THE ROLE OF GLUTAMATE IN AXONAL PHYSIOLOGY
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The information within the spinal cord and peripheral nervous system propagates along the nerves in the form of Compound Action Potentials (CAP). Although CAPs were always considered to be steady signals, with constant amplitude and velocity as determined by the conductive properties of the nerves our data show that exogenous glutamate increased the CAP. This increase in CAP was blocked after the addition of the general glutamate receptor antagonist kynurenic acid, the specific glutamate receptor antagonist MK801 and CNQX and prevented when these experiments were performed in a calcium-free medium. The goal of this thesis was to examine the changes in axonal physiology in response to electrical stimulation and to pharmacological manipulation. We found that high frequency stimulation, or addition of exogenous glutamate (100 µM) increases the amplitude of compound action potentials (CAPs) in sciatic nerve preparations. These results were further extended and supported by immunohistochemical experiments showing that axolemma contains glutamate receptors (NMDA, AMPA/kainate and mGluR2), the excitatory amino acid transporter responsible for glutamate uptake (Excitatory Amino Acid Transporter-EAAT), and voltage-gated sodium and calcium channels. Thus, the axolemma of peripheral nerves expresses several proteins important for neuronal communication and modulation of the membrane excitability. Apparently, these proteins embedded into the axonal membrane, can under the influence of electrical stimulation or exogenous glutamate change membrane permeability and ionic conductance leading to an increase in the amplitude of the compound action potentials observed in our experiments. Our results demonstrate of existence of axonal plasticity expressed as a change in the amplitude of the action potential following periods of changed activity accompanied by release of neurotransmitters. Therefore we suggest a mechanism of the process whereby electrical stimulation leads to increased axonal activity and subsequent release of glutamate that through activation of the glutamate receptors results in changes in the amplitude of CAPs. We term this phenomenon as axonal plasticity, which would represent one of the forms of neuronal plasticity. Neuronal plasticity is defined as a treatment-induced change in the neuronal response in spite of unchanged strength of the test stimulation. This observation was long described as a property of central synapses and thought to be the basis of learning (Malenka, 1994). Axonal plasticity , would constitute exclusive property of the axon and could contribute together with synaptic plasticity to modification of the efficiency of neuronal connections. This type of plasticity would be fundamentally different from the synaptic plasticity expressed in CNS in the form of Long-Term Potentiation-LTP (Bliss and Collingridge, 1993), Long-Term Depression-LTD (Dudek et al; 1992), and Spike Timing Dependent Plasticity (STDP) (Markram et al., 1997) which has been intensively investigated for last several decades. We assume that high frequency electrical stimulation induces the release of glutamate from stimulated axons. Subsequent increase in the extracellular glutamate concentration would be responsible for observed increase in CAP. Increase in the amplitude of CAP may be a result of: An increase in the number of activated axons (recruitment), 2) and/or increase in the amplitudes of individual potentials generated by single axons. The mechanisms responsible for each of these changes are very different. In the case of recruitment one can suggest paracrine action of glutamate which released from group of axons would enhance the CAP of their neighbors. The increase in the action potential generated by individual axon could be due to a change in the threshold of this individual axon. Our novel data together with published results clearly indicate that in spite of prevailing notion about "all-or-nothing" property of the action potential, axons and action potentials are capable of conveying the information in an analog manner (Clark and Hausser 2006). Presented results convincingly demonstrate that the amplitude of subsequently generated action potentials can change in a way correlated with the frequency of stimulation, or pharmacological treatment. In both cases the change occurred gradually with each evoked action potential slightly larger than its predecessor. This indicates that the effect was building step by step as the intraaxonal mechanisms have been recruited to contribute to the final effect. We have also observed reduced latency and increased area of CAP after glutamate application. The most obvious explanation for both phenomena would be a recruitment of additional, fast conducting axons which would shorten the latency and increase the area of CAP. Simultaneously this would increase the duration of the entire CAP, as slower conducting axons which contributed to CAP before the treatment would be activated as well.
The Role of Striatal Neuropeptides on Glutamate and Methamphetamine-Induced Neurotoxicity in the Murine Brain
ABSTRACT THE ROLE OF STRIATAL NEUROPEPTIDES ON GLUTAMATE AND METHAMPHETAMINE-INDUCED NEUROTOXICITY IN THE MURINE BRAIN by Lauriaselle Afanador Adviser: Dr. Jesus A. Angulo
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The rising worldwide epidemic in addiction to methamphetamine (METH) and the well-documented neurological detriments it causes emphasizes the importance of elucidating the mechanisms by which METH causes widespread and prolonged damage. Also, METH's pathophysiology resembles a number of neurodegenerative diseases. Therefore a better understanding of the mechanisms involved would provide more effective therapeutic targets for the treatment of these neurological disorders.
