Neto1 and Neto2 are CUB domain-containing transmembrane proteins that are expressed in the mammalian brain. Previous studies showed that Neto1 is a NMDAR-associated protein with important roles in synaptic plasticity and learning/memory (Ng et al., 2009). To establish the functions of Neto2, I first searched for its binding partners. Using yeast two-hybrid analysis, GST pull-down and co-immunoprecipitation studies, I found that Neto2 can bind to the PDZ domain-containing protein GRIP. In the brain, GRIP regulates the synaptic trafficking and stability of AMPA and kainate receptors (KARs) (Hirbec et al., 2003). To determine whether Neto2 is required for the synaptic expression of KARs and/or AMPARs, I examined whether Neto2 was associated with these receptors at the postsynaptic membrane. Coimmunoprecipitation studies showed that while Neto2 is a component of postsynaptic KAR protein complexes, it is not associated with AMPARs. In the cerebellum, Neto2-null mice showed a 44% (n=3;p<0.01) decrease in the abundance of postsynaptic KARs with no change in the level of total KARs, thus suggesting a specific deficit in KAR synaptic localization. Unexpectedly, loss of Neto2 had no effect on the abundance of hippocampal KARs (n=3; p>0.05), or on neurotransmission by them (n=12; p>0.05). To determine whether this normal KAR function might be due to compensation by Neto1, which also interacts with KARs, I examined KAR abundance in Neto1-null, and Neto1/2-double null hippocampus. Loss of Neto1 resulted in a 53% decrease in postsynaptic levels of GluK2-KARs (n=3;p<0.01). However, in double null animals, the reduction was indistinguishable from Neto1 single null mice, suggesting that Neto2 is not involved in the postsynaptic localization of hippocampal KARs. In Neto1-null mice, KAR-mediated currents showed smaller amplitude (61% of wild-type;n=14;p<0.01), and faster decay kinetics (40% of wild-type;n=14;p<0.001). Together, these findings establish both Neto1 and Neto2 as auxiliary proteins of native KARs: Neto1 regulates the synaptic abundance and kinetics of KARs in the hippocampus, while Neto2 mediates the synaptic localization of cerebellar KARs. Additionally, the results presented here, in conjunction with previous findings, reveal a unique ability of Neto1 in controlling synaptic transmission by serving as an auxiliary protein for two different classes of ionotropic glutamate receptors.

The mechanisms underlying the transition to seizure are still unresolved. Proposed mechanisms include excitatory GABAergic drive, loss of interneuron-mediated inhibition, and glutamatergic input potentiation. The objective of this thesis was to investigate the relative contributions of synchronized glutamatergic and GABAergic inputs and their functional roles during ictogenesis in the epileptic neonatal (postnatal days 6-12) mouse hippocampus, induced with 0.25mM Mg2+/5mM K+ perfusion. Simultaneous field and whole-cell patch-clamp recordings were obtained from CA3 stratum-oriens interneurons and pyramidal cells. The antagonists for GABAA and glutamate receptors abolished the preictal and ictal discharges, respectively, suggesting that the preictal state is mediated by the coherent discharges of GABAergic inhibitory interneurons, whereas the recurrent excitatory inputs are required for ictogenesis. Synaptic charge transfers underlying the synchronized discharges showed a dynamic change in the balance between the inputs: GABAergic currents markedly diminished by ictal onset whereas glutamatergic currents dominated at ictal onset and throughout the ictus.

