CONFORMATIONAL CHANGES IN THE EXTRACELLULAR DOMAIN OF GLUTAMATE RECEPTORS

The family of membrane protein called glutamate receptors play an important role in the central nervous system in mediating signaling between neurons. Glutamate receptors are involved in the elaborate game that nerve cells play with each other in order to control movement, memory, and learning. Neurons achieve this communication by rapidly converting electrical signals into chemical signals and then converting them back into electrical signals. To propagate an electrical impulse, neurons in the brain launch bursts of neurotransmitter molecules like glutamate at the junction between neurons, called the synapse. Glutamate receptors are found lodged in the membranes of the post-synaptic neuron. They receive the burst of neurotransmitters and respond by fielding the neurotransmitters and opening ion channels. Glutamate receptors have been implicated in a number of neuropathologies like ischemia, stroke and amyotrophic lateral sclerosis. Specifically, the NMDA subtype of glutamate receptors has been linked to the onset of Alzheimer’s disease and the subsequent degeneration of neuronal cells. While crystal structures of AMPA and kainate subtypes of glutamate receptors have provided valuable information regarding the assembly and mechanism of activation; little is known about the NMDA receptors. Even the basic question of receptor assembly still remains unanswered. Therefore, to gain a clear understanding of how the receptors are assembled and how agonist binding gets translated to channel opening, I have used a technique called Luminescence Resonance Energy Transfer (LRET). LRET offers the unique advantage of tracking large scale conformational changes associated with receptor activation and desensitization. In this dissertation, LRET, in combination with biochemical and electrophysiological studies, were performed on the NMDA receptors to draw a correlation between structure and function. NMDA receptor subtypes GluN1 and GluN2A were modified such that fluorophores could be introduced at specific sites to determine their pattern of assembly. The results indicated that the GluN1 subunits assembled across each other in a diagonal manner to form a functional receptor. Once the subunit arrangement was established, this was used as a model to further examine the mechanism of activation in this subtype of glutamate receptor. Using LRET, the correlation between cleft closure and activation was tested for both the GluN1 and GluN2A subunit of the NMDA receptor in response to agonists of varying efficacies. These investigations revealed that cleft closure plays a major role in the mechanism of activation in the NMDA receptor, similar to the AMPA and kainate subtypes. Therefore, suggesting that the mechanism of activation is conserved across the different subtypes of glutamate receptors.

Long-term synaptic facilitation of tail sensorimotor connections in {\it Aplysia\/}

Long-term sensitization in Aplysia is a well studied model for the examination of the cellular and molecules mechanisms of long-term memory. Several lines of evidence suggest long-term sensitization is mediated at least partially by long-term synaptic facilitation between the sensory and motor neurons. The sensitization training and one of its analogues, serotonin (5-HT), can induce long-term facilitation. In this study, another analogue to long-term sensitization training has been developed. Stimulation of peripheral nerves of pleural-pedal ganglia preparation induced long-term facilitation at both 24 hr and 48 hr. This is the first report that long-term facilitation in Aplysia persists for more than 24 hr, which is consistent with the observation that long-term sensitization lasts for more than one day. Thus, the data support the hypothesis that long-term facilitation is an important mechanism for long-term sensitization.^ One of the major differences between short-term and long-term facilitation is that long-term facilitation requires protein synthesis. Therefore, the effects of anisomycin, a protein synthesis inhibitor, on long-term facilitation was examined. Long-term facilitation induced by nerve stimulation was inhibited by 2 $\mu$M anisomycin, which inhibits $\sim$90% of protein synthesis. Nevertheless, at higher concentration (20 $\mu$M), anisomycin induced long-term facilitation by itself, which raises an interesting question about the function of anisomycin other than protein synthesis inhibition.^ Since protein synthesis is critical for long-term facilitation, a major goal is to identify and functionally characterize the molecules whose mRNA levels are altered during the formation of long-term facilitation. Behavioral training or its analogues (nerve stimulation and 5-HT) increases the level of mRNA of calmodulin (CaM). Thus, the role of Ca$\sp{2+}$-CaM-dependent protein kinase II (CaMKII), a major substrate of CaM, in long-term facilitation induced by nerve stimulation was examined. KN-62, a specific CaMKII inhibitor, did not block either the induction or the maintenance of long-term facilitation induced by nerve stimulation. These data indicate that CaMKII may not be involved in long-term facilitation. Another protein whose mRNA level of a molecule was increased by the behavioral training and the treatment of 5-HT is Aplysia tolloid/BMP-1-like protein 1 (apTBL-1). Tolloid in Drosophila and BMP-1 in human tissues are believed to be secreted as a metalloprotease to activate TGF-$\beta.$ Thus, the long-term effects of recombinant human TGF-$\beta1$ on synaptic strength were examined. Treatment of ganglia with TGF-$\beta1$ produced long-term facilitation, but not short-term or intermediate-term facilitation ($\le$4 hr). In addition, TGF-$\beta1$ and 5-HT were not additive in producing long-term facilitation, which indicates an interaction between two cascades. Moreover, 5-HT-induced facilitation (at both 24 hr and 48 hr) and nerve stimulation-induced facilitation (at 24 hr) were inhibited by TGF-$\beta$ sRII, a TGF-$\beta$ inhibitor. These results suggest that TGF-$\beta$ is part of the cascade of events underlying long-term sensitization, and also indicate that a signaling molecule used in development may also have functions in adult neuronal plasticity. ^