METH toxicity is a complex interplay of various factors however a number of necessary components have been identified such as dopamine overflow (DA), glutamate signaling, and oxidative stress. Although METH-induced DA overflow is the initiating event, it is not the direct cause of damage. Oxidative stress is thought to be the mediator of METH toxicity and nitric oxide (NO) as a contributor.
We have found that substance P (SP) exacerbates METH-induced NO. Inhibition of SP signaling mitigated NO synthesis and conferred protection. Considering the role SP is playing in METH toxicity we wanted to investigate the role that other striatal neuropeptides play in these events, notably the inhibitory peptides neuropeptide Y (NPY) and somatostatin (SST).
We hypothesized that SP is augmenting NMDA signaling and thus magnifying NO production. Whereas NPY and SST would serve as a counteracting force thus dampening oxidative stress and conferring protection. Overall, our data demonstrated that SP does augment NMDA signaling as inhibition of the neurokinin-1 receptor (NK-1R) decreased NMDA-induced striatal cell loss. We found that SP was potentiating NMDA-induced NO production. Although the predominant source of NO was the inducible form of nitric oxide synthase (NOS).
In support of our hypothesis, NPY and SST proved to attenuate NO. Also, they were protective from METH-induced cell death although SST failed to protect DA terminals. However, an agonist for the NPY-Y2 receptor was successful in maintaining DA terminal viability. Of interest is that neither NPY nor SST modulated NMDA-induced NO or cell loss suggesting that their protective mechanism does not include modulation glutamate signaling within the striatum.
Dopamine-glutamate interaction in the actions of typical antipsychotic drugs
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Typical antipsychotic drugs (APD) are currently the most effective psychoactive agents for the treatment of schizophrenia. Studies suggest that besides their conventional action of blocking dopamine (DA) D2 receptors, these drugs also interact with glutamatergic N-methyl-D-aspartate (NMDA) receptors. In addition, blockade of DA D2 receptors is believed to result in DA cell depolarization block (DB) and movement disorders (catalepsy) in animals. Since it has been hypothesized that drug's antipsychotic potency may be predicted by its ability to produce DB and catalepsy, using CD rats in behavioral, microdialysis and receptor binding studies we investigated whether typical APD induce DB and catalepsy though action on the dopaminergic system, glutamatergic system, or through the interaction between the two systems. Focus of this project was on striatum (STR) and frontal cortex (FC), two brain regions implicated in the DA-glutamate interplay. Our behavioral results show that haloperidol, a potent APD and postsynaptic DA D2 receptor blocker is a strong catalepsy inducer. Receptor binding study showed that chronic administration of this drug caused a decrease in maximal binding at the NMDA receptors in STR and FC but no significant changes in the DA D2 receptor densities were seen in the two brain areas. In contrast, metoclopramide, another DA D2 receptor blocker but not an APD, within the therapeutic doses (5 mg/kg-10 mg/kg) did not produce catalepsy in experimental animals. The maximal binding parameters for DA D2 and NMDA receptors in STR and FC after repeated administration of metoclopramide were significantly elevated as compared to haloperidol. However, when animals were pre-treated with metoclopramide (10 mg/kg) it sensitized the brain to haloperidol and enhanced catalepsy. Additionally, our receptor binding studies showed that psychotomimetic agents, PCP and ketamine that cause schizophrenia-like symptoms have several-fold higher binding affinity at NMDA receptors as compared to DA D2 receptors, indicating that pharmacological effect of these drugs may be mainly mediated by blockade of NMDA receptors. Finally, studying the neurochemical mechanism for DA cell DB we saw a decrease in striatal DA release after chronic cocaine treatment compared to controls. In a series of follow-up experiments we compared the effect of low dose (0.5 mg/kg) haloperidol and high dose (3.0 mg/kg) haloperidol by acute injection to the chronic cocaine treated rats and to the control animals. Low dose haloperidol significantly increased straital DA release compared to respective controls, while the high dose haloperidol significantly reduced it compared to the low dose. On the other hand, high dose haloperidol drastically increased striatal DA release in chronic cocaine-treated rats compared to controls. These results suggest that the mechanism for catalepsy is based on the concurrent DA D2 receptor antagonism and activation of glutamatergic NMDA transmission. Similarly, the mechanism for DA cell DB is mediated through blockade of dopaminergic D2 receptors and stimulation of NMDA receptors. Thus, catalepsy as well as antipsychotic activity appears to be mediated through modifications of dopaminergic and glutamatergic transmissions.
