No description available.

Activation of ventral tegmental area (VTA) dopaminergic (DA) neurons by ethanol has been implicated in the rewarding and reinforcing actions of ethanol. GABAergic transmission is thought to play an important role in regulating the activity of DA neurons. While at most central synapses ethanol generally increases inhibitory synaptic transmission, no studies have explored the effect of acute ethanol on GABAergic transmission in the VTA. Here we investigated how ethanol modulates GABAergic transmission in the VTA in relation to the overall action of ethanol on VTA-DA neuron activity. We demonstrated that ethanol dose-dependently enhances action potential-dependent and -independent GABA release onto VTA-DA neurons. Utilizing whole-cell voltage clamp recording techniques, ethanol increased both spontaneous and miniature inhibitory postsynaptic current (s/mIPSC) frequency while having minimal effect on s/mIPSC amplitude. The ethanol enhancement in GABA release was independent of GABAB auto-receptor inhibition of release. Intra-terminal calcium levels regulate neurotransmitter release, thus we investigated how modulation of calcium levels would affect the ethanol-enhancement in GABA release. Ethanol enhanced mIPSC frequency in the presence of the voltage-gated calcium channel blockers, cadmium chloride and nicardipine. However, blockade of intracellular calcium stores with 2-APB and cyclopiazonic acid eliminated the ethanol-enhancement of mIPSC frequency. Intracellular calcium stores are regulated via Gq protein-coupled receptors such as the 5-HT2C receptor. 5-HT2C receptor activation robustly enhanced mIPSC frequency whereas blockade inhibited the ethanol-enhancement in mIPSC frequency. These observations suggest that increased calcium release from intracellular stores via 5-HT2C receptor activation is involved in the ethanol-enhancement of GABA release onto VTA-DA neurons. Utilizing cell-attached current-clamp recordings, we demonstrated that the ethanol-enhancement of VTA-DA neuron activity is modulated by the concurrent enhancement in GABA release. Blockade and activation of GABAA receptors enhanced and reversed, respectively, the stimulatory effect of ethanol on VTA-DA neurons. Mu-opioid receptors (MORs) on GABAergic interneurons have been demonstrated to modulate both basal and ethanol-enhanced VTA-DA activity in vivo, though we failed to demonstrate such an effect in vitro. Overall, the results of this study suggest that the 5-HT2C receptor and intra-terminal calcium-dependent ethanol enhancement in GABA release acts to regulate the overall stimulatory effect of ethanol on VTA-DA activity. / text

<p>The development and function of the nervous system relies on complex regulation of gene expression programs. MicroRNAs (miRNAs) are small RNAs that have diverse functions in mammalian development and disease. In concert with the RNA-induced silencing complex, miRNAs repress translation by binding to target mRNAs. The nervous system contains the largest proportion of miRNAs, yet few have been functionally characterized <italic>in vivo</italic>. </p><p>miR-133b is a highly conserved miRNA embedded in the sequence of 7H4, a noncoding RNA that is enriched at the neuromuscular junction (NMJ), a large synapse that is essential for eliciting muscle contraction and movement. I have found that, like 7H4, miR-133b expression is enriched at the NMJ and upregulated postnatally, coinciding with important events in synaptic maturation, including synaptic growth and elimination. Knockdown of miR-133b in postnatal muscle by electroporation of modified antisense oligonucleotides gave rise to abnormally large synapses, indicating a role for miR-133b in synaptic maturation. To specifically remove miR-133b <italic>in vivo</italic>, I generated a mouse containing a targeted deletion of the miR-133b stemloop. NMJ maturation and synapse elimination proceeded normally in miR-133b knockout mice, suggesting that miR-133b may have other functions at the synapse. The expression of 7H4 and miR-133b is upregulated following nerve transection, consistent with a role in synaptic regeneration. Indeed, NMJ reinnervation is delayed in miR-133b KO mice following nerve crush, but not nerve cut. These data suggest that miR-133b may have a specific protective function at the synapse that could be relevant to disease states, including amyotrophic lateral sclerosis (ALS), where NMJ denervation occurs following motor neuron cell death. However, loss of miR-133b did not affect survival or disease progression in the SOD1(G93A) mouse model, differentiating its role from that of miR-206, another miRNA found in 7H4.</p><p>miR-133b has recently been proposed to regulate the development and maintenance of midbrain dopaminergic (mDA) neurons. mDA neurons have critical functions in the control of movement and emotion, and their degeneration leads to motor and cognitive defects in Parkinson's disease. miR-133b is enriched in the midbrain and regulates mDA neuron differentiation <italic>in vitro</italic> by targeting Pitx3, a transcription factor required for appropriate development of substantia nigra DA neurons. However, the function of miR-133b in the intact midbrain has not been determined. miR-133b KO mice have normal numbers of midbrain dopaminergic neurons during development and aging. Moreover, dopamine neurotransmitter levels are unchanged in the striatum and other brain regions, while expression of dopaminergic genes including Pitx3 is also unaffected. Finally, miR-133b null mice display normal motor coordination and activity, suggesting that miR-133b does not play a significant role in the development or maintenance of the mDA neuron population.</p> / Dissertation

