No description available.

Thesis (Ph. D.)--Lehigh University, 2000. / Includes bibliographical references and vita.

The effects of adenosine agonists and antagonists were examined on population excitatory postsynaptic potential (population EPSP) slopes, population spike (PS) amplitudes and the relationship between the two i.e. EPSP-spike (E-S) coupling. Activation or blockade of adenosine A2A receptors responses, evoked by stimulation in the stratum radiatum, had no effect on these parameters. However, activation of the adenosine A1 receptor using N6-cyclopentyladenosine (CPA) resulted in a significant decrease in both population EPSP and population spike. When adenosine A1 receptors were activated using CPA (50 nM) the decrease in population spike amplitude was greater than could be accounted for by the decrease in population EPSP, resulting in a dissociation in the EPSP-spike relationship as measured by a right-shift in the E-S curve or a decrease in the ratio PS/EPSP. When adenosine A1 and A2A receptors were activated at the same time using CPA and 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680) respectively, the depression of the E-S relationship was significantly attenuated. This was due to a mainly postsynaptic effect whereby activation of A2A receptors significantly attenuated the postsynaptic elevation of action potential threshold by adenosine A1 receptor activation. The use of a variety of signal transduction modulators suggested that adenylate cyclase, protein kinases or nitric oxide were not involved in the interaction between A2 and A1 receptors. Blocking potassium channels with barium or glibenclamide reduced the inhibitory effects of CPA on population spike amplitude but not population EPSP slope and attenuated the inhibitory effects of adenosine A1 receptor activation on E-S coupling in a similar manner to adenosine A2A receptor activation. The increases in the ratio PS/EPSP seen when A2A receptors were activated with CGS 21680 or when potassium channels were blocked with barium were not significantly different, suggesting that a change in postsynaptic potassium conductance may underlie the antagonistic effect of adenosine A2A receptor activation on A1-mediated responses. Application of ATP 10 μM to hippocampal slices initially decreased population spike amplitude and then produced long-term potentiation (LTP). 2.5 μM ATP also resulted in LTP of responses although did not inhibit the potentials during perfusion. The stable analogue of ATP, αβ-methyleneATP had no long-term effects on potentials. Blocking P2 receptors (with suramin) or N-methyl-D-aspartate (NMDA) receptors prevented the induction of LTP. When adenosine A1 receptors were blocked or the slices superfused with adenosine deaminase no inhibition of responses was seen during perfusion with ATP, nor was LTP induced. Adenosine itself did not induce LTP. These results suggest that induction of LTP following ATP perfusion requires the activation of both P2 and A1 receptors. During superfusion with ZM 241385, ATP 10 μM still induced LTP but no longer caused the inhibition of responses during perfusion. Following electrically induced LTP (100 Hz for 1 second), perfusion of ATP caused no further potentiation of responses, suggesting that electrically and ATP induced LTP share common mechanisms. Perfusion of a preparation of 5'adenylic acid deaminase (AMPase) from Aspergillus species for 10 min resulted in an initial increase in population spike amplitude which gave way to a decrease in potential size and EPSP slope that remained depressed for at least 30 min following washout of the enzyme, resulting in long-term depression (LTD). A decrease in EPSP-spike coupling was observed 30 min following washout of enzyme. Superfusion of AMPase on slices in which transmission had been potentiated using high frequency stimulation resulted in depotentiation of responses. The LTD caused by AMPase could not be inhibited by the allosteric inhibitor of the enzyme 2,3-diphosphoglyceric acid. Further investigation also ruled out the involvement of nitric oxide, protein kinase, and cyclooxygenase in the activity of the AMPase. The use of AMPase extracted from rabbit muscle resulted in no LTD of responses. Analysis of the crude enzyme extract from Aspergillus revealed that the preparation was not pure. Separation of the various components in the crude preparation led to the discovery that the AMPase was not the active factor causing depression of evoked responses. Further purification of the active protein and subsequent sequencing showed that the protein was similar to a β-glucosidase. However, perfusion of β-glucosidase extracted from almonds did not result in LTD of responses. Overall, this thesis extends knowledge of how synaptic transmission can be modulated by purines, and raises the possibility that a new family of proteins, related structurally to glucosidases, may also affect synaptic plasticity.

No description available.

The aim of this project was to determine the temporal and spatial pattern of expression of GDNF family receptors in the developing embryo, with particular emphasis on expression in the peripheral nervous system, and to investigate how expression of receptor mRNAs is regulated in developing neurons. It was hoped that the data obtained would prove useful in further characterizing the role that the GDNF family of neurotrophic factors play in embryonic development. Semi- quantitative PCR revealed that GFRα-1, GFRα-2, GFRα-4 and ret mRNAs are widely distributed with both complementary and overlapping, though distinct, patterns of expression in the chicken embryo during development. Different populations of PNS neurons display different levels of responsiveness to GDNF and NTN and their sensitivity to these factors change throughout development. Examination of receptor expression by quantitative RT-PCR revealed that neurons that are more sensitive to GDNF express higher levels of GFRα-1 mRNA than GFRα-2 mRNA, and neurons that are more sensitive to NTN express higher levels of GFRα-2 mRNA compared to GFRα-1 mRNA. However, developmental changes in responsiveness of a population of neurons to these factors are not consistently paralleled by changes in the relative levels of GFRα transcripts. Furthermore, all neuronal populations express relatively high levels of ret mRNA. These results indicate the responsiveness of PNS neurons to GDNF and NTN is in part governed by the relative levels of expression of their GPI-linked receptors. To determine how the expression of the GDNF family receptors is regulated, embryonic neurons were cultured under different experimental conditions. I found that GFRα-1, GFRα-2, GFRα-4 and ret mRNAs are not significantly regulated by GDNF and/or NTN. However, depolarizing levels of KC1 cause marked changes in the expression of GFRα mRNAs. The effects of KCl are inhibited by L-type Ca2+ channel antagonists, suggesting that they were mediated by elevation of intracellular free Ca2+. KCl treatment increases the response of neurons to GDNF and decreases their response to NTN. There is no marked effect of depolarization on ret mRNA expression.

