Rebecca Prosser - US grants

Daily or circadian rhythms controlled by endogenous clocks and synchronized to the solar cycle are characteristic of all organisms. The primary mammalian circadian clock, in the suprachiasmatic nuclei (SCN), survives in vitro where its phase can be monitored through its 24 hr rhythm of neuronal activity. Changes in the phase of this rhythm reliably reflect phase changes in the underlying clock. In the SCN, phase is modulated by afferents from the retina, intergeniculate leaflet of the lateral geniculate nucleus, and raphe nuclei. The primary neurotransmitters for these inputs are an excitatory amino acid (possibly glutamate), neuropeptide Y (NPY) and gamma-aminobutyric acid (GABA), and serotonin (5-HT), respectively. These afferents synapse primarily onto vasoactive intestinal polypeptide (VIP)-containing SCN cells, and possibly converge onto the exact same cells. Thus, in addition to individually modulating clock phase, it is likely that these afferent systems influence each others effects on SCN clock phase. While previous studies indicate that each of these afferent neurotransmitters can phase-shift the SCN pacemaker, both in vivo and in vitro, when applied individually, there is scant information concerning possible interactions between these inputs, and even less is known about the mechanisms through which these interactions might occur. This proposal represents the first phase of a broad investigation into afferent modulation of SCN clock phase. These experiments investigate 1. How 5-HT, NPY, GABA, glutamate, and optic chiasm stimulation affect the SCN in vitro when applied individually, determining in particular A) if and when they phase-shift the clock, and B) what their acute effects on SCN neuronal activity are when applied during the day and night; and 2. How 5-HT, NPY, glutamate, GABA, and optic chiasm stimulation modulate each other's effects in the SCN, investigating whether applying these stimuli together affects A) each other's pattern of phase-shifting, and B) their acute effects on the firing rates of SCN cells. This will be the first systematic investigation of interactions between SCN afferents in vitro, and as such it should provide critical information concerning the basic mechanisms underlying the mammalian circadian system. In addition, the increased understanding of how the SCN circadian pacemaker can be manipulated by external stimuli should produce rapid advances in our ability to alleviate problems that have been linked to circadian rhythm disorders, including sleeplessness, narcolepsy, and manic depression, as well as the medical and performance problems associated with jet lag and shift work schedules.

Daily or circadian rhythms controlled by endogenous clocks and synchronized to the solar cycle are characteristic of all organisms. The primary mammalian circadian clock in the suprachiasmatic nuclei (SCN), survives in vitro where it continues to produce 24 rhythms. SCN clock phase is modulated by afferents from the retina, intergeniculate leaflet of the lateral geniculate nucleus, and raphe nuclei. The primary neurotransmitters for these inputs are an excitatory amino acid (possibly glutamate), neuropeptide Y (NPY) and gamma-aminobutyric acid (GABA), and serotonin (5-HT), respectively. These afferents synapse onto vasoactive intestinal polypeptide (VIP)-containing SCN cells, and possibly converge onto the exact same cells. Thus, modulation of clock phase may involve an interaction among these afferent systems, possibly at the level of the VIP cells. I am currently investigating interactions between these afferent systems in terms of their ability to phase shift the clock and to modulate SCN cell firing. As part of this ongoing effort, I propose to investigate how these afferent neurotransmitters affect VIP release in the SCN in vitro. This will be the first systematic investigation of how these afferent systems affect VIP release in the SCN. As such it could provide critical information concerning how afferent signals are integrated within the SCN, and ultimately how they modulate SCN clock phase. The increased knowledge of how the SCN circadian pacemaker can be manipulated by external stimuli should produce rapid advances in our ability to alleviate problems that have been linked to circadian rhythm disorders, including sleeplessness, narcolepsy, and manic depression, as well as the medical and performance problems associated with jet lag and shift work schedules.

This is a proposal to provide equipment for an undergraduate neurobiology course and for undergraduate independent research involving neurobiology techniques. Each year the laboratory course will impact 12 students, with at least 2 more students able to use the equipment for independent research. An additional 171 students enrolled in other courses will also benefit through the equipment being used in laboratory exercises or lecture demonstrations. The course helps fill a significant gap in the undergraduate education at the University of Tennessee, Knoxville, in that there are no laboratory courses devoted to the rapidly growing area of neuroscience. The proposed course will compliment the lecture course, Foundations in Neurobiology (BCMB 415), currently being taught by the PI and Co-PI. The proposed laboratory course is an intensive semester-long analysis of the neural circuitry controlling feeding behavior in the snail, Lymnaea stagnalis. During the course, students will be exposed to a variety of neuroanatomical and electrophysiological techniques while they learn basic principles associated with the field of neurobiology. In addition to completing 5 formal exercises, students will also be given the opportunity to conduct their own hypothesis-driven experiments. Required written reports and an oral presentation of the results of their independent project will provide students with further training necessary for advancing in the field of neuroscience. Finally, lab exercises will be disseminated electronically via the development of a neurobiology laboratory page accessible via the World Wide Web.

