Eric M. Mintz - US grants

The hypocretins (orexins) are a recently discovered neuropeptide family thought to play a role in the regulation of sleep, feeding, and neuroendocrine secretion. Neurons that manufacture hypocretins are located exclusively in and around the perifornical nucleus and lateral hypothalamus. Most behavioral studies to date involving hypocretins have focused on their effects on sleep and arousal or feeding. This proposal aims to examine a possible role for the hypocretins in the regulation of circadian rhythms. There is very dense hypocretin innervation of several brain regions known to be involved in the regulation of circadian rhythms, including the midbrain raphe nuclei and the intergeniculate leaflet of the thalamus. In addition, hypocretins are thought to be released directly into the ventricular system, raising the possiblilty that hypocretins could act in brain regions that don't display hypocretin-immunoreactive fibers. Since hypocretins appear to stimulate arousal /waking, and induced arousal and sleep deprivation are capable of shifting the phase of the circadian clock, this proposal will investigate hypocretin as a potential mediator of the effects of arousal state on circadian activity rhythms. Microinjections of hypocretin-1 and hypocretin-2 will be given into several brain nuclei or the lateral ventricle during the middle of the subjective day. This is the time of the circadian day when behavioral stimuli have maximal effects on circadian clock phase. These brain regions will include the suprachiasmatic nucleus, the intergeniculate leaflet, the dorsal raphe, the median raphe, and several other regions with dense hypocretin innervation. This will tell us whether hypocretin is capable of shifting the circadian clock. In addition, the neural pathways between the hypocretin-containing cells and the suprachiasmatic nucleus will be determined using a combination of anterograde and retrograde tracers. Overall, this proposal will begin the process of determining the mechanisms by which hypocretin may be involved in the regulation of circadian rhythms, and lead to greater understanding of interactions between sleep and circadian rhythmicity.

DESCRIPTION (provided by applicant): The suprachiasmatic nucleus of the hypothalamus (SCN) is the location of the primary circadian clock in mammals. This clock drives daily rhythms and behavior using both humoral and neural outputs. The clock receives numerous inputs from other brain regions, but the most prominent of these are glutamatergic projections from the retina, serotonergic projections from the midbrain raphe, and neuropeptide Y (NPY) containing projections from the intergeniculate leaflets of the thalamus. Photic information from the retina helps to synchronize the timing of the circadian clock with the environment, but these signals can be modulated by changes in the amount of serotonin or NPY being released in the SCN. This proposal focuses on using reverse microdialysis perfusion of drugs into the SCN region of the Syrian hamster to test specific hypotheses about the function of serotonin and NPY in the hamster circadian system. These hypotheses are: 1) Do serotonin and NPY inhibit both the phase delaying and phase advancing effects of light? 2) Does the sensitivity of the SCN to serotonin and NPY vary with circadian time, and 3) Are variations in sensitivity of the SCN to exogenous stimulation by serotonin and NPY driven by variation in endogenous stimulation levels? These hypotheses will be tested by infusing specific serotonin and NPY agonists and antagonists into the SCN region, and measuring how these injections alter the phase shifting effects of light. Finally, we will begin to examine the hypothesis that the effects of serotonin and NPY on circadian rhythmicity are mediated by GABA. The experiments of this proposal will lead to a greater understanding of how the serotonin and NPY neurotransmitter systems modulate circadian rhythms, and lead to future investigations of the functional relevance of these neurotransmitters in the normal function of the circadian system.

