Most current methods of gene delivery for primary cultured hippocampal neurons are limited by toxicity, transient expression, the use of immature neurons, and/or low efficiency. We performed a direct comparison of seven serotypes of adeno-associated virus (AAV) vectors for genetic manipulation of primary cultured neurons in vitro. Serotypes 1, 2, 7, 8 and 9 mediated highly efficient, nontoxic, stable long-term gene expression in cultured cortical and hippocampal neurons aged 0-4 weeks in vitro; serotypes 5 and 6 were associated with toxicity at high doses. AAV1 transduced over 90% of all cells with approximately 80% of the transduced cells being neurons. The method was readily adapted to a high-throughput format to demonstrate neurotrophin-mediated neuroprotection from glutamate toxicity in cultured neurons at 2 weeks in vitro. These vectors should prove highly useful for efficient overexpression or downregulation of genes in primary neuronal cultures at any developmental stage.

Cellular phenotype is the conglomerate of multiple cellular processes involving gene and protein expression that result in the elaboration of a cell's particular morphology and function. It has been thought that differentiated postmitotic cells have their genomes hard wired, with little ability for phenotypic plasticity. Here we show that transfer of the transcriptome from differentiated rat astrocytes into a nondividing differentiated rat neuron resulted in the conversion of the neuron into a functional astrocyte-like cell in a time-dependent manner. This single-cell study permits high resolution of molecular and functional components that underlie phenotype identity. The RNA population from astrocytes contains RNAs in the appropriate relative abundances that give rise to regulatory RNAs and translated proteins that enable astrocyte identity. When transferred into the postmitotic neuron, the astrocyte RNA population converts 44% of the neuronal host cells into the destination astrocyte-like phenotype. In support of this observation, quantitative measures of cellular morphology, single-cell PCR, single-cell microarray, and single-cell functional analyses have been performed. The host-cell phenotypic changes develop over many weeks and are persistent. We call this process of RNA-induced phenotype changes, transcriptome-induced phenotype remodeling.neuron ͉ transcriptome-induced phenotype remodeling ͉ single cell ͉ Waddington I n multicellular organisms, all cells contain nearly identical copies of the genome but exhibit drastically different phenotypes. Even a single neuron has a set of phenotypic characteristics that distinguish it from other neurons as well as other cell types, such as the nearby astrocytes. Indeed, as Waddington proposed in his classical epigenetic landscape model, genetically predetermined cells can follow any specific permitted trajectories that eventually lead to different cellular phenotypes (1). From this point of view, the genome serves as a repository of dynamic control information whose state can be reprogrammed to match the stable phenotypic states.Emerging evidence has demonstrated the reversibility and flexibility of the cellular phenotype. Gurdon et al. first showed that the ability to obtain fertile adult male and female frogs by injecting endoderm nuclei into enucleated eggs (2). This result not only forms the foundation of the field in nuclear transplantation but also provides evidence that the cytoplasmic components of a differentiated cell can support nuclear reprograming. Generation of induced pluripotent stem (iPS) cells by transfection of transcription factors into dividing fibroblasts (3), followed by cell selection, represents a new strategy to globally revert a mature cell into a different cell type (4-9). The need for redifferentiation of these embryonic stem cell-like-iPS cells into desired cell types adds a layer of complexity that is difficult to control (10, 11). Nevertheless, studies of nuclear reprogramming from genomic and epigenetic modification, as seen from so...

Summary:Purpose: Previous studies in neonatal (postnatal day 10) and adult rats suggest that status epilepticus (SE) induces changes in the α1 subunit of the GABA A receptor (GABRA1) in dentate granule neurons (DGNs) that are age dependent and vary inversely with the likelihood of epilepsy development. In the present study, we examined GABRA1 expression after SE at postnatal day 20 (P20), an intermediate age when only a subset of SE-exposed animals develop epilepsy.Methods: SE was induced with lithium-pilocarpine or kainate at P20. Animals were video-EEG monitored after SE to determine the presence or absence of spontaneous seizures. GABRA1 mRNA and protein levels were determined 7 days or 3 months later in SE-exposed and control animals by using a combination of aRNA amplification, Western blotting, and immunohistochemistry techniques.Results: GABRA1 mRNA levels in DGNs of SE-exposed rats that did not become epileptic were higher than those in control rats, but were not different from DGNs in epileptic SE-exposed rats. GABRA1 protein levels in dentate gyrus were significantly increased in both epileptic and nonepileptic SE-exposed rats compared with controls. GABRA1 mRNA changes were region specific and did not occur in CA1 or CA3 areas of hippocampus. GABRA1 alterations were present by 1 week after P20 SE and were similar whether pilocarpine or kainate was used to induced SE.Conclusions: P20 SE results in persistent increases in GABRA1 levels selectively in dentate gyrus. These changes preceded the onset of epilepsy, were not model specific, and occurred in both epileptic and nonepileptic animals.