[unreadable] DESCRIPTION (provided by applicant): The proposed experiments investigate the effects of ethanol on the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Alcohol abuse and withdrawal have pro profo found und effects on circadian rhythms and sleep. These two phenomena are likely interrelated, since disruptions in circadian functioning are a major cause of many sleep irregularities. Since sleep problems have been linked to both the development of alcoholism and the likelihood of relapse in recovering alcoholics, it is important to better understand the neural processes that contribute to these sleep problems. Known cellular targets of ethanol include glutamate N-methyl-D-aspartate (NMDA) receptor receptors. s. NMDA receptor activation in the SCN is necessary for the circadian clock to properly synchronize to the environment. Our preliminary data show that acute eth ethanol tre anol treatment blocks glutamate stimulation of the SCN circadian clock. We propose to follow up thes these preliminary experiments by pursuing the following specific aims: AIM 1: Does acute or chronic ethanol treatment affect photic phase shifts in vivo? Our preliminary data involve an in vitro preparation, and it is important to determine wheth whether ethanol has similar effects in vivo. Furthermore, given the many differences in acute vs. chronic ethanol exposure, we will also investigate how chronic ethanol treatment affects circadian rhythms in general, and photic phase shifting in particular. AIM 2: Does acute ethanol treatment in vitro block glutamate-induced phase shifts through direct or indirect actions on glutamatergic NM c NMDA receptors? These experiments will DA in investigate vestigate the mechanisms through which ethanol inhibits the phase-shifting effects of glutamate on the circadian clock. In this initial stage we AIM 3: Does acute ethanol treatment affect non-photic phase shifts in vitro? While many of ethanol's effects appear to center a around NM round NMDA receptors, other DA neuroturotransmitters, such as serotonin, are also affected by alcohol. Thus, we will investi investigate gate whether acute ethanol alters in vitro phase shifts induced by the neurotransmitter, serotonin. Future Directions: The results of these studies will provide the basis of a more extensive investigation into the effects of acute and chronic ethanol, and ethanol withdrawal, on the mammalian circadian clock. [unreadable] [unreadable] [unreadable]

SPECIFIC AIMS This proposal continues our investigation of ethanol effects on the mammalian circadian clock located in the suprachiasmatic nucleus (SCN). Alcohol use, abuse and withdrawal have profound effects on sleep and circadian rhythms. Alcohol effects on these two processes likely are closely intertwined since disruptions in circadian functioning are major causes of many sleep irregularities. The long-term goal of our research is to better understand the neural processes contributing to the etiology of these sleep problems, specifically focusing on the role circadian rhythmsOur collaborative research shows that ethanol inhibits photic phase resetting of the SCN circadian clock in vivo and in vitro. The objective of this RO1 proposal is to identify the cellular mechanisms underlying the acute effects of ethanol, and to expand our research to investigate the effects of chronic alcohol and alcohol withdrawal. The major advantage of our collaboration is our ability to interweave complementary in vivo and in vitro experiments that take advantage of the unique aspects of each approach. Our central hypothesis is that ethanol inhibits glutamate signaling in core SCN neurons, which over time leads to up-regulated glutamatergic activity. Our rationale for the proposed studies is that identifying the cellular mechanisms through which ethanol modulates the circadian clock will lead to new insights on treatments that may prevent or overcome this debilitating disease. Therefore, we proposed to pursue the following specific aims: Specific Aim 1. Determine time-dependent changes in ethanol effects on photic/ glutamate signaling in the SCN: acute vs. tolerance vs. chronic vs. withdrawal. These experiments will characterize the effects of ethanol on the SCN circadian clock through monitoring behavioral and electrophysiological (in vivo and in vitro) rhythms, and how these effects vary depending on the duration of ethanol exposure. Specific Aim 2. Determine the cellular mechanisms of ethanol actions in the SCN. These experiments will assess the effects of ethanol on in vivo SCN neuropeptide release patterns, and investigate the cellular mechanisms through which ethanol inhibits glutamate-induced phase resetting in vitro. Specific Aim 3. Determine how ethanol affects non-photic phase resetting. These experiments will assess the effects of acute and chronic ethanol, and ethanol withdrawal, on serotonergic, GABAergic, and NPYergic phase resetting in vivo and in vitro, and probe the mechanisms and locations through which these effects occur.