BDNF signaling in the VTA links the drug-dependent state to drug withdrawal aversions.

Drug administration to avoid unpleasant drug withdrawal symptoms has been hypothesized to be a crucial factor that leads to compulsive drug-taking behavior. However, the neural relationship between the aversive motivational state produced by drug withdrawal and the development of the drug-dependent state still remains elusive. It has been observed that chronic exposure to drugs of abuse increases brain-derived neurotrophic factor (BDNF) levels in ventral tegmental area (VTA) neurons. In particular, BDNF expression is dramatically increased during drug withdrawal, which would suggest a direct connection between the aversive state of withdrawal and BDNF-induced neuronal plasticity. Using lentivirus-mediated gene transfer to locally knock down the expression of the BDNF receptor tropomyosin-receptor-kinase type B in rats and mice, we observed that chronic opiate administration activates BDNF-related neuronal plasticity in the VTA that is necessary for both the establishment of an opiate-dependent state and aversive withdrawal motivation. Our findings highlight the importance of a bivalent, plastic mechanism that drives the negative reinforcement underlying addiction.

The aggregation and inheritance of damaged proteins determines cell fate during mitosis.

Recent evidence suggests that proliferating cells polarize damaged proteins during mitosis to protect one cell from aging, and that the structural conformation of damaged proteins mediates their toxicity. We report that the growth, resistance to stress, and differentiation characteristics of a cancer cell line (PC12) with an inducible Huntingtin (Htt) fused to enhanced green fluorescent protein (GFP) are dependent on the conformation of Htt. Cell progeny containing inclusion bodies have a longer cell cycle and increased resistance to stress than those with diffuse Htt. Using live imaging, we demonstrate that asymmetric division resulting from a cell containing a single inclusion body produces sister cells with different fates. The cell that receives the inclusion body has decreased proliferation and increased differentiation compared with its sister cell without Htt. This is the first report that reveals a functional consequence of the asymmetric division of damaged proteins in mammalian cells, and we suggest that this is a result of inclusion body-induced proteasome impairment.

The asymmetric segregation of damaged proteins is stem cell-type dependent.

Asymmetric segregation of damaged proteins (DPs) during mitosis has been linked in yeast and bacteria to the protection of one cell from aging. Recent evidence suggests that stem cells may use a similar mechanism; however, to date there is no in vivo evidence demonstrating this effect in healthy adult stem cells. We report that stem cells in larval (neuroblast) and adult (female germline and intestinal stem cell) Drosophila melanogaster asymmetrically segregate DPs, such as proteins with the difficult-to-degrade and age-associated 2,4-hydroxynonenal (HNE) modification. Surprisingly, of the cells analyzed only the intestinal stem cell protects itself by segregating HNE to differentiating progeny, whereas the neuroblast and germline stem cells retain HNE during division. This led us to suggest that chronological life span, and not cell type, determines the amount of DPs a cell receives during division. Furthermore, we reveal a role for both niche-dependent and -independent mechanisms of asymmetric DP division.

Infusion of brain-derived neurotrophic factor into the ventral tegmental area switches the substrates mediating ethanol motivation.

Recent work has shown that infusion of brain-derived neurotrophic factor (BDNF) into the ventral tegmental area (VTA) promotes a switch in the mechanisms mediating morphine motivation, from a dopamine-independent to a dopamine-dependent pathway. Here we showed that a single infusion of intra-VTA BDNF also promoted a switch in the mechanisms mediating ethanol motivation, from a dopamine-dependent to a dopamine-independent pathway (exactly opposite to that seen with morphine). We suggest that intra-VTA BDNF, via its actions on TrkB receptors, precipitates a switch similar to that which occurs naturally when mice transit from a drug-naive, non-deprived state to a drug-deprived state. The opposite switching of the mechanisms underlying morphine and ethanol motivation by BDNF in previously non-deprived animals is consistent with their proposed actions on VTA GABAA receptors.

