by Poon Yim-chu Daisy. / Bibliography: leaves 128-148 / Thesis (M.Phil.) -- Chinese University of Hong Kong, 1983

Neurotransmission is essential for neuronal communication. At the presynapse, synaptic vesicles (SVs) undergo exocytosis to release neurotransmitter in response to incoming action potentials, and endocytosis to maintain the supply of SVs needed for further rounds of exocytosis. A key event during SV endocytosis is the efficient sorting and localisation of SV proteins at the plasma membrane. This ensures that nascent SVs that are formed have the correct molecular composition to participate in subsequent exocytic events. The sorting of SV proteins at the plasma membrane is usually facilitated by adaptor proteins (e.g. AP-2) which recognise binding motifs present on key SV proteins and facilitate their internalisation during endocytosis. In addition to this, certain SV proteins possess the ability to chaperone each other as part of an endocytic transport complex throughout the SV recycling process. In conjunction with AP-2-facilitated sorting, the transport of complexed SV proteins during endocytosis provides further mechanistic insight into how SVs are generated with consistent high fidelity for functional viability. Using pHluorins as a tool to visualise SV protein trafficking in hippocampal cultures, the relationship between two key SV proteins, synaptic vesicle protein 2A (SV2A) and synaptotagmin I (SYT1), was investigated. SYT1 predominantly acts as the Ca2+ sensor for fast synchronous release at the presynapse, whilst the exact function of SV2A remains unknown to this day. In this study, the ablation of the AP-2 binding site in SV2A (Y46A) resulted in increased SYT1 surface expression and accelerated SYT1 retrieval compared to WT SV2A. No additive defects were observed when a second point mutation (T84A) was introduced to SV2A that disrupts the phosphorylation-dependent interaction between SV2A and SYT1, thus confirming that SYT1 localisation and retrieval is dependent on normal SV2A retrieval by AP-2. The hypothesis that disruption of the SV2A-SYT1 interaction may provide an underlying mechanism for motor onset seizures in epilepsy was also investigated. An epilepsy-related mutation (R383Q) in SV2A also resulted in increased SYT1 surface expression and accelerated SYT1 retrieval mirroring the defects caused by the Y46A mutation. Introduction of Y46A or T84A mutation into SV2A R383Q resulted in no additive defects compared to the single mutant, suggesting that the observed defects in SYT1 localisation and retrieval kinetics in the epilepsy-related mutant may be caused by the ablation of normal SV2A internalisation. GST pulldown assays, mass spectrometry and western blotting data indicate that presence of the mutation disrupts normal binding of the SV2A cytosolic loop with actin, tubulin and certain subunits of V-ATPase. Finally, a link between SV2A-dependent presynaptic dysfunction and epilepsy was examined through studies utilising the anti-epileptic drug, levetiracetam (LEV). SV2A contains a binding site for LEV, suggesting that it may act as a carrier for the drug into the presynapse. Hippocampal neuronal cultures were treated with LEV at various concentrations in the presence of specific patterns of neuronal activity. No observed effects of the drug on synaptophysin, vesicular glutamate transporter 1 (VGLUT1) and SYT1 recycling were observed, suggesting that LEV is unlikely to function as a modulator of excitatory presynaptic activity or by influencing SV2A function. In conclusion, this work demonstrates that SV2A is essential for accurate SYT1 trafficking and a link has been established between defective SV2A internalisation and subsequent downstream effects on SYT1 localisation and retrieval during SV recycling.