Biodiversity and Ethnography of Tea Management Systems in Yunnan, China
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This study investigates biodiversity and cultural practices associated with tea (Camellia sinensis (L.) O. Kuntze; Theaceae) management systems in Yunnan Province of southwestern China. Surveys were conducted in smallholder communities of six sociolinguistic groups (Akha, Bulang, Han, Hmong, Lahu, and Yao) that manage tea resources in forests, agro-forests, mixed crop fields, and terrace gardens. Interviews were carried out between 2006 - 2010 to identify the influence of socio-economic and policy variables on tea production and consumption patterns. Ecological plot sampling and ethnobotanical inventories were employed to characterize the composition, structure, and uses of tea management systems. Tea leaf samples were randomly selected within each plot for: (1) video morphometrics to measure six shape and size attributes, (2) high performance liquid chromatography (HPLC) to quantify nine catechin and methylxanthine compounds and, (3) amplified fragment length polymorphisms (AFLP) molecular marker analysis to assess genetic diversity. Results indicate a relationship between the perceived value of a commodity and a change of management practices, ecological knowledge, and land use. Findings demonstrate how variable management practices result in the loss, conservation, or enhancement of plant species richness and genetic diversity, and how smallholders variably benefit from diversity in their agro-ecosystems. Plant species richness was found in the order agro-forest edge > forest > agro-forest > mixed crop field > terrace gardens. Statistically significant variation was found in morphological, phytochemical, and genetic characters between the different types of tea management systems. Morphological diversity was found in the order agro-forest > mixed crop field > forest > terrace gardens, whereas genetic diversity was found in the order mixed crop field > agro-forest > forest > terrace gardens. HPLC data show that tea samples from agro-forests and mixed crop fields had greater mean Total Catechin Content (TCC) and mean Total Methylxanthine Content (TMC) compared to forests and terrace gardens. Results further demonstrate that management, processing, and preparation methods are related to the phytochemical profile, anti-oxidant activity, and flavor of tea. This study provides useful baseline data to examine long-term change linked to expanded market integration and an engagement of ecosystem ecology with anthropology.
MAG does not Require NgR1, PirB or Sialic Acid Binding to Inhibit Neurite Outgrowth
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The Role of Gangliosides, NgR1, NgR2 and PirB Receptors in MAG Inhibition of Neurite Outgrowth by Najat Al-bashir Thesis Advisor: Dr. Marie T. Filbin Following injury, axons in the central nervous system (CNS) do not spontaneously regenerate, and this is due to several factors, one of which is the presence of myelin- associated inhibitors. There are three major myelin-associated inhibitors that have been identified, Nogo-66, myelin associated glycoprotein (MAG), and Oligodendrocyte myelin glycoprotein (OMgp). MAG is a member of immunoglobulin (IgG) super-family and contains 5 Ig-like domains in its extracellular domain. Like Nogo-66 and OMgp, MAG binds to a receptor complex consisting of NgR1- p75NTR-Lingo-1 to inhibit neurite outgrowth. MAG is also a sialic acid binding protein and specifically binds to gangliosides GT1b and GD1a. Recently, NgR2 was also shown to be a sialic acid- dependent binding receptor for MAG. Recently, paired immunoglobulin B (PirB) was also identified as a novel receptor for MAG, Nogo-66 and OMgp. Previously, we showed that the sialic acid binding activity of MAG is not necessary for its inhibitory effects. We mapped the sialic acid binding site on MAG to Arg 118 in the first Ig-domain. When this site is mutated, sialic acid binding is lost but MAG, when expressed by CHO cells, still retains its ability to inhibit neurite outgrowth. Also, we showed that a soluble form of MAG consisting of the MAG extracellular domain fused to the Fc portion of human IgG (MAG-Fc), and a truncated soluble form of MAG consisting iii only of the first three Ig-like domains (MAG (d1-3)-Fc), both bind to neurons in a sialic acid-dependent manner; however, only MAG-Fc inhibits neurite outgrowth. In addition, MAG mutated at Arg118 (MAG (R118A)-Fc), does not bind to neurons and could not inhibit neurite outgrowth. Recently, we mapped the inhibition site on MAG to Ig-domain 5, which is distinct from the sialic acid binding site. Others have reported that gangliosides are functional binding partners for MAG and are necessary for inhibition by MAG when expressed in immobilized membranes. They reported that neurons from mice deficient in the B1, 4-N-actylgalactosaminyltransferase (GalNAcT) gene, which lack all complex gangliosides including GT1b and GD1a, are not inhibited by MAG in immobilized membrane. Others have also shown that clustering gangliosides with antibodies in the absence of MAG is sufficient to inhibit neurite outgrowth via a mechanism engaging p75 NTR receptor. Here we show that clustering MAG (d1-3)-Fc can inhibit neurite outgrowth in neurons from wild type mice but not from GalNAcT deficient mice. We also show that MAG can inhibit neurite outgrowth independent of NgR1, PirB, and sialic acid binding. We show that neurons from GalNAcT deficient mice are inhibited by MAG as effectively as neurons from wild type mice. Also, we show that neurons from NgR1 deficient mice are inhibited by full length MAG and mutated MAG (MAG R118A) that cannot bind sialic acid residues. In addition, in the presence of PirB antibodies, both MAG- and mutated MAG (R118A)-expressing CHO were able to inhibit neurite outgrowth of neurons from NgR1 deficient mice and wild type mice. Taking all these results together, MAG interacts with another as yet unknown receptor(s), in addition to NgR's, PirB and sialic acid to inhibit neurite outgrowth.
