), placed under the control of either the nestin enhancer (E/nestin) or the Talpha1 tubulin promoter (P/Talpha1), two regulatory regions that direct transcription in neural progenitor cells. The resultant P/Talpha1:hGFP+ and E/nestin:enhanced (E)GFP+ cells expressed betaIII-tubulin or microtubule-associated protein-2; many incorporated bromodeoxyuridine, indicating their genesis in vitro. Using fluorescence-activated cell sorting, the E/nestin:EGFP+ and P/Talpha1:hGFP+ cells were isolated to near purity, and matured antigenically and physiologically as neurons. Thus, the adult human hippocampus contains mitotically competent neuronal progenitors that can be selectively extracted. The isolation of these cells may provide a cellular substrate for re-populating the damaged or degenerated adult hippocampus.