Excitatory synaptic transmission and plasticity mediated by glutamate receptors are important for many brain functions, including learning and memory. Various molecular and cellular models have been established to study multiple forms of synaptic plasticity that coexist in the hippocampal CA1 region. Metabotropic glutamate receptor-dependent long-term depression (mGluR-dependent LTD) is a form of long lasting synaptic plasticity thought to play critical roles in diverse physiological and pathological processes. The GluR2 subunit of AMPA receptors has been a focus of neuroscience research over the last decade due to its important roles in endocytic trafficking and Ca2+ permeation in many forms of activity-dependent synaptic plasticity and homeostatic plasticity. However, the underlying mechanisms of mGluR-dependent LTD and the possible involvement of GluR2 in this form of plasticity remain unknown. In this project, I utilized GluR2 knockout (KO) mice and tested the requirement of GluR2 in multiple forms of hippocampal synaptic plasticity at different developmental stages. The results showed that although GluR2 is dispensable for long lasting synaptic plasticity in juvenile mice, it is essential for the expression of mGluR-dependent LTD in adult animals. Next, I examined the involvement of a number of GluR2-specific functions in mGluR-dependent LTD and found that GluR2 N-terminal interaction with the cell adhesion molecule N-cadherin is a key process required for GluR2 to regulate the expression of mGluR-dependent LTD. Furthermore, using a combination of approaches including electrophysiology, biochemical assays, and virus-mediated expression of several mutant GluR2 constructs, I identified a signaling cascade involving N-cadherin/β-catenin complex, Rac1 Rho GTPase, LIM-kinase 1 and cofilin, through which GluR2 exerts its effect on actin regulation and mGluR-dependent LTD. Importantly, the impaired LTD in GluR2 KO mice can be fully rescued by manipulating GluR2-N-cadherin N-terminus interaction or cofilin-mediated actin reorganization. Lastly, I showed that this signaling cascade also plays a critical role in the regulation of dendritic spine plasticity during mGluR-dependent LTD. Together, these results reveal a novel signaling process by which GluR2 regulates long lasting synaptic plasticity and provide insights into how functional and structural plasticity are coordinated in the mammalian central nervous system.

Excitatory synaptic transmission and plasticity mediated by glutamate receptors are important for many brain functions, including learning and memory. Various molecular and cellular models have been established to study multiple forms of synaptic plasticity that coexist in the hippocampal CA1 region. Metabotropic glutamate receptor-dependent long-term depression (mGluR-dependent LTD) is a form of long lasting synaptic plasticity thought to play critical roles in diverse physiological and pathological processes. The GluR2 subunit of AMPA receptors has been a focus of neuroscience research over the last decade due to its important roles in endocytic trafficking and Ca2+ permeation in many forms of activity-dependent synaptic plasticity and homeostatic plasticity. However, the underlying mechanisms of mGluR-dependent LTD and the possible involvement of GluR2 in this form of plasticity remain unknown. In this project, I utilized GluR2 knockout (KO) mice and tested the requirement of GluR2 in multiple forms of hippocampal synaptic plasticity at different developmental stages. The results showed that although GluR2 is dispensable for long lasting synaptic plasticity in juvenile mice, it is essential for the expression of mGluR-dependent LTD in adult animals. Next, I examined the involvement of a number of GluR2-specific functions in mGluR-dependent LTD and found that GluR2 N-terminal interaction with the cell adhesion molecule N-cadherin is a key process required for GluR2 to regulate the expression of mGluR-dependent LTD. Furthermore, using a combination of approaches including electrophysiology, biochemical assays, and virus-mediated expression of several mutant GluR2 constructs, I identified a signaling cascade involving N-cadherin/β-catenin complex, Rac1 Rho GTPase, LIM-kinase 1 and cofilin, through which GluR2 exerts its effect on actin regulation and mGluR-dependent LTD. Importantly, the impaired LTD in GluR2 KO mice can be fully rescued by manipulating GluR2-N-cadherin N-terminus interaction or cofilin-mediated actin reorganization. Lastly, I showed that this signaling cascade also plays a critical role in the regulation of dendritic spine plasticity during mGluR-dependent LTD. Together, these results reveal a novel signaling process by which GluR2 regulates long lasting synaptic plasticity and provide insights into how functional and structural plasticity are coordinated in the mammalian central nervous system.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 131-147).