Confocal analysis of synaptic connectivity of the rod pathway in the rabbit retina

The retina is a specialized neuronal structure that transforms the optical image into electrical signals which are transmitted to the brain via the optic nerve. As part of the strategy to cover a stimulus range as broad as 10 log units, from dim starlight to bright sunlight, retinal circuits are broadly divided into rod and cone pathways, responsible for dark and light-adapted vision, respectively. ^ In this dissertation, confocal microscopy and immunocytochemical methods were combined to study the synaptic connectivity of the rod pathway from the level of individual synapses to whole populations of neurons. The study was focused on synaptic interactions at the rod bipolar terminal. The purpose is to understand the synaptic structure of the dyad synapse made by rod bipolar terminals, including the synaptic components and connections, and their physiological functions in the rod pathway. In addition, some additional components and connections of the rod pathway were also studied in these experiments. The major results can be summarized as following: At the dyad synapse of rod bipolar terminals, three postsynaptic components—processes of All amacrine cells and the varicosities of S1 or S2 amacrine cells express different glutamate receptor subunits, which may underlie the functional diversity of these postsynaptic neurons. A reciprocal feedback system is formed by rod bipolar terminals and S1/S2 amacrine cells. Analysis showed these two wide-field GABA amacrine cells have stereotyped synaptic connections with the appropriate morphology and distribution to perform specific functions. In addition, S1 and S2 cells have different coupling patterns and, in general, there is no coupling between the two types. Besides the classic rod pathway though rod bipolar cells and All amacrine cells, the finding of direct connections between certain types of OFF cone bipolar cells and rods indicates the presence of an alternative rod pathway in the rabbit retina. ^ In summary, this dissertation presents a detailed view of the connection and receptors at rod bipolar terminals. Based on the morphology, distribution and coupling, different functional roles were identified for S1 and S2 amacrine cells. Finally, an alternative to the classic rod pathway was found in the rabbit retina. ^

Spectroscopic and functional investigations of the AMPA subtype of ionotropic glutamate receptors

Ion channels play a crucial role in the functioning of different systems of the body because of their ability to bridge the cell membrane and allow ions to pass in and out of the cell. Ionotropic glutamate receptors are one class of these important proteins and have been shown to be critical in propagating synaptic transmission in the central nervous system and in other diverse functions throughout the body. Because of their wide-ranging effects, this family of receptors is an important target for structure-function investigations to understand their mechanism of action. ^ α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are one subtype of glutamate receptors and have been shown to be the primary receptors involved in rapid excitatory signaling in the central nervous system. Agonist binding to the extracellular ligand binding domain of these receptors causes various conformational changes that culminate in formation of the ion channel. Previous structural investigations have provided important information about their mechanism of action, including uncovering a relationship between the degree of cleft closure in the binding domain and activation of the receptor. However, what question remains unanswered is how specific interactions between the agonist and the protein interplay with cleft closure to mediate receptor activation. ^ To investigate this question, I applied a multiscale approach to investigate the effects of agonist binding on various levels. Vibrational spectroscopy was utilized to investigate molecular-level interactions in the binding pocket, and fluorescence resonance energy transfer (FRET) was employed to measure cleft closure in the isolated ligand binding domain. The results of these studies in the isolated binding domain were then correlated to activation of the full receptor. These investigations showed a relationship between the strength of the interaction at the α-amine group of the agonist and extent of receptor activation, where a stronger interaction correlated to a larger activation, which was upheld even when the extent of cleft closure did not correlate to activation. These results show that this interaction at the α-amine group is critical in mediating the allosteric mechanism of activation and provide a bit more insight into how agonist binding is coupled to channel gating in AMPA receptors. ^