Misidentification of OLGA-PH-J/92, believed to be the only crustacean cell line.

Continuous cell lines from aquatic invertebrate species are few and the development of crustacean cell lines remains an elusive goal. Although a crayfish cell line derived from neural ganglia of Orconectes limosus was reported in 2000, this cell line OLGA-PH-J/92 failed to be authenticated as such. In this report, we describe our attempts to identify the taxonomic identity of the cell line through immunological and molecular techniques. Immunohistochemical screening for the expression of a suite of invertebrate neuropeptides gave negative results, precluding an invertebrate neural origin. PCR amplification and DNA sequencing for the mitochondrial cytochrome c oxydase I, and 18S ribosomal RNA genes that had been widely used to confirm species identity, could not confirm the OLGA-PH-J/92 cells as originating from crayfish. Subsequent attempts to identify the cells provided moderate homology (82%) to Gephyramoeba sp. (AF293897) following PCR amplification of an 18S rDNA fragment after a BLAST search. A literature search provided morphological evidence of the similarity of OLGA-PH-J/92 to the Gephyramoeba distributed by the American Type Culture Collection as ATCC 50654, which also had been misidentified and was renamed Acramoeba dendroida (Smirnov et al., Eur J Protistol 44:35-44, 2008). The morphology of the OLGA-PH-J/92 cells which remains identical to the original report (Neumann et al., In Vivo 14:691-698, 2000) and matched corresponding micrographs that were available from the ATCC before the cell line was dropped from their catalog (ATCC CRL 1494) is very similar to A. dendroida and could thus belong to the Acramoebidae. These results unequivocally indicate that the OLGA-PH-J/92 cell line is not derived from the crayfish O. limosus, and the search for an immortal crustacean cell line continues.

Ventral tegmental area BDNF induces an opiate-dependent-like reward state in naive rats.

The neural mechanisms underlying the transition from a drug-nondependent to a drug-dependent state remain elusive. Chronic exposure to drugs has been shown to increase brain-derived neurotrophic factor (BDNF) levels in ventral tegmental area (VTA) neurons. BDNF infusions into the VTA potentiate several behavioral effects of drugs, including psychomotor sensitization and cue-induced drug seeking. We found that a single infusion of BDNF into the VTA promotes a shift from a dopamine-independent to a dopamine-dependent opiate reward system, identical to that seen when an opiate-naïve rat becomes dependent and withdrawn. This shift involves a switch in the gamma-aminobutyric acid type A (GABAA) receptors of VTA GABAergic neurons, from inhibitory to excitatory signaling.

Establishment of long term cultures of neural stem cells from adult sea bass, Dicentrarchus labrax.

Long term cell cultures could be obtained from brains of adult sea bass (Dicentrarchus labrax) up to 5 days post mortem. On three different occasions, sea bass brain tissues were dissected, dispersed and cultured in Leibovitz's L-15 media supplemented with 10% fetal bovine serum. The resulting cellular preparations could be passaged within 2 or 3 weeks of growth. The neural cells derived from the first trial (SBB-W1) have now been passaged over 24 times within two years. These cells have been cryopreserved and thawed successfully. SBB-W1 cells are slow growing with doubling times requiring at least 7 days at 22 degrees C. These long term cell cultures could be grown in suspension as neurospheres that were immunopositive for nestin, a marker for neural stem cells, or grown as adherent monolayers displaying both glial and neural morphologies. Immunostaining with anti-glial fibrillary acidic protein (a glial marker) and anti-neurofilament (a neuronal marker), yielded positive staining in most cells, suggesting their possible identity as neural stem cells. Furthermore, Sox 2, a marker for neural stem cells, could be detected from these cell extracts as well as proliferating cell nuclear antigen, a marker for proliferating cells. SBB-W1 could be transfected using pEGFP-N1 indicating their viability and suitability as convenient models for neurophysiological or neurotoxicological studies.