Synaptic alterations in the nucleus accumbens (NAc) are crucial for the aberrant reward-associated learning that forms the foundation of drug dependence. Glutamatergic synaptic plasticity in the NAc has been implicated in several behavioral responses to psychomotor stimulating agents, such as cocaine and amphetamine, yet no studies, at present, have investigated its modulation by ethanol. We demonstrated that both in vitro and in vivo ethanol treatment significantly disrupts normal synaptic functioning in medium spiny neurons (MSNs) of the NAc shell. Utilizing whole-cell voltage clamp recording techniques, synaptic conditioning (low frequency stimulation with concurrent postsynaptic depolarization) reliably depressed (NAc-LTD) AMPA-mediated excitatory postsynaptic currents (EPSCs). Acute ethanol exposure inhibited the depression of AMPA EPSCs differentially with increasing concentrations, but this inhibitory action of ethanol was reversed by a D1-like dopamine receptor agonist. When examined 24 hours following a single bout of in vivo chronic intermittent ethanol (CIE) vapor exposure, NAc-LTD was absent and instead synaptic potentiation (LTP) was reliably observed. We further investigated CIE-induced modulation of NAc-LTD by distinguishing between the two subpopulations of MSNs in the NAc, D1 receptor-expressing (D1+) and D2 receptor-expressing (D1-). We determined that NAc-LTD is expressed solely in D1+ but not D1- MSNs. In addition, 24 hours following a repeated regimen of in vivo CIE exposure NAc-LTD is completely occluded in D1+ MSNs, while D1- MSNs are able to express LTD. Complete recovery of normal synaptic plasticity expression in both D1+ and D1- MSNs does not occur until two weeks of withdrawal from CIE vapor exposure. To our knowledge, this is the first demonstration of a reversal in the cell type-specificity of synaptic plasticity in the NAc shell, as well as, the gradual recovery of the pre-drug exposure plasticity state following extended withdrawal. This study suggests that NAc-LTD is cell type-specific and highly sensitive to both acute and chronic ethanol exposure. We believe these observations also highlight the adaptability of NAc MSNs to the effects of long-term ethanol exposure. A change in these synaptic processes may constitute a neural adaptation that contributes to the induction and/or expression of alcohol dependence. / text

One of the central questions of neuroscience research has been how the cellular and molecular components of the brain give rise to complex behaviours. Three major breakthroughs from the past sixty years have made the study of learning and memory central to our understanding of how the brain works. First, the psychologist Donald Hebb proposed that information storage in the brain could occur through the strengthening of the connections between neurons if the strengthening were restricted to neurons that were co-active (Hebb, 1949). Second, Milner and Scoville (1957) showed that the hippocampus is required for the acquisition of new long-term memories for consciously accessible, or declarative, information. Third, Bliss and Lømo (1973) demonstrated that the synapses between neurons in the dentate gyrus of the hippocampus could indeed be potentiated in an activity-dependent manner. Long-term potentiation (LTP) of the glutamatergic synapses in area CA1, the primary output of the hippocampus, has since become the leading model of synaptic plasticity due to its dependence on NMDA receptors (NMDARs), required for spatial and temporal learning in intact animals, and its robust pathway specificity. Using whole-cell recording in hippocampal slices from adult rats, I find that the efficacy of synaptic transmission from CA3 to CA1 can in fact be enhanced without the induction of classic LTP at the glutamatergic inputs. Taking care not to directly stimulate inhibitory fibers, I show that the induction of GABAergic plasticity at feedforward inhibitory inputs in CA1 results in the reduced shunting of excitatory currents, producing a long-term increase in the amplitude of Schaffer collateral-mediated postsynaptic potentials which is dependent on NMDAR activation and is pathway specific. The sharing of these fundamental properties with classic LTP suggests the possibility of a previously unrecognized target for therapeutic intervention in disorders linked to memory deficits, as well as a potentially overlooked site of LTP expression in other areas of the brain.