Depolarization-dependent synaptic potentiation and hyperexcitability induced by a Ca2+-independent trigger

Despite vast research efforts since Cajal's seminal thoughts on the adaptation of the nervous system, researchers have only recently begun to understand the diversity of forms of neuronal plasticity and its mechanisms. All known forms of activity-dependent neuronal plasticity utilize alterations in [Ca 2+]i as a signal of changes in the membrane voltage. Ca 2+ sensors trigger modifications in excitability or synaptic strength that last from seconds to weeks and presumably years. Intriguingly, Kunjilwar et al., (unpublished observations) discovered in peripheral sensory axons of Aplysia that the induction of depolarization-dependent long-term axonal hyperexcitability does not require Ca2+ transients. Here we show that induction of depolarization-dependent intermediate-term and long-term synaptic potentiation in Aplysia occurs in conditions that prevent Ca2+ entry through voltage-gated channels and elevation of [Ca2+]i. We found that the intermediate-term synaptic potentiation induced under conditions expected to prevent Ca 2+ transients is associated with increased excitability of sensory neuron axons near presynaptic terminals, suggesting that the synaptic potentiation involves a presynaptic locus. The Ca2+-independent intermediate- and long-term synaptic potentiation appeared similar to previously reported Ca2+-dependent modifications in Aplysia. ^

Catchment area of oral cleft cases for two major hospitals in Harris County

Background. Orofacial clefts are among the most common birth defects and considered to be of complex etiology with both genetic and environmental factors.^ Objectives. The purpose of this study was to describe maternal and infant characteristics, examine the catchment area, and determine if there are any geospatial patterns among infants with an orofacial cleft delivered at two major hospitals in Harris County, The Woman's Hospital of Texas and Memorial Hermann Hospital-Texas Medical Center, from January 1, 2003 through December 31, 2007.^ Methods. Data were obtained from two major hospitals in Harris County and included all babies delivered in the period from 2003 through 2007 with an orofacial cleft. Residential addresses were mapped using MapInfo GIS software and the cluster analysis performed with SaTScan software.^ Results. Ninety-nine cases were identified spanning nine counties. 26% of cases resided within a 5-mile radius of the Texas Medical Center. Birth rates ranged from 1.4 to 16.5 per 10,000 total births. A cluster was identified in southwest Harris County, however, it was not significant (p=0.066).^ Conclusion. This study encourages further focus on linking cleft cases to environmental factors in order to determine potential risks. ^

Spatial and temporal aspects of synaptic plasticity

The notion that changes in synaptic efficacy underlie learning and memory processes is now widely accepted even if definitive proof of the synaptic plasticity and memory hypothesis is still lacking. When learning occurs, patterns of neural activity representing the occurrence of events cause changes in the strength of synaptic connections within the brain. Reactivation of these altered connections constitutes the experience of memory for these events and for other events with which they may be associated. These statements summarize a long-standing theory of memory formation that we refer to as the synaptic plasticity and memory hypothesis. Since activity-dependent synaptic plasticity is induced at appropriate synapses during memory formation, and is both necessary and sufficient for the information storage, we can speculate that a methodological study of the synapse will help us understand the mechanism of learning. Random events underlie a wide range of biological processes as diverse as genetic drift and molecular diffusion, regulation of gene expression and neural network function. Additionally spatial variability may be important especially in systems with nonlinear behavior. Since synapse is a complex biological system we expect that stochasticity as well as spatial gradients of different enzymes may be significant for induction of plasticity. ^ In that study we address the question "how important spatial and temporal aspects of synaptic plasticity may be". We developed methods to justify our basic assumptions and examined the main sources of variability of calcium dynamics. Among them, a physiological method to estimate the number of postsynaptic receptors as well as a hybrid algorithm for simulating postsynaptic calcium dynamics. Additionally we studied how synaptic geometry may enhance any possible spatial gradient of calcium dynamics and how that spatial variability affect plasticity curves. Finally, we explored the potential of structural synaptic plasticity to provide a metaplasticity mechanism specific for the synapse. ^