Perturbations of the complex intrinsic and extrinsic factors that contribute to cellular differentiation can have many consequences ranging from dedifferentiation to cell death. The overall objective of my research is to investigate the factors that contribute to the maintenance of mature midbrain neurons and glia. In order to address this objective, I first carried out a detailed immunocytochemical analysis to demonstrate that histone deacetylase inhibitor (HDACI) treatment of differentiated midbrain neurons in culture results in an overall destabilization of neuronal phenotype, which leads to caspase-independent cell death. GFAP positive astrocytes are refractory to the effects of HDACI treatment, suggesting that inhibition of HDACs has differential effects on neurons and glia. HDACI treatment alone was not sufficient to induce neuronal dedifferentiation as evidenced by RT-PCR analysis of stem/progenitor markers, and recovery experiments. Finally, I demonstrate that cortical neurons do not undergo cell death in response to HDACI treatment, suggesting that there may be microenvironmental factors that promote the susceptibility of midbrain neurons to the neurotoxic effects of HDACI. In the second part of this thesis I determined the molecular mechanism that was at least partly responsible for the effects of HDACI treatment on midbrain neurons. Gene expression profiling of HDACI treated midbrain cultures revealed a strong down-regulation of immune related factors. This observation is supported by the loss of microglia in HDACI treated midbrain cultures. I also provide evidence that Toll-like receptor (TLR) signaling, likely through the activation of Interleukin-6 (IL-6) expression, mediates HDAC-dependent neuronal survival. These data provide new evidence that the neuroimmune system is an extrinsic regulator for the homeostasis and survival of neurons.

