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

Asymmetric Division of Damaged Proteins in Proliferating Cells

This thesis explores the unequal partitioning of damaged proteins during mitosis and its implications for cell fate. Initially described in unicellular organisms, it was unclear if this method was used in vivo in multicellular organisms and had functional consequences in mammalian cells. To determine if this asymmetry was conserved in multicellular organisms, I studied three stem/progenitor populations in Drosophila: the larval neuroblast, adult female germline stem cell, and adult intestinal stem cell. Each cell type was found to asymmetrically segregate damaged proteins identified by the 2,4-hydroxynonenal (HNE) modification, which are associated with oxidative stress and age. Both the larval neuroblast and female germline stem cell were found to retain damaged proteins during division, whereas the intestinal stem cell segregated damaged proteins to differentiating progeny. I suggest that functional lifespan, and not cell type, determines the cell that receives the majority of damaged proteins during division. In each cell type, damaged proteins were associated with DE-Cadherin, a common component of the stem cell niche and removal from the niche was associated with reduced damaged protein polarization. Interestingly, when larval neuroblasts were mechanically dissociated from their niche and placed in culture, the internal polarization of damaged proteins was found to increase with progression through the cell-cycle. Therefore, I suggest that both the niche and intrinsic factors play a role in the asymmetric division of damaged proteins. To determine if an asymmetric division of damaged proteins influenced cell fate, I used a mammalian cell line with inducible expression of misfolded Huntingtin, which shares similar properties to damaged proteins. This study also revealed that the conformation of damaged proteins impacts cell fate: cells with diffuse Huntingtin displayed greater proliferation and reduced resistance to stress. Tracking cells containing an aggregate with live-imaging revealed that the cell that inherits the aggregate has a longer cell-cycle and an enhanced capacity to differentiate. Therefore, the asymmetric inheritance of damaged proteins impacts cell fate. In the final chapter of this thesis, I discuss the implications of an asymmetric division of damaged proteins on cell fate and how this information can be applied to cancer treatments.Ph

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

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