Familial component of epilepsy in cleft lip and palate

It is well recognized that offspring of women with epilepsy who are taking anticonvulsant medications have an increased incidence of clefting abnormalities. This increase has been attributed to the teratogenic effects of anticonvulsant medications but an alternative explanation involving a genetic association of epilepsy and clefting has also been proposed. Five family studies attempting to resolve this controversy have been inconclusive either because of study design or analytic limitations. This family study was designed to determine whether epilepsy aggregates in families ascertained by an individual with a clefting disorder. The Mayo Clinic medical linkage registry was used to identify individuals with cleft lip with or without cleft palate and cleft palate in southeast Minnesota from 1935-1986. Only those cases who were 15 years or younger during this period were included in the study. The proband's parents and descendants of their parents, including the proband's sibs, children, grandchildren, niece/nephews, grandnieces/nephews, halfsibs and spouses were also identified and all of their medical records were reviewed for seizure disorders. The standardized morbidity ratios for epilepsy of 0.9 (95% CI 0.2-2.6) observed for first degree relatives (excluding parents) and 0.0 for second degree relatives were not increased. The SMRs ranged from 0.7-2.2 for the individual relative types (parents 1.5, sibs 0.7, children 2.2, probands 1.1, spouses 2.0) and were also not increased. These results do not support the suggestions of some that clefting and epilepsy aggregate together in families. ^

Spatial distribution of orofacial cleft birth defects and uranium-radium concentrations in tap water in Atascosa, Bee, Brooks, Calhoun, Duval, Goliad, Hidalgo, Jim Hogg, Jim Wells, Karnes, Kleberg, Live Oak, McMullen, Nueces, San Patricio, Refugio, Starr, Victoria, Webb, and Zavala Counties, Texas

Background Past and recent evidence shows that radionuclides in drinking water may be a public health concern. Developmental thresholds for birth defects with respect to chronic low level domestic radiation exposures, such as through drinking water, have not been definitely recognized, and there is a strong need to address this deficiency in information. In this study we examined the geographic distribution of orofacial cleft birth defects in and around uranium mining district Counties in South Texas (Atascosa, Bee, Brooks, Calhoun, Duval, Goliad, Hidalgo, Jim Hogg, Jim Wells, Karnes, Kleberg, Live Oak, McMullen, Nueces, San Patricio, Refugio, Starr, Victoria, Webb, and Zavala), from 1999 to 2007. The probable association of cleft birth defect rates by ZIP codes classified according to uranium and radium concentrations in drinking water supplies was evaluated. Similar associations between orofacial cleft birth defects and radium/radon in drinking water were reported earlier by Cech and co-investigators in another of the Gulf Coast region (Harris County, Texas).50, 55 Since substantial uranium mining activity existed and still exists in South Texas, contamination of drinking water sources with radiation and its relation to birth defects is a ground for concern. ^ Methods Residential addresses of orofacial cleft birth defect cases, as well as live births within the twenty Counties during 1999-2007 were geocoded and mapped. Prevalence rates were calculated by ZIP codes and were mapped accordingly. Locations of drinking water supplies were also geocoded and mapped. ZIP codes were stratified as having high combined uranium (≥30μg/L) vs. low combined uranium (<30μg/L). Likewise, ZIP codes having the uranium isotope, Ra-226 in drinking water, were also stratified as having elevated radium (≥3 pCi/L) vs. low radium (<3 pCi/L). A linear regression was performed using STATA® generalized linear model (GLM) program to evaluate the probable association between cleft birth defect rates by ZIP codes and concentration of uranium and radium via domestic water supply. These rates were further adjusted for potentially confounding variables such as maternal age, education, occupation, and ethnicity. ^ Results This study showed higher rates of cleft births in ZIP codes classified as having high combined uranium versus ZIP codes having low combined uranium. The model was further improved by adding radium stratified as explained above. Adjustment for maternal age and ethnicity did not substantially affect the statistical significance of uranium or radium concentrations in household water supplies. ^ Conclusion Although this study lacks individual exposure levels, the findings suggest a significant association between elevated uranium and radium concentrations in tap water and high orofacial birth defect rates by ZIP codes. Future case-control studies that can measure individual exposure levels and adjust for contending risk factors could result in a better understanding of the exposure-disease association.^