The homeobox gene Pitx3 has been implicated as a key regulator for lens development because homozygous mutant aphakia mice, which are hypomorph for Pitx3, fail to develop lenses. One aim of my thesis is to investigate the underlying cellular and molecular mechanism of Pitx3 mediated lens defect by studying knockout mice lacking Pitx3. Chimeric embryos, generated by aggregating the wild type embryos with Pitx3 heterozygous or Pitx3 homozygous mutant ES cells, have been used to analyse lens development. Pitx3 null cells failed to colonise the lens epithelium in Pitx3 null wild type chimeric lens, suggesting that Pitx3 is cell-autonomously required for lens epithelial cells. Further study of Pitx3 null mice revealed an earlier downregulation of the lens epithelial markers PDGFR-alpha and E-cadherin in E11.5 lens epithelium, suggesting the loss of lens epithelial identity in Pitx3 deficient mice. Furthermore, cell cycle inhibitors p27KIP1 and p57KIP2 were ectopically expressed throughout the morphologically normal Pitx3 mutant lens vesicle, suggesting that inactivation of Pitx3 leads to cell cycle exit of epithelial lens cells. In addition, precocious activation of the fibre cell-specific proteins beta- and gamma-crystallins was observed in Pitx3 null lens. Beta-crystallin expression could be observed as early as E10.5 throughout the entire Pitx3 null lens vesicle and gamma-crystallin was detected in the malformed Pitx3 deficient lens at E11.5. RNA in situ hybridisation study revealed that the expression of the transcription factor Foxe3 was lost in Pitx3 null lens at E10.5, suggesting that Pitx3 maintains the lens epithelial cells partly via the regulation of transcription factor Foxe3 during lens development. Accordingly, this study provides the cellular and molecular basis for the lens defect observed in Pitx3 null and Pitx3 hypomorph aphakia mice. Pitx3 is a key transcription factor for the maintenance of lens epithelium and its absence leads to premature activation of fibre cell differentiation programme of lens epithelial cells. In the other part of my PhD, I have further developed the Pitx3-GFP knockin ES cell system with a goal to use this tool for the identification of determinants of midbrain dopaminergic (mDA) neurons, the type of cells lost in Parkinson’s disease (PD) patients. Experimental cell therapy and clinical trials have shown that foetal midbrain tissues, but not tissues from other DA neuron containing regions, can functionally restore the lost mDA neurons when transplanted in Parkinson’s disease patients. Therefore, it is essential to coax mDA properties on stem cell-derived neurons when considering therapeutic development. Within the central nervous system, Pitx3 is expressed exclusively in mDA neurons. Using a Pitx3-GFP knockin mouse line previously generated in the laboratory I have derived heterozygous and homozygous Pitx3-GFP ES cells from mouse blastocysts. In keeping with previous findings in our laboratory, the heterozygous Pitx3-GFP (Pitx3GFP/+) ES cell-derived GFP positive cells of neuronal morphology can be detected after in vitro differentiation using the PA6 coculture system. Furthermore, I have shown that these cells express tyrosine hydroxylase and midbrain markers Engrailed-1 and Nurr-1, demonstrating their midbrain characteristics. I have also generated supertransfectable Pitx3GFP/+ ES cells to offer a rapid and efficient way to express a transgene episomally. The Cre-mediated inducible system of Pitx3-GFP reporter ES cells has also been developed in our laboratory and I have shown that they have high induction efficiency thus allows transgene activation in a temporally controlled manner. The Pitx3 null ES cells showed impaired potential to differentiate into mDA neurons thus they may be used to evaluate candidate Pitx3 downstream target by gain-of-function test. In summary, I have developed a Pitx3-GFP reporter ES cell system to identify mDA regulators functionally by in vitro differentiation.

Perturbations of the complex intrinsic and extrinsic factors that contribute to cellular differentiation can have many consequences ranging from dedifferentiation to cell death. The overall objective of my research is to investigate the factors that contribute to the maintenance of mature midbrain neurons and glia. In order to address this objective, I first carried out a detailed immunocytochemical analysis to demonstrate that histone deacetylase inhibitor (HDACI) treatment of differentiated midbrain neurons in culture results in an overall destabilization of neuronal phenotype, which leads to caspase-independent cell death. GFAP positive astrocytes are refractory to the effects of HDACI treatment, suggesting that inhibition of HDACs has differential effects on neurons and glia. HDACI treatment alone was not sufficient to induce neuronal dedifferentiation as evidenced by RT-PCR analysis of stem/progenitor markers, and recovery experiments. Finally, I demonstrate that cortical neurons do not undergo cell death in response to HDACI treatment, suggesting that there may be microenvironmental factors that promote the susceptibility of midbrain neurons to the neurotoxic effects of HDACI. In the second part of this thesis I determined the molecular mechanism that was at least partly responsible for the effects of HDACI treatment on midbrain neurons. Gene expression profiling of HDACI treated midbrain cultures revealed a strong down-regulation of immune related factors. This observation is supported by the loss of microglia in HDACI treated midbrain cultures. I also provide evidence that Toll-like receptor (TLR) signaling, likely through the activation of Interleukin-6 (IL-6) expression, mediates HDAC-dependent neuronal survival. These data provide new evidence that the neuroimmune system is an extrinsic regulator for the homeostasis and survival of neurons.