Neural Effects of Exposure to the Environmental Chemical, Bisphenol A, During Development
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Exposure to Bisphenol A (BPA), an environmental chemical, has been linked to changes in physiology, neural development, and behavior. The focus of this study was to determine the effects of BPA exposure, during a short developmental window, on physiology, activity, anxiety, cognition, and neurochemistry. In prenatal study, dams were administered 100 mcg/kg/day orally, from gestational day 16 to parturition. Postnatal study pups received subcutaneous injection of 60 or 100 mcg/kg BPA from postnatal day 0 to 6. All pups were weighed, examined for evidence of vaginal opening, and, at adulthood, performed behavioral tasks measuring locomotor activity, anxiety, and visual and spatial memory. Brain monoamines were measured using high performance liquid chromatography in the postnatal group. Prenatal BPA contributed to low juvenile body weight in both sexes and adult overweight in male subjects. Hyperactivity and memory deficits were observed in both sexes of BPA treated subjects. Postnatal 100 mcg/kg BPA females experienced delayed vaginal opening, less anxiety behavior in elevated plus maze, and spatial memory impairments. BPA treated subjects of both sexes had increased norepinephrine and dopamine turnover in basolateral amygdala and hippocampus, areas which are implicated in anxiety and cognition, respectively. The data suggests that BPA exposure during perinatal life causes disruptions in physiology, behavior, memory and neurochemistry that persist to adulthood. In addition, postnatal effects of BPA may be mediated by alterations in central monoaminergic function.
The Vocal Behaviors of Captive North American River Otters (Lontra canadensis) Individual differences and shared repertoires
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The current information on the vocal repertoire of the North American River Otter is very limited. To date there have been no direct studies conducted on their repertories. In this study, I examined the vocal behavior of 12 captive river otters. The discriminant function analysis suggests that river otters have 4 distinct call types with 7 sub-call types and one call the whistle is unique to one group of pups. The results of the Kruskal-Wallis comparing acoustical structures shows strong evidence for the presence of individuality with some individuals showing greater differences in comparison to the others. I also examined the differences in sexes and age groups, and the results show that unique calls are present, and there are significant differences across groups when comparing acoustical structures. Finally, I examined the uses of vocalizations, and the results show a positive correlation between the duration, max frequency, and max power of the call and the arousal state of the individual producing the call. Specific call types also showed tendencies to be produced when the individual was in a particular interaction (asocial or social) and when in a particular arousal state.
The Ecology of the Woodlands of Central Park, New York City
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A quantitative ecological inventory was conducted in the 54.6-hectare (ha) urban woodlands of Central Park, New York City. Fifteen sites were selected and woody stems greater than or equal to one centimeter (cm) diameter were surveyed using the point-centered quarter transect method. Total area surveyed was 1.091 hectares. The survey tallied 1,271 stems from 82 species in 31 families and 50 genera. Stem diameters ranged from 1 cm to 218 cm. In terms of ecological dominance, Prunus serotina Ehrh. was the dominant taxon followed by Quercus rubra L. The largest trees were Quercus rubra, Prunus serotina, Morus alba L., Phellodendron amurense Rupr., Platanus occidentalis L., Liriodendron tulipifera L., Quercus palustris Münchh., Ulmus americana L., and Styphnolobium japonicum (L.) Schott, ranging in diameter from 100 cm to 218 cm. Lower diameter at breast height (DBH) quartile stem sizes were dominated by Acer platanoides L., Prunus serotina, Celtis occidentalis L. and Q. palustris. As a fully human-made park under continual management, these woodlands contain a high percentage of non-native and horticultural species. A survey of the biodiversity of the park, however, shows the significant role even a highly managed park can play in wildlife habitat. Invasive plants are a serious threat to native plants and wildlife habitat everywhere. Numerous invasive species are present in Central Park. This study evaluates management practices to control these species and makes further recommendations. It analyzes the potential of other non-native species, as well as native species, to become invasive. This study can help park managers decide which plants to highlight and preserve and which to manage and control.