The calpain family of cysteine proteases has a well-established causal role in neuronal cell death following acute brain injury. However, the relative contribution of calpain isoforms to the various forms of injury has not been determined as available calpain inhibitors are not isoform-specific. In this study, we evaluated the relative role of m-calpain and l-calpain in a primary hippocampal neuron model of NMDA-mediated excitotoxicity. Baseline mRNA expression for the catalytic subunit of m-calpain (capn2 ) was found to be 50-fold higher than for the l-calpain catalytic subunit (capn1) based on quantitative real-time PCR. Adeno-associated viral vectors designed to deliver short hairpin RNAs targeting capn1 or capn2 resulted in 60% and 90% knockdown of message respectively.Knockdown of capn2 but not capn1 increased neuronal survival after NMDA exposure at 21 days in vitro. Nuclear translocation of calpain substrates apoptosis inducing factor, p35/p25 and collapsin response mediator protein (CRMP) 2-4 was not detected after NMDA exposure in this model. However, nuclear translocation of CRMP-1 was observed and was prevented by capn2 knockdown. These findings provide insight into potential mechanisms of calpain-mediated neurodegeneration and have important implications for the development of isoform-specific calpain inhibitor therapy.

Summary:Purpose: To determine the fate of newborn dentate granule cells (DGCs) after lithium-pilocarpine-induced status epilepticus (SE) in an immature rat.Methods: Postnatal day 20 (P20) rats were injected with lithium and pilocarpine to induce SE, and then with bromodeoxyuridine (BrdU) 4, 6, and 8 days later (P24, 26, and 28), and killed 1 day (P29), 1 week (P34), and 3 weeks (P50) after the last dose of BrdU for cell counts. Immunohistochemistry and TUNEL staining were performed to assess the fate of newborn DGCs.Results: Pilocarpine-treated animals had significantly more BrdU-labeled DGCs than did littermate controls at all times. The day after the final BrdU injection (P29), sixfold more cells were found in pilocarpine-treated animals than in controls, which was reduced to threefold, 3 weeks later. A decrease in the BrdUlabeled cell density was noted from P29 to P50 in the control and pilocarpine-treated animals. Evidence of DGC cell death was seen in pilocarpine and control animals, with threefold more TUNEL-positive cells in the pilocarpine-treated than in the control animals at P29. The surviving newborn DGCs became mature neurons; expressing the neuronal marker NeuN in both control and pilocarpine-treated animals.Conclusions: These findings suggest that SE during postnatal development increases the birth and death of DGCs. A subset of the newborn DGCs survive and mature into dentate granule neurons, resulting in an increased population of immature DGCs after SE that may affect hippocampal physiology. Key Words: Neurogenesis-Cell death-Hippocampus-Status epilepticus.The generation of a large number of new neurons throughout life is a distinctive feature of the hippocampal dentate gyrus (DG) (1,2). Limited studies have suggested that immature dentate granule cells (DGCs) have unique physiologic properties, and the birth of new DGCs is necessary for the establishment of certain types of memory in the rodent (3-5). As the total numbers of DGCs do not increase throughout life, a concomitant loss of DGCs must occur. The gradual loss of DGCs over weeks to months has been demonstrated in several species; however, why certain cells die is not well understood (6,7).Several growth factors have been implicated in DG neurogenesis (8-10), and alterations in the environment, drugs, hormones, age, and seizures all influence the rate of neurogenesis (11)(12)(13)(14). Multiple types of seizures have been shown to modify the birth rate of DGCs, increasing the rate from one to as much as tenfold (14-17), although also see (18). Along with the increase in the birth of newborn DGCs, an increase of injured and dying DGCs (19,20). Many of the dying cells are found at the dentate/hilar border, the area of DGC birth, suggesting that immature DGCs may be particularly susceptible to dying, although the number and phenotype of the dying cells has not been extensively characterized. Because SE increases both the birth and death rate of DGCs, it remains unclear how many newborn DGCs survive long after SE and thus could contribute to...