1985 — 1991 |
Pilar, Guillermo R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Ganglionic Synaptic Transmission @ University of Connecticut Storrs
Two mechanisms involved in transmission at a ganglionic synapse will be studied: 1) the function of presynaptic autoreceptors, and 2) the release of transmitter by the neuronal cellbody. Both could be involved in modulating ACh secretion and action. These experiments will be done in the avian ciliary ganglion. The co-existence of substance P and enkephalines in this ganglion also provides the opportunity to analyze the postulated role of these peptides in neurotransmission. The interesting possibility of interaction between opiates and ACh autoreceptors in regulating ACh release will be examined. Neuronal soma release is a new concept in synaptic physiology. It may provide feedback regulation of presynaptic release or it may serve as a sefl-biasing device in regulating membrane excitability. Since these mechanisms involve ionically and chemically activated channels, a study with the membrane patch clamp technique will be initiated. One issue of particular interest will be the investigation of the stability of cholinergic channels in neurons and how this property is dependent upon the interaction of the neuron with its synaptic target and its synaptic input. Cholinergic neurons in the CNS are directly involved in movement, memory, learning, etc. Understanding ACh dynamics could be of importance in revealing mechanisms involved in these fundamental CNS functions, and this subject is indeed of clinical relevance because of the recent discovery that dysfunction and eventual death of a group of cholinergic neurons in the human brain may well be the primary cause of ALzheimer's Disease. The patch clamp technique is best used on a preparation where neuronal elements can be visualized, therefore a new "in vitro" preparation will be used. The presynaptic cell forming the bird's accessory nucleus will be grown in culture and caused to synapse with dissociated ciliary cells added to the culture. These preparations will open new avenues of inquiry such as direct study of calcium channels in presynaptic terminals and how they are regulated by neurotransmitters, membrane potentials, and second messengers.
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0.936 |
1995 — 1997 |
Pilar, Guillermo R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Ganglionic Synaptic Transmission Developmental Studies @ University of Connecticut Storrs
The objective of this proposal is to learn what mechanisms of cell death operate during in vivo development of the nervous system, as only the broadest outlines of the mechanism(s) of cell death are known in higher vertebrates. Naturally occurring cell death, as it occurs during development of the nervous system, has different morphological characteristics, nuclear or cytoplasmic, as we have previously described in avian ciliary ganglion (CG) neurons . We will investigate whether the different types of cell death are mediated by similar or different mechanisms, and will characterize the two morphological types of death during CG development in vitro (tentatively referred to as "apoptosis" and "necrosis"). We have developed a tissue culture system that mimics the conditions of target competition in vivo, and will compare the mechanisms operating in this system with those in neurons deprived o trophic support (CIPE, CNTF, and GPA) in vitro, and with cell death in the chick embryo. Perturbations of these mechanisms in vivo and in vitro will allow us to define and clarify the conflicting hypotheses that have been proposed regarding the mechanisms of cell death (apoptosis vs. necrosis). A novel aspect of this proposal is that we will study the cell death process using morphological, electrophysiological and molecular techniques. Furthermore, these techniques will be used in the same cell(s) at critical times (ie. measurements of intracellular Ca++will be made with Fura-2, followed by electrophysiological measurement of Ca++ currents and completed by histological observations of DNA fragmentation). We will characterize DNA degradation using agarose electrophoresis, and the TUNEL and COMET techniques, correlating these and ultrastructural changes with some of the functional mechanisms operating during cell death, such as ionic membrane permeability changes (Na+ and Ca++) and changes in intracellular Ca++ levels. We will then begin to characterize some of the genes that might be expressed, such as bcl-2, and which are perhaps required for survival in vertebrates. These questions are central to determining whether the neuronal degenerative processes occurring in such diseases as Alzheimer's and parkinson's is due to apoptosis (programmed death) or necrosis (death due to injury or toxicity). This information may be ued to develop new treatments or drugs to ameliorate the symptoms of these diseases which affect a large number of patients.
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