Katumi Sumikawa, PhD - US grants

A complete functional nicotinic acetylcholine receptor (AChR) consisted of five subunits of four different types arranged as alpha2, beta, gamma and delta. Each of the four subunits is coded by a separate and discrete mRNA. Thus, the synthesis of a complete functional AChR molecule involves complex processes including assembly of the four different types of subunits in a particular order. The mechanisms involved in a multi-subunit AChR assembly will be studied by expressing normal, and site directed mutant, AChR subunits from cloned cDNAs in various combinations in the large oocyte cells of the frog. Specific topics to be investigated include: (1) The requirement of post-translational modifications for assembly and membrane insertion. (2) The order in which different subunits are put together. (3) Identification and localization of incomplete AChR molecules in the surface membrane. (4) The intracellular compartments in which AChR assembly occurs. (5) The presence of signals on the AChR subunits required for assembly. This research should substantially improve our understanding of the mechanisms involved in the assembly and membrane insertion of the nicotinic AChR in particular and of multi-subunit membrane proteins in general.

Desensitization is an intrinsic property of many neurotransmitter receptors, characterized by a decline in response to maintained activation by neurotransmitters and by changes in affinity to agonists and a variety of drugs. Although the phenomenon of desensitization has long bene known, its significance in normal and disordered brain function, as well as in learning and memory is still unknown. The nicotinic acetylcholine receptor (AChR) has been used widely to study desensitization, and it is known that phosphorylation at a specific site on a particular subunit is critical, or whether an increase in overall level of phosphorylation is more important in modulating the AChR desensitization. Evidence also suggests that Ca2+ ions modulate the AChR desensitization. The goal of this research is to understand the mechanisms involved in desensitization. To pursue this goal, I will use the AChR as a model system. The initial aim is to identify phosphorylation sits on the particular receptor subunits which are responsible for determining the rate of desensitization. The studies will be made by eliminating potential phosphorylation sites on the AChR at the DNA level, and then expressing site directed mutant AChRs in Xenopus oocytes. The large size of these cells facilitate many procedures including microinjection, voltage-clamp recording of ACh-activated currents, and manual isolation of the surface membrane. The interaction of Ca2+ with the phosphate groups and negatively charged amino acids, introduced at phosphorylation sites by site directed mutagenesis, is the AChRs in oocytes will also be studies by internal injection of EGTA or Ca2+. Subsequently, I will focus on the identification and characterization of the regions of the alpha, gamma and epsilon subunits of AChRs involved in controlling the rate of desensitization. Extrajunctional AChRs of mammalian muscles expressed in oocytes appear to desensitize more slowly than Torpedo AChRs, also synthesized in oocytes. This appears to be a consequence of the structural differences in the gamma subunits of the respective AChRs. Similarly, junctional muscle AChRs expressed in Xenopus oocytes, in which the gamma subunit is replaced by the epsilon subunit, desensitize more rapidly than extrajunctional AChRs. Furthermore, the hybrid AChR consisting of the muscle alpha and Torpedo beta, gamma and delta subunits desensitizes much faster than the normal Torpedo AChR. By contrast the hybrid AChR formed by the Torpedo alpha and muscle, beta, gamma and delta subunits desensitizes more slowly than the normal muscle AChR. Therefore, the alpha, gamma and epsilon subunit cDNAs will be very important tools to study the mechanisms involved in desensitization, chimeric alpha, gamma or epsilon subunit cDNAs with different combinations of Torpedo and muscle counterparts will be made and expressed in oocytes.

DESCRIPTION (provided by applicant): The long-term objective of this research is understanding how long-term nicotine use and subsequent nicotine withdrawal alter the normal functioning of synapses in the hippocampus. Cigarette smoking and acute and chronic administration of nicotine can enhance cognitive function, a property that has been linked with the continued use of cigarettes. Our studies have demonstrated that acute and chronic nicotine exposure facilitate the induction of N-methyl-D-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP; considered to be a cellular substrate for learning and memory) in the hippocampus. Following nicotine withdrawal, however, the threshold for LTP induction fluctuates and nicotine is no longer effective in facilitating the induction of LTP. These nicotine effects may represent the cellular basis of nicotine-mediated cognitive enhancement and unpleasant withdrawal symptoms, contributing to cigarette-seeking behavior. Nicotine enhances NMDAR responses in pyramidal cells via three different pathways, two involving disinhibition of pyramidal cells and the other involving activation of muscarinic acetylcholine receptors (AChRs). Our working hypothesis is that long-term nicotine exposure and withdrawal differentially alter these pathways, affecting NMDAR responses and thereby LTP induction. The proposed experiments will test this hypothesis by identifying altered pathways in hippocampi from chronic-nicotine-treated and withdrawn rats with combinations of electrophysiological, histochemical, and molecular biological approaches. The specific aims are to determine: (1) which pathways of nicotine action, leading to the enhancement of NMDAR responses, are altered, (2) if the normal functioning of a7 and non-a7 nicotinic AChRs on GABAergic interneurons are affected, (3) if feedforward GABAergic inhibition is altered, and (4) if muscarinic AChR-mediated signaling is affected. Results from these studies are expected to provide significant insights into mechanisms that underlie nicotine dependence, which may aid in the development of novel therapeutic strategies.