Discrete electrolytic lesions were placed in the mesencephalic dorsal noradrenergic (DNE) bundle of 22 male Sprague-Dawley rats, and sham operations were performed on 14 control animals. Eight components of copulatory behavior were compared in 2 preoperative and 2 postoperative heterosexual mating tests. A significant postlesion decrease in the postejaculatory interval (PEI), number of intromissions, number of incomplete mounts and the ejaculation latency from the first intromission (ELI) occurred. Norepinephrine levels were significantly reduced in the hippocampus, amygdala and hypothalamus, but not in the preoptic area. The only statistically significant correlations between NE concentrations and behavior in the lesioned animals were negative (hippocampal NE with PEI and ELI). The results support the hypothesis that rostral midbrain lesions disinhibit some components of male rodent copulatory behavior, but suggest that a system or systems other than the DNE bundle may be responsible for this disinhibition.

<p> Schizophrenia is treated with haloperidol, an antipsychotic drug. Although highly effective in treating the positive symptoms of this disease, extrapyramidal side effects also accompany haloperidol treatment, including parkinsonism. Previous investigations revealed that dopamine receptor blockade by haloperidol was not temporally correlated with the appearance of parkinsonian side effects, which begin approximately 3 weeks after haloperidol treatment. In fact, by using tyrosine hydroxylase as a marker for dopamine, TH-immunoreactivity was significantly decreased 5 minutes after haloperidol administration and further downregulation was seen after 10 minutes. Microglial activation has also been implicated in Parkinson's disease models. Haloperidol also induces maximal microglial activation at 5 minutes after administration, with activation increasing by 2 minutes. In this respect, microglial activation may precede TH downregulation, thereby mediating the downregulation. In order to test this possibility, minocycline, a microglial inhibitor, was administered to Sprague-Dawley rats. Minocycline successfully inhibited microglial activation and showed partial protection over TH levels. Caffeine and nicotine have also been implicated as neuroprotective agents in Parkinson's disease. Epidemiological evidence has indicated that both caffeine and nicotine protect against Parkinson's disease. Therefore, caffeine and nicotine were independently tested and found to both prevent TH downregulation and inhibit microglial activation. Overall, microglial activation has been found to correlate with TH downregulation induced by haloperidol. Minocycline, nicotine and caffeine have all been found to inhibit microglial activation, preventing neurotoxicity associated with haloperidol administration. </p> / Thesis / Master of Science (MSc)

No description available.

We have previously shown that the arcuate organization of cell fates within the ventral midbrain critically depends upon the morphogen, Sonic Hedgehog (SHH), which is secreted from a signaling center located along the ventral midline, called the floor plate (FP). Thus, it is ultimately the specification of the FP that is responsible for the patterning and specification of ventral midbrain cell fates. Interestingly, we have found that the chick midbrain FP can be divided into medial (MFP) and lateral (LFP) regions on the basis of gene expression, mode of induction and function. Overexpression of SHH alone is sufficient to recapitulate the entire pattern of ventral cell fates, although remarkably it cannot induce MFP, consistent with the observation that the MFP is refractory to any perturbations of HH signaling. In contrast, overexpression of the winged-helix transcription factor FOXA2/HNF3[beta]robustly induced the MFP fate throughout ventral midbrain while blocking its activity resulted in the absence of the MFP. Thus, by analyzing the differences between SHH and FOXA2 blockade and overexpression, we were able to attribute functions to each the LFP and the MFP. Notably, we observed that FOXA2 overexpression caused a bending of the midbrain neurepithelium that resembled the endogenous median hinge-point observed during neurulation. Additionally, FOXA2 misexpression led to a robust induction of DA progenitors and neurons that was never observed after SHH expression alone. In contrast, we found that all other ventral cell types required HH signaling directly, at a distance and early on in the development of the midbrain when its tissue size is relatively small. Additionally, HH blockade resulted in increased cell-scatter of the arcuate territories and in the disruption of the regional boundaries between the ventral midbrain and adjacent tissue. Thus, we bring new insight into the mechanism by which midbrain FP is specified and ascribe functional roles to its subregions. We propose that while the MFP regulates the production of dopaminergic progenitors and the changes in cellshape required for bending and shaping the neural tube, the LFP appears to be largely responsible for cell survival and the formation of a spatially coherent pattern of midbrain cell fates. / text