Neto1 and Neto2 are CUB domain-containing transmembrane proteins that are expressed in the mammalian brain. Previous studies showed that Neto1 is a NMDAR-associated protein with important roles in synaptic plasticity and learning/memory (Ng et al., 2009). To establish the functions of Neto2, I first searched for its binding partners. Using yeast two-hybrid analysis, GST pull-down and co-immunoprecipitation studies, I found that Neto2 can bind to the PDZ domain-containing protein GRIP. In the brain, GRIP regulates the synaptic trafficking and stability of AMPA and kainate receptors (KARs) (Hirbec et al., 2003). To determine whether Neto2 is required for the synaptic expression of KARs and/or AMPARs, I examined whether Neto2 was associated with these receptors at the postsynaptic membrane. Coimmunoprecipitation studies showed that while Neto2 is a component of postsynaptic KAR protein complexes, it is not associated with AMPARs. In the cerebellum, Neto2-null mice showed a 44% (n=3;p<0.01) decrease in the abundance of postsynaptic KARs with no change in the level of total KARs, thus suggesting a specific deficit in KAR synaptic localization. Unexpectedly, loss of Neto2 had no effect on the abundance of hippocampal KARs (n=3; p>0.05), or on neurotransmission by them (n=12; p>0.05). To determine whether this normal KAR function might be due to compensation by Neto1, which also interacts with KARs, I examined KAR abundance in Neto1-null, and Neto1/2-double null hippocampus. Loss of Neto1 resulted in a 53% decrease in postsynaptic levels of GluK2-KARs (n=3;p<0.01). However, in double null animals, the reduction was indistinguishable from Neto1 single null mice, suggesting that Neto2 is not involved in the postsynaptic localization of hippocampal KARs. In Neto1-null mice, KAR-mediated currents showed smaller amplitude (61% of wild-type;n=14;p<0.01), and faster decay kinetics (40% of wild-type;n=14;p<0.001). Together, these findings establish both Neto1 and Neto2 as auxiliary proteins of native KARs: Neto1 regulates the synaptic abundance and kinetics of KARs in the hippocampus, while Neto2 mediates the synaptic localization of cerebellar KARs. Additionally, the results presented here, in conjunction with previous findings, reveal a unique ability of Neto1 in controlling synaptic transmission by serving as an auxiliary protein for two different classes of ionotropic glutamate receptors.

The mechanisms underlying the transition to seizure are still unresolved. Proposed mechanisms include excitatory GABAergic drive, loss of interneuron-mediated inhibition, and glutamatergic input potentiation. The objective of this thesis was to investigate the relative contributions of synchronized glutamatergic and GABAergic inputs and their functional roles during ictogenesis in the epileptic neonatal (postnatal days 6-12) mouse hippocampus, induced with 0.25mM Mg2+/5mM K+ perfusion. Simultaneous field and whole-cell patch-clamp recordings were obtained from CA3 stratum-oriens interneurons and pyramidal cells. The antagonists for GABAA and glutamate receptors abolished the preictal and ictal discharges, respectively, suggesting that the preictal state is mediated by the coherent discharges of GABAergic inhibitory interneurons, whereas the recurrent excitatory inputs are required for ictogenesis. Synaptic charge transfers underlying the synchronized discharges showed a dynamic change in the balance between the inputs: GABAergic currents markedly diminished by ictal onset whereas glutamatergic currents dominated at ictal onset and throughout the ictus.

Thesis (M.S.)--Montana State University--Bozeman, 2007. / Typescript. Chairperson, Graduate Committee: Michael Babcock. Includes bibliographical references (leaves 43-51).

As the major excitatory neurotransmitter, glutamate (Glu) is physiologically important in brain function. Excessive Glu release, however, is a critical underlying pathological mechanism in neurodegenerative disease, especially stroke. Strategies to protect neurons from cell death under these conditions are scarce; in part because of incomplete understanding of inherent neuroprotective mechanisms. The suprachiasmatic nucleus (SCN) is a region of the brain that exhibits endogenous resistance to Glu excitotoxicity. A previous study demonstrated that SCN2.2 cells (an immortalized SCN cell line) were resistant to Glu excitotoxicity as compared to GT1-7 neurons (from the neighboring hypothalamus). This thesis explored the cellular mechanisms underlying this endogenous neuroprotection in SCN2.2 cells. Extracellular regulated kinase (ERK) is expressed in the SCN, activated by Glu, and is anti-apoptotic in other systems. Therefore, this thesis was designed to test the following central hypothesis: SCN2.2 cells are dependent on ERK signaling for survival in the presence of an excitotoxic insult. Glu increased ERK activity in SCN2.2 cells and importantly, resistance to Glu excitotoxicity in SCN2.2 cells was compromised by pre-treatment with an ERK inhibitor (PD98059; PD). ERK inhibition + Glu mediated SCN2.2 cell death in an N-methyl-D-aspartate receptor (NMDAR)-dependent manner; specifically via the NMDAR 2B (NR2B) subunit. Glu treatment increased expression of NR2B, phosphorylated NR2B and NR1 proteins and decreased NR2A and NR2D mRNA in the GT1-7 cells. Glu-treated SCN2.2 cells showed decreased NR2B, phosphorylated NR2B, increased NR2C proteins and increased NR2A and NR2D mRNA levels. These data are consistent with varied NMDAR responses to Glu in GT1-7 vs. SCN2.2 cells, which might underlie the different physiological responses to Glu in the two cell types. Further experiments investigated the role of several signaling kinases, e.g. protein kinase A (PKA), protein kinase C (PKC), calcium/calmodulin-dependent kinase II (CaMK-II) and c-Jun N-terminal kinase-II (JNK-II) in regulation of ERK activation and on SCN2.2 cell fate. PKA and PKC inhibition together, CaMK-II inhibition and JNK-II inhibition resulted in SCN2.2 cell death in the presence of Glu. PKA + PKC inhibition and CaMK-II inhibition resulted in a corresponding decrease in Glu-induced ERK phosphorylation. Combined inhibition of ERK, CaMK-II and JNK-II resulted in exacerbation of cell death as compared to when the inhibitors were used individually. These results suggest that ERK activity is regulated by a number of different kinases. Glu treatment resulted in a persistent increase in ERK phosphorylation (activation) for up to 48 h in the SCN2.2 cells whereas the pro-apoptotic p38 was phosphorylated (activated) in the GT1-7 cells exposed to Glu. JNK-II was transiently phosphorylated (activated) in the SCN2.2 cells. This suggests an activation of a short-term stress response which can result in activation of a long-term neuroprotective response in these cells. Pro-apoptotic Bid mRNA and cleaved Bid protein levels were increased in the Glu-treated GT1-7 cells. The effect of Glu treatment on the expression of several downstream effector molecules of ERK activation was also explored. Neuritin mRNA was increased with Glu treatment in the SCN2.2, but not in the GT1-7 cells. However, there was no change in the neuritin protein levels in either cell type with Glu treatment. Bcl2 levels remained unchanged in the Glu-treated GT1-7 cells. Although there was no change in the Bcl2 mRNA levels in the SCN2.2 cells, Bcl2 protein was significantly increased with Glu treatment, thus suggesting a post-translational mechanism of neuroprotection involving Bcl2. Taken together, these results are consistent with activation of an apoptotic mechanism in the GT1-7 cells exposed to Glu as opposed to a pro-survival effect in similarly treated SCN2.2 cells. Future studies should be able to take advantage of these mechanisms in developing therapeutic strategies in the treatment of neurodegenerative disorders.