The diet and foraging ecology of gray seals, Halichoerus grypus, in United States waters
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Once extinct in U.S. waters, there are now more than 7,000 gray seals (Halichoerus grypus) that breed and forage in the waters of Maine and Massachusetts. This is the first long-term study of the diet and foraging behavior of this species in its U.S. range. I used hard parts in 305 seal scats and 49 stomachs, and fatty acid profiles in 45 seal blubber cores, to 1) reconstruct the diet of gray seals in U.S. waters, and 2) investigate regional, temporal, and intraspecific variation in the diet. I compared species in the diet with those most abundant in the seals' range, as measured by bottom trawl surveys. I analyzed the tracks of 6 satellite-tagged seals, and asked which prey species were most abundant in areas where foraging activity occurred. I recovered a total of 3,798 otoliths, and 7,005 prey individuals from 34 prey taxa. Sand lance (Ammodytes spp.) dominated the diet by weight (53.3% of total) and number (66.3% of total). Sand lance, winter flounder (Pseudopleuronectes americanus), red/white hake (Urophycis spp.) and Atlantic cod (Gadus morhua) together made up 82% of the diet by weight. Cod comprised 6.4% of the diet by weight, although this varied seasonally. Fatty acid profiles were best able to classify seals by age (young-of-the-year pups vs. yearlings, Wilks-Lambda = 0.27, F25,19 = 2.07, p <0.054), suggesting that diet differences were most pronounced between these two groups. Consistent 2:1 ratios of 22:6n3 and 20:5n3 fatty acids occurred in seal blubber (10.12/5.00 = 2.02). These ratios are similar to those in smooth skate (Malacoraja senta, 20.87/10.02 = 2.08) and alewife (Alosa pseudoharengus, 15.04/7.48 = 2.01), indicating that these species were important in the diet. Seals consumed abundant species, and tracked interannual trends in sand lance abundance, but the diet could not be predicted from prey availability alone. Satellite telemetry of seals revealed area restricted search behavior and central place foraging activity in areas with high abundance of sand lance and winter flounder, and these taxa comprised over 72% of the diet estimated from scats.
Neuromodulatory and cytoprotective roles of zinc in the vertebrate retina
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There is increasing evidence that the role of Zn2+ in the central nervous system is more complex and widespread than originally thought. Chelatable Zn2+ is co-localized with glutamate in the terminals of mossy fiber hippocampal and first order retinal neurons. The co-release of Zn2+ with glutamate in a stimulation-dependent manner has been shown in the hippocampus and the distal retina, while the electrophysiological effects of photoreceptor-released Zn2+ suggest a neuromodulatory role at the first visual synapse. This dissertation examines the neuromodulatory and cytoprotective roles of zinc in the vertebrate retina. When endogenous Zn2+ release is chelated in a skate eyecup preparation, the photoreceptor-generated a-wave of the electroretinogram doubles. This treatment also depolarizes horizontal cells and enhances their light response, suggesting that in the absence of Zn2+ , tonic release of glutamate from photoreceptors onto postsynaptic neurons is increased. Live cell imaging demonstrates that Zn2+ is released from photoreceptor terminals following Ca2+ entry through synaptic voltage-gated calcium channels. In isolated photoreceptors from salamander, chelation of extracellular Zn2+ significantly increases Ca2+ entry at the terminal and this effect is abolished when voltage-gated calcium channels are blocked pharmacologically. In the skate, removal of retinal Zn2+ via intraocular injections of chelators severely damages the inner retina. In the absence of Zn2+ , the retina develops classic signs of glutamate excitotoxicity; cell and tissue swelling, pyknosis and spongy appearance of the inner plexiform layer. Similar tissue characteristics are observed with injections of kainate, a well-known and potent excitotoxic agent. Additionally, neurons in the ganglion cell layer become necrotic with either kainate or chelator treatments, suggesting they are particularly sensitive to overactivation of glutamate receptors. Taken together, these experiments show the importance of Zn2+ as a neuromodulatory agent at the first visual synapse, where control of glutamate release affects the transmission of the visual message and provides broad protection of the retina from excitotoxicity. Understanding the role of Zn2+ in the retina may provide novel insights into retinal diseases and contribute to our growing knowledge of zinc's important functions elsewhere in the CNS.