Charles N. Allen - US grants

DESCRIPTION (provided by applicant): Disturbances in circadian rhythms are known to contribute to a variety of diseases and to impair mental and physical performance. Within individual suprachiasmatic nucleus (SCN) neurons, circadian rhythms are generated by molecular clocks consisting of gene transcription feedback loops. Yet not all SCN neurons have rhythmic clock gene expression, a finding that raises the question of how neurons