DESCRIPTION (provided by applicant): Smoking during pregnancy is an important public health problem associated with a wide range of adverse developmental effects. The offspring of smokers have an elevated risk of tobacco smoking, cognitive deficits, and impaired learning and memory during adolescence, but little is known about the mechanisms underlying these effects. Animal studies support the view that nicotine, the principle neuroactive component of tobacco, is responsible for these effects. The effects of nicotine are mediated by its interaction with nicotinic acetylcholine receptors (nAChRs). Thus, inappropriate stimulation of nAChRs is most likely responsible for the development of adverse effects during adolescence. During early postnatal development, 3-aminobutyric acid (GABA)ergic interneurons play a critical role in neural circuit formation. Our central hypothesis is that maternal nicotine exposure causes inappropriate GABA release in the hippocampus, a brain region associated with memory formation, via nAChRs, resulting in a long-lasting disturbance of circuit operation extending into adolescence. Hippocampal CA1 pyramidal cells, which provide the major output of the hippocampus, receive two major excitatory synaptic inputs either directly or indirectly from the neocortex. Nicotine modulates synaptic transmission and long-term potentiation (LTP;one form of synaptic plasticity considered to be a cellular substrate of learning and memory) induction in opposite directions at these pathways via activation of the 12 nAChR subtype. This subtype, the most sparsely expressed nAChR subtype in the brain, shows a distinct localization in a subset of GABAergic interneurons in the hippocampus and its activation also modulates LTP induction in these interneurons. These observations suggest that this subtype is an important component in hippocampal circuitry involved in cognitive function. Because this nAChR subtype is continuously activated in the presence of nicotine, maternal nicotine-induced adverse effects may be due to altered functioning of 12* nAChR-expressing interneurons. The goal of this project is to determine whether maternal nicotine exposure influences the functioning of 12* nAChR-expressing interneurons, affecting normal circuit operation and, therefore, information processing in the hippocampus. To achieve this goal, we will deliver a chronic nicotine dose during early postnatal development, and subsequently examine synaptic functioning in hippocampal slices prepared from the rats at various developmental stages, using electrophysiological and optical recording techniques, as well as morphological and molecular biological techniques. The specific aims are to determine whether maternal nicotine exposure affects: 1) the expression of 12* nAChRs and the number of 12* nAChR- expressing interneurons, 2) the operation of circuits, 3) the operation of inhibitory circuits, 4) the nicotinic modulation of N-methyl-D-aspartate receptor responses in pyramidal cells, and 5) the induction of LTP in 12* nAChR-expressing interneurons and at the two major excitatory synapses. Results from these studies will help determine not only the cellular basis of maternal smoking-induced cognitive impairments, but may also aid in the development of an effective prevention against maternal smoking-induced cognitive impairments by targeting the 12 nAChR subtype. PUBLIC HEALTH RELEVANCE: Maternal smoking during pregnancy elevates the risk of attentional and cognitive deficits during adolescence. The proposed experiments will test the hypothesis that maternal nicotine exposure causes inappropriate stimulation of nicotinic acetylcholine receptors in the hippocampus, a brain region associated with memory formation, which results in a long-lasting disturbance of neural circuit operation, and therefore affecting the mechanisms underlying learning and memory during adolescence. Results from the proposed experiments will help determine the cellular basis of maternal smoking-induced cognitive deficits in children.