One of the central questions of neuroscience research has been how the cellular and molecular components of the brain give rise to complex behaviours. Three major breakthroughs from the past sixty years have made the study of learning and memory central to our understanding of how the brain works. First, the psychologist Donald Hebb proposed that information storage in the brain could occur through the strengthening of the connections between neurons if the strengthening were restricted to neurons that were co-active (Hebb, 1949). Second, Milner and Scoville (1957) showed that the hippocampus is required for the acquisition of new long-term memories for consciously accessible, or declarative, information. Third, Bliss and Lømo (1973) demonstrated that the synapses between neurons in the dentate gyrus of the hippocampus could indeed be potentiated in an activity-dependent manner. Long-term potentiation (LTP) of the glutamatergic synapses in area CA1, the primary output of the hippocampus, has since become the leading model of synaptic plasticity due to its dependence on NMDA receptors (NMDARs), required for spatial and temporal learning in intact animals, and its robust pathway specificity. Using whole-cell recording in hippocampal slices from adult rats, I find that the efficacy of synaptic transmission from CA3 to CA1 can in fact be enhanced without the induction of classic LTP at the glutamatergic inputs. Taking care not to directly stimulate inhibitory fibers, I show that the induction of GABAergic plasticity at feedforward inhibitory inputs in CA1 results in the reduced shunting of excitatory currents, producing a long-term increase in the amplitude of Schaffer collateral-mediated postsynaptic potentials which is dependent on NMDAR activation and is pathway specific. The sharing of these fundamental properties with classic LTP suggests the possibility of a previously unrecognized target for therapeutic intervention in disorders linked to memory deficits, as well as a potentially overlooked site of LTP expression in other areas of the brain.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 109-117).

archives@tulane.edu / Previous work has revealed a presynaptic cytosolic Na+-dependent regulation on vesicular glutamate content and mEPSC amplitude via activating vacuolar Na+/H+ exchangers (NHEs) expressed on the synaptic vesicles, suggesting a presynaptic determinant of quantal size for synaptic strength. However, it remains unknown how spike activities control intracellular Na+ at the axon terminals and how the fluctuation of presynaptic Na+ during activities modulates quantal content and contributes to synaptic strength. I studied these questions using the calyx of Held, a giant glutamatergic synapse. With two-photon Na+ imaging, I found that presynaptic Na+ substantially accumulated during spike firing in a frequency and duration-dependent manner. This spike-induced elevation of presynaptic Na+ gradually increased EPSC amplitude by solely affecting vesicular glutamate filling, which was further confirmed as increased amplitude of asynchronous released vesicles, but without affecting the size of readily releasable pool or neurotransmitter release probability. This Na+-dependent modulation of EPSC amplitude resulted in a change of the reliability of transferring presynaptic spike to postsynaptic firing. Finally, blockade of NHEs reduced both EPSC amplitude and reliability of synaptic signaling, suggesting that NHEs are required for presynaptic Na+ regulation of synaptic transmission. Recent studies demonstrated that a non-inactivation cation channel NALCN (Na+ leak channel, non-selective), characterized as a major Na+ leak channel, is widely expressed in the central nervous system. Immunostaining revealed the expression of NALCN channel at the calyceal terminals. In line with a role of NALCN in controlling the cell excitability, calyces with conditional knockout (cKO) of NALCN exhibited a more hyperpolarized resting membrane potential compared with the wildtype (WT) calyces. Blockade of NALCN with a non-specific blocker gadolinium (Gd3+) induced a reduction of basal Na+ level and mEPSC amplitude in the WT but not in cKO group, suggesting the involvement of presynaptic NALCN channels in regulating the vesicular glutamate content. More importantly, two-photon Ca2+ imaging showed that NALCN channels were permeable to Ca2+, and Gd3+ decreased the basal Ca2+ level in WT but not cKO calyces. The Ca2+ permeability was further confirmed by reduced sensitivity of mEPSC frequency in response to increased extracellular Ca2+ concentration in cKO and reduced initial release probability in response to application of Gd3+ to block NALCN channels in WT group. Finally, Gd3+ induced a stronger reduction of EPSC amplitude in WT group compared to cKO group. Overall, these data indicate that NALCN channels regulate glutamate transmission through modulation of both quantal size and initial release probability. / 1 / Dainan Li