The main aim of this project was to define the neurotrophin survival requirements of sensory neurons during the early stages of their development both in vivo and in vitro. The in vitro survival of neural crest-derived but not placode-derived cranial sensory neurons is promoted by several different neurotrophins early in their development. Neural crest-derived neurons subsequently lose responsiveness to all neurotrophins except NGF. Loss of responsiveness of neural crest-derived sensory neurons to BDNF and NT3 is associated with a marked shift in the dose responses of these neurons to higher neurotrophin concentrations. Analysis of the timing of cell death in the trigeminal ganglia of mouse embryos that are homozygous for null mutations in the TrkA, TrkB and TrkC genes which encode high affinity receptors for NGF, BDNF and NT3 respectively, show that there is an early peak of apoptosis in TrkB and TrkC knockouts which is consistent with the early survival response of trigeminal neurons to BDNF and NT3 in vitro. The elevated peak of apoptosis in TrkA knockouts occurs at the same development stages as in wild type embryos which is consistent with the later response of trigeminal neurons to NGF in vitro. Furthermore, there is a high level of expression of TrkC mRNA in early trigeminal neurons which accords with the early survival response of these neurons to NTS. It is also shown that subsets of trigeminal neurons discriminate between neurotrophins at very high concentrations during the period of cell death, indicating that neurotrophin responses can be far more highly specific than previously thought. Taken together, these results show that neurotrophin switching is a physiologically relevant phenomenon in certain populations of developing sensory neurons.

The main aim of this project was to determine the neurotrophic actions of glial cell line-derived neurotrophic factor (GDNF) and neurturin, two novel members of the transforming growth factor-beta superfamily of proteins, on neurons from the peripheral nervous system and to identify their receptors. It is found that GDNF promotes the survival of multiple populations of chicken sensory and autonomic neurons in culture throughout development. Whereas sympathetic, parasympathetic and propioceptive neurons become less responsive to GDNF with age, enteroceptive and sensory cutaneous neurons become more responsive to GDNF. GDNF mRNA is expressed in the tissues innervated by these neurons, and developmental changes in its expression in several tissues mirror the changing responses of the innervating neurons to GDNF. These results have changed the previous notion that GDNF is a highly specific neurotrophic factor for motoneurons and dopaminergic neurons. It is shown that neurturin, which is structurally related to GDNF, also promotes the in vitro survival of embryonic chicken sensory and autonomic neurons. Thus, GDNF and neurturin compose a novel subfamily of homologous neurotrophic factors with a similar pattern of activity. The cloning of chicken GDNF receptor-α (GDNFR-α) and a novel receptor termed neurturin receptor-α (NTNR-α) is reported. GDNFR-α and NTNR-α are homologous receptors linked to the membrane via a glycosyl- phosphatidylinositol linkage. It is shown that ectopic co-expression in neurons of GDNFR-α with RET (rearranged during transfection), a transmembrane receptor tyrosine kinase, confers a survival response to GDNF, but not neurturin, and that co-expression of NTNR-α with RET confers a survival response to neurturin, but not GDNF. GDNFR-α and NTNR-α mRNAs are widely expressed in the nervous system, including GDNF and neurturin responsive neurons, and in non-neuronal tissues. These findings indicate that GDNF and neurturin promote neuronal survival by signalling via similar multicomponent receptors that consist of a common transducing receptor tyrosine kinase and a member of a newly emerging family of glycosyl-phosphatidylinositol-linked receptors that confer ligand- specificity.

No description available.

Anti-estrogen drugs such as Tamoxifen and Raloxifene are widely prescribed for breast cancer patients. While they are effective, they also have serious side effects. Alternative drugs are therefore being developed. In the drug discovery process, the in vitro binding of estrogen receptors and lead compounds were studied. The binding strength was conventionally quantified in terms of equilibrium dissociation constants (K0 ). However, the binding kinetic rates and especially off-rates (k0 ff) were recently shown to be better indicators of drug potency. In this thesis, we identified a few dietary estrogens as candidate lead compounds. We studied the binding of full-length human recombinant ERa with these dietary estrogens. In particular, we measured for the first time their binding kinetics rate constants. We also measured the change in the receptor-ligand binding kinetics upon its recruitment of co-activators, as a means to gauge agonist/antagonist propensity ofthe ligand. Our results showed that the following dietary estrogens, a-Zearalenol, Zearalenone, and Coumestrol bind favorably to the estrogen receptor alpha.

No description available.