A grant has been awarded to Kent State University under the direction of Dr. Mintz for the purchase of two instruments dedicated to measuring cell-specific gene expression. The first instrument is a laser capture microscope, which allows researchers to extract individual cells or groups of cells from tissues in order to analyze their contents. The second instrument is an Affymetrix GeneChip instrument system, which will allow researchers to analyze the expression patterns of thousands of genes simultaneously from just a few cells extracted using the laser capture microscope. With these instruments, researchers can examine differences in gene activity between different types of cells that are all mixed together in one tissue. This is of critical importance in understanding how individual cells serve different roles within one tissue.
Many projects at Kent State in diverse areas of the life sciences will benefit from this instrumentation. Although these instruments are often used in genetic and molecular biology studies, we seek mainly to apply their power to ask questions in the areas of neuroendocrinology, reproductive physiology, environmental biology, and biochemistry. The Kent State Department of Biological Sciences has an ongoing collaboration with the departments of Physics, Mathematics, and Computer Science in the fields of computational neuroscience and bioinformatics, and these instruments will add many new possible directions for that collaboration.
In addition to the benefits to Kent State's research programs, this instrumentation will have a significant impact on the educational experience we can give our students. Experience with this modern equipment through individual research mentoring by faculty will strongly benefit both our graduate and undergraduate students, and significantly enhance their ability to obtain positions after completing their educations. In addition, the instrumentation will be incorporated into our summer cell and molecular biology workshop, used specifically to give formal training in techniques to interested students. Overall, this award will enhance the scientific productivity of both faculty and students at Kent State University and support new and exciting directions in research.

Biological rhythms in physiology and behavior, such as the sleep/wake cycle, are regulated by an internal biological clock located at the base of the brain in the hypothalamus. This clock (located in a brain region called the suprachiasmatic nucleus, or SCN) has a well-defined structure that has many features that are conserved across mammalian species. The ways in which the complexities of this brain region contribute to the function of organisms are not fully understood. The SCN appears to have regions devoted to processing sensory input and generating rhythmic output. Using a gene-expression based approach, this project aims to distinguish the functions of these subregions of the clock and how they contribute to the overall function of the organism. It is anticipated that the results of these experiments will reveal novel information about how the structure of the biological clock relates to its function, and how different portions of the clock carry out different functions. This project will significantly impact our current understanding of clock function, which has common mechanisms in nearly all animals. Gene expression data will be deposited in the National Library of Medicine's Gene Expression Omnibus where it will be available to other researchers and the public.

PROJECT SUMMARY Circadian rhythms are critical to the maintenance of good physical and mental health, and their disruption, such as that induced by night or rotating shiftwork, jet lag, or various pathological conditions, is known to negatively affect numerous health outcomes and an individual?s quality of life. While a variety of drugs, both medicinal and recreational, have been investigated for their impacts on biological rhythms, mostly with regard to the sleep/wake cycle, one class of drug that has not received much attention are the cannabinoids. This is a significant oversight, as recent data suggests that the endogenous cannabinoid system plays a significant role in regulating the master circadian clock located in the suprachiasmatic nucleus of the hypothalamus (SCN), thus making it likely that exogenous cannabinoids too would affect the clock. Unfortunately, little to nothing is known about how cannabinoids interact with the clock at the molecular, systems, or behavioral levels, which given recent changes to state laws that have resulted in a rise in the use of both medical and recreational marijuana, is a timely and important question that should be answered. The goals of this project are to examine how cannabinoids affect rhythmicity and to identify the neural substrates that mediate these effects, particularly the ability of the body to properly synchronize internal rhythms to external environmental cues. The proposed experiments are designed to test the central hypothesis that cannabinoid activity can alter the timing of behavioral circadian rhythms, and that this activity occurs through action on the SCN. The first aim of this study will be to determine the consequences of the loss of signaling through cannabinoid receptor 1 to the expression of behavioral circadian rhythms. The second aim will identify the molecular components of the cannabinoid system that are present in the SCN, and whether they are rhythmically expressed. The third and final aim will whether the lack of CB1 receptors in knockout mice have organizational effects on the brain that alter circadian clock function. With increased use of marijuana in the United States, it is critically important that we understand the biological mechanisms of the cannabinoid system to be able to evaluate potential impacts of cannabinoid use on human health. Evaluation of the contribution of cannabinoid signaling to circadian clock function will be an important step in this process.