FZD6, MATN2 AND SLC25A32, POSSIBLE CANDIDATE GENES IN NONSYNDROMIC CLEFT LIP AND PALATE

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect with a multifactorial etiology. Despite decades of research, the genetic underpinnings of NSCLP still remain largely unexplained. A genome wide association study (GWAS) of a large NSCLP African American family with seven affected individuals across three generations found evidence for linkage at 8q21.3-24.12 (LOD = 2.98). This region contained three biologically relevant candidate genes: Frizzled-6 (FZD6) (LOD = 2.8), Matrilin-2 (MATN2) (LOD = 2.3), and Solute Carrier Family 25, Member 32 (SLC26A32) (LOD = 1.6). Sequencing of the coding regions and the 5’ and 3’ UTRs of these genes in two affected family members identified a rare intronic variant, rs138557689 (c.-153+432A>C), in FZD6. The rs138557689/C allele segregated with the NSCLP phenotype; in silico analysis predicted and EMSA analysis showed that the 138557689/C allele creates new DNA binding sites. FZD6 is part of the WNT pathway, which is involved in craniofacial development, including midface development and upper lip fusion. Our novel findings suggest that an alteration in FZD6 gene regulation may perturb this tightly controlled biological pathway and in turn contribute to the development of NSCLP in this family. Studies are underway to further define how the rs138557689/C variant affects expression of FZD6.

THE FUNCTIONAL ORGANIZATION OF SYNAPTIC VESICLE POOLS IN A RETINAL BIPOLAR NEURON

Ribbon synapses are found in sensory systems and are characterized by ‘ribbon-like’ organelles that tether synaptic vesicles. The synaptic ribbons co-localize with sites of calcium entry and vesicle fusion, forming ribbon-style active zones. The ability of ribbon synapses to maintain rapid and sustained neurotransmission is critical for vision, hearing and balance. At retinal ribbon synapses, three vesicle pools have been proposed. A rapid pool of vesicles that are docked at the plasma membrane, and whose fusion is limited only by calcium entry, a releasable pool of ATP-primed vesicles whose size also correlates with the number of ribbon-tethered vesicles, and a reserve pool of non-ribbon-tethered cytoplasmic vesicles. However evidence of vesicle fusion at sites away from ribbon-style active zones questions this organization. Another fundamental question underlying the mechanism of vesicle fusion at these synapses is the role of SNARE (Soluble N-ethylmaleimide sensitive factor Attachment Protein Receptor) proteins. Vesicles at conventional neurons undergo SNARE complex-mediated fusion. However a recent study has suggested that ribbon synapses involved in hearing can operate independently of neuronal SNAREs. We used the well-characterized goldfish bipolar neuron to investigate the organization of vesicle pools and the role of SNARE proteins at a retinal ribbon synapse. We blocked functional refilling of the releasable pool and then stimulated bipolar terminals with brief depolarizations that triggered the fusion of the rapid pool of vesicles. We found that the rapid pool draws vesicles from the releasable pool and that both pools undergo release at ribbon-style active zones. To assess the functional role of SNARE proteins at retinal ribbon synapses, we used peptides derived from SNARE proteins that compete with endogenous proteins for SNARE complex formation. The SNARE peptides blocked fusion of reserve vesicles but not vesicles in the rapid and releasable pools, possibly because both rapid and releasable vesicles were associated with preformed SNARE complexes. However, an activity-dependent block in refilling of the releasable pool was seen, suggesting that new SNARE complexes must be formed before vesicles can join a fusion-competent pool. Taken together, our results suggest that SNARE complex-mediated exocytosis of serially-organized vesicle pools at ribbon-style active zones is important in the neurotransmission of vision.