L'homéostasie glutamatergique dans les fentes synaptiques régule la neurotransmission et préserve de l'excitotoxicité. Cela est particulièrement important dans l'oreille interne où il y a une libération soutenue de neurotransmetteur. Pour la plupart des cellules ciliées cochléaires et vestibulaires, la clairance du glutamate est assurée par les transporteurs du glutamate EAAT1 (GLAST) exprimés par les cellules de soutien. Un tel mécanisme n'est pas possible pour les cellules ciliées vestibulaires de type I car leur terminaison synaptique en calice empêche tout accès à la fente synaptique. Nous avons donc postulé qu'un ou plusieurs transporteurs du glutamate devaient être présents au niveau des cellules ciliées de type I ou du calice ou des deux.Grâce à des enregistrements électrophysiologiques, nous avons démontré qu'un courant anionique induit par le glutamate et bloqué par le DL-TBOA est présent dans les cellules ciliées de type I. Les techniques d'hybridation in situ et d'immunohistochimie ont révélé la présence d'EAAT4 et EAAT5. Ces deux transporteurs du glutamate, qui pourraient êtres à l'origine des courants enregistrés, sont exprimés par les cellules ciliées de type I et de type II. De plus, des expériences de RT-PCR et de microscopie électronique ont confirmé ces résultats et suggéré que ces transporteurs pourraient aussi être exprimés postsynaptiquement par le calice. Ces travaux de thèse montrent qu'EAAT4 et EAAT5, considérés respectivement comme spécifiques des tissus cérébelleux et rétiniens, ont une distribution plus large. Ces résultats posent la question des rôles potentiels de ces transporteurs dans l'homéostasie glutamatergique vestibulaire. / Glutamate homeostasis in synaptic clefts shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear where there is a continually high rate of neurotransmitter release. In the case of most cochlear and vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glial glutamate transporter. A similar mechanism is unlikely to work in vestibular type I hair cells because the presence of calyx endings separates supporting cells from the synaptic zone. Based on this arrangement, we postulated that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrated that a glutamate-activated anion current blocked by DL-TBOA is expressed in type I hair cells. In situ hybridization and immunohistochemistry revealed that EAAT4 and EAAT5, two glutamate transporters that could support the anion current, are expressed in both type I and type II hair cells. Furthermore, RT-PCR and immunogold investigations confirmed those results and added that although preferentially expressed presynaptically, the transporters may also be present in the postsynaptic calyx membrane. Previously thought to be exclusively expressed in the cerebellum and retina respectively, this thesis work shows that EAAT4 and EAAT5 have a wider distribution. The potential role of these transporters in the glutamatergic homeostasis of the calyx synapse is then discussed.

A significant number of individuals begin drinking alcohol early in life during adolescence, a period in which their brain is developing. Drinking alcohol at an early age is linked to a greater likelihood that a person will become an alcoholic later in life. Levels of Glutamate (GLU), a major neurotransmitter, in the medial prefrontal cortex (mPFC) has been directly linked to the expression of alcohol-use disorders. Thus, a better understanding of how childhood drinking produces alterations in the brain, thereby contributing to alcoholism, is needed. The current research utilized an animal model of alcoholism to examine the long range consequences of alcohol consumption during adolescence on GLU functioning within the mPFC in adulthood. It was hypothesized, adolescent drinking would lead to a higher levels of GLU in the mPFC in adulthood. Two groups of alcohol-preferring (P) rats received either free-access to alcohol (15% v/v) and water or water alone in their home cage (24 hrs a day; 7 days a week) during their adolescent period. At the end of the adolescent period, alcohol was removed and all animals were provided only water to drink for approximately 21 days. Next, animals were implanted with guide cannula aimed at infralimbic and prelimbic regions of the mPFC and provided one-week to recover before undergoing quantitative microdialysis, a method that allows for the direct sampling of GLU from brain tissue. During testing, samples were collected every 10 minutes and animals were first exposed to artificial cerebral spinal fluid (aCSF) followed by aCSF containing three GLU concentrations (1 µM, 5 µM , and 10 µM; presented in randomized order across rats). By exposing the animals to different levels of GLU, the average brain level of GLU can be established as well as how fast the brain is removing/clearing GLU. Samples were analyzed using high-pressure liquid chromatography a method that quantifies GLU levels in each sample. Analyses revealed a significantly lower level of GLU removal/clearance in the prelimbic region of the mPFC of the alcohol-drinking group compared to the water group. Analyses also revealed a significantly higher average level of GLU in the alcohol-drinking group compared to the water drinking control group. There were no differences between groups in average GLU levels or GLU clearance in the infralimbic region of the mPFC. Overall, the data from the current study suggest that the consumption of alcohol during adolescence may produce a long-lasting reduction of GLU removal/clearance thereby resulting in increased GLU levels within the prelimbic region of the mPFC. The current findings may represent a long-lasting change that happens in the brain when an individual consumes alcohol during adolescence which could then contribute to the development of an alcohol-use disorder later in life.

The retinogeniculate synapse, the connection between retinal ganglion cells (RGCs) and thalamic relay neurons, undergoes extensive remodeling and refinement in the first few postnatal weeks. While many studies have focused on this process, little is known about the factors that influence excitatory transmission during this dynamic period. A major goal of my dissertation research was to identify mechanisms that regulate glutamate release and clearance at the developing synapse. First, we investigated the role of glutamate transporters and metabotropic glutamate receptors (mGluRs) in shaping excitatory transmission. Early in development, we found presynaptic group II/III mGluRs are present and are activated by glutamate released from RGCs following optic tract stimulation at natural frequencies. This response was found to diminish with age, but glutamate transporters continued to shape synaptic currents throughout development. The finding that glutamate is able to escape the synaptic cleft and bind extrasynaptic high-affinity mGluRs led us to speculate that glutamate might also diffuse to neighboring synapses and bind ionotropic glutamate receptors opposing quiescent release sites. Excitatory currents recorded from immature, but not mature, retinogeniculate synapses display a prolonged decay timecourse. We found evidence that both asynchronous release of glutamate as well as spillover of glutamate between neighboring synapses contributes to these slowly decaying synaptic currents. Furthermore, we uncovered and characterized a novel, purely spillover-mediated current from immature relay neurons, which strongly supports the presence of glutamate spillover between boutons of different RGCs. The results of my studies indicate that far more RGCs contribute to relay neuron firing than would be predicted by the anatomy alone. Finally, in an ongoing study, we investigated the functional role of the neuronal glutamate transporter GLT-1 at the immature retinogeniculate synapse. While GLT-1 has been found in both neurons and glia, excitatory currents at the retinogeniculate synapse were largely unaffected in mice lacking neuronal GLT-1, suggesting non-neuronal glutamate transporters are responsible for the majority of glutamate removal from the developing synapse. Taken together, these results provide insight into the synaptic environment of the developing retinogeniculate synapse and identify a number of mechanisms that shape excitatory transmission during this period of synaptic maturation and refinement.

No description available.