DESCRIPTION (provided by applicant): Smoking during pregnancy is an important public health problem associated with a wide range of adverse neonatal and developmental effects. The offspring of smokers display attentional and cognitive deficits and impaired learning and memory, but little is known about the potential mechanisms of these effects. Animal studies support the view that nicotine, the principle neuroactive component of tobacco, is responsible for these effects. The effects of nicotine are mediated by its interaction with nicotinic acetylcholine receptors (nAChRs). Thus, inappropriate stimulation of nAChRs is most likely responsible for the development of synaptic and behavioral deficits later in life. During early postnatal development, GABAergic interneurons play a critical role in neural circuit formation. Our central hypothesis is that maternal nicotine exposure causes inappropriate GABA release in the hippocampus, a brain region associated with memory formation, via sustained activation of a specific nAChR subtype on interneurons, resulting in a long-lasting disturbance of circuit operation. Hippocampal CA1 pyramidal cells, which provide the major output of the hippocampus, receive two major excitatory synaptic inputs directly or indirectly from the neocortex. Nicotine modulates synaptic transmission and long-term potentiation (LTP;one form of synaptic plasticity and considered to be a cellular substrate of learning and memory) induction in opposite directions at these pathways via the activation of the a2 nAChR subtype. The expression of this subtype in GABAergic interneurons is upregulated during early postnatal development. The goals of the proposed project are to explore whether the sustained activation of the a2 nAChR subtype on GABAergic interneurons in fetal brains affects the functional development of hippocampal circuits. To achieve these goals, we will deliver a chronic nicotine dose during early development, and subsequently examine synaptic function in hippocampal slices prepared from adolescent wild-type and a2 knockout mice using electrophysiological and optical recording techniques. The specific aims are to determine whether maternal nicotine exposure affects: 1) synaptic transmission and LTP induction at the two pathways in an a2 nAChR-dependent manner, and 2) nicotinic modulation of synaptic transmission and LTP induction at the two pathways in an a2 nAChR-dependent manner. Results from these studies will help determine not only the cellular basis of maternal smoking- induced cognitive impairments, but may also aid in the development of an effective prevention against maternal smoking-induced cognitive impairments by targeting a2 nAChRs. The offspring of smokers display attentional and cognitive deficits and impaired learning and memory, but little is known about the potential mechanisms of these effects. During early postnatal development, GABAergic interneurons play a critical role in neural circuit formation. The proposed experiments will test the hypothesis that maternal nicotine exposure causes inappropriate GABA release in the hippocampus, a brain region associated with memory formation, via sustained activation of a specific nicotinic acetylcholine receptor (nAChR) subtype on interneurons, resulting in a long-lasting disturbance of circuit operation. This project will help determine not only the cellular basis of maternal smoking-induced cognitive impairments, but also may aid in the development of an effective prevention against maternal smoking-induced cognitive impairments by targeting a specific nAChR subtype.

Nicotine abuse is one of the major health problems in the world today. The use of nicotine via cigarette smoking forms lifelong memories that are recalled in response to environmental cues associated with previous nicotine use. This recall increases drug craving and is a factor for the continued use of nicotine. Understanding how the long-lasting memories are formed may aid in developing therapies for preventing the relapse, and thereby promote smoking cessation. Increasing evidence indicates that nicotine produces addictive behavior by causing long-lasting changes at synapses. The long-term objective of this project is to determine how nicotine promotes long-lasting changes at synapses in the hippocampus, a brain region associated with memory formation. The behavioral and cellular effects of nicotine are mediated by its interaction with nicotinic acetylcholine receptors (nAChRs) that are normally activated by the neurotransmitter acetylcholine. When nicotine is administered systemically, it is slowly delivered to the brain where it bathes neurons for a relatively long time. Under these conditions, some nAChR subtypes are continuously activated, whereas other subtypes are inactivated by desensitization. We hypothesize that these effects of nicotine set up a condition, which promotes long-lasting changes at synapses. The specific aim of this proposed research is to determine whether continuous activation of a distinct nAChR subtype in the hippocampus during nicotine exposure modulates circuit operation to facilitate different forms of synaptic plasticity. The proposed research will be carried out using electrophysiological and optical recording techniques, as well as morphological techniques. Animals lacking a particular nAChR subtype will also be used. Results from these studies will help determine not only the cellular basis of nicotine-mediated enhancement of learning and memory, but may also aid in the development of effective treatments for long-lasting memory of nicotine abuse, by identifying a nAChR subtype involved in the effects of nicotine.

DESCRIPTION (provided by applicant): Nicotine addiction is a major social and health problem in the world today. Nicotine produces extremely stable changes in the brain that underlie long-lived addictive behavior. The long-term objective of this research is to understand how nicotine produces such long-lasting neural changes in the hippocampus, a brain region involved in the formation of memories and the context-dependent learning of drug-associated behaviors. Such knowledge will likely lead to a better understanding of addiction processes and may aid in developing therapies for preventing the relapse, and thereby promoting smoking cessation. Increasing evidence indicates that addictive drugs such as nicotine produce addictive behavior by causing long- lasting modifications of synapses. The N-methyl-D-aspartate receptor (NMDAR) is critical for such long-lasting modifications of synapses. Our study shows that in vivo exposure to nicotine causes the enhancement of NR2B-containing NMDAR-mediated responses via tyrosine phosphorylation, leading to long-lasting long-term potentiation (LTP; considered to be a cellular substrate of learning and memory) in the hippocampus. The effect of nicotine on NMDAR is mimicked by anti-acetylcholinesterase drugs for Alzheimer's disease and prevented by coadministration of a muscarinic receptor antagonist. These observations suggest not only a common signaling pathway stimulated by cholinergic memory enhancing drugs, but also that NR2B-NMDARs are a point of convergence of cholinergic and glutamatergic pathways involved in learning and memory. We hypothesize that nicotine produces long-lasting neural changes by usurping this signaling pathway. The proposed experiments will test this hypothesis by elucidating the nicotine-induced signaling cascade that leads to NR2B-NMDAR-dependent long-lasting synaptic modifications. The specific aims are to determine the roles of: 1) nicotinic acetylcholine receptors in the proposed pathway, 2) acetylcholine and muscarinic receptors in the nicotine-induced enhancement of NR2B-NMDAR responses; 3) muscarinic receptor-Src tyrosine kinase signaling pathway in the effect of nicotine; and 4) nicotine-induced enhancement of NR2B-NMDAR responses in long-lasting synaptic modifications. The proposed research will be carried out using nicotine- and other drug- exposed animals with a combination of electrophysiological, molecular biological, and morphological approaches. In addition to muscarinic receptor knockout mice, recombinant adenovirus vectors will be used to alter the activity of proteins in the proposed signaling pathway. Results from these studies will not only help determine the cellular basis of nicotine-mediated neuroplasticity, but also aid in developing effective treatments for smoking cessation. Nicotine abuse is one of the major health problems in the world today. Increasing evidence indicates that addictive drugs such as nicotine produce addictive behavior by causing long-lasting neural changes in brain pathways subserving learning and memory. The goal of this project is to determine how nicotine produces such changes in the hippocampus, a brain region involved in context-dependent learning of drug-associated behaviors. Results from this study will likely lead to a better understanding of addiction processes and may aid in developing therapies for preventing the relapse, and thereby promoting smoking cessation.

Cigarette smoking during pregnancy can have severe impacts on the mental and physical health of offspring, including long-lasting impairments in IQ and memory. Still, as of 2010 an estimated 12.3% of expectant mothers in the United States continued smoking. Cigarette smoke has been shown to contain more than 8,000 chemicals, but among these nicotine is thought to be the leading neuroteratogen. Several studies using rodent models of perinatal nicotine treatment have demonstrated that exposure to nicotine during early development causes long-lasting deficits in learning and memory. However, the outstanding question in the field remains which cellular and molecular changes induced by nicotine underlie this cognitive impairment. The first two weeks of postnatal development of rodents, which is roughly equivalent to the third trimester of human development, is a critical time for neurogenesis and synaptogenesis in the hippocampus, a brain region associated with memory formation. In order to identify long-lasting cellular, molecular and circuitry changes in the hippocampus that may underlie nicotine-induced cognitive impairments, we use a model of early postnatal nicotine exposure in rodents that targeted this critical period of hippocampal development. We have tested our model to show that it results in impaired memory, and used electrophysiological, pharmacological and voltage sensitive dye imaging techniques to identify nicotine-induced changes in hippocampal function. We found several changes in hippocampal network activity, synaptic plasticity and nicotinic modulation of hippocampal function, all associated with the ?2 nicotinic acetylcholine receptor (nAChR) subtype, that could be the cause of memory deficits. The ?2* nAChR, the most sparsely expressed nAChR subtype in the brain, has long been ignored in the study of nicotine's central action. However, we have shown that this subtype, which is selectively expressed in GABAergic interneurons in the stratum oriens/alveus, is an important component in the hippocampal circuitry gating information flow and long-term potentiation (LTP; considered to be a cellular substrate of learning and memory). Furthermore, our results suggest the importance of ?2* nAChRs in hippocampus- dependent memory, and lay the foundation for further studies of the mechanisms underlying cognitive impairment after maternal smoking. In the research proposed here, we will test the hypotheis that developmental nicotine exposure, by inappropriately activating ?2* nAChRs, causes functional alterations of ?2* nAChRs-expressing interneurons, and that these changes could be tied to maternal-nicotine-induced hippocampal memory impairments. Our major goals are to determine whether the ?2* nAChR indeed plays a causal role in maternal-nicotine-induced memory impairments, and whether the adverse effects can be rescued by pharmacological and optogenetic manipulations of ?2* nAChR-expressing interneurons. Given the very limited expression of ?2* nAChRs in the hippocampus, determinating their role in nicotine-induced cognitive deficits could yield a unique and powerful therapeutic target for children whose mothers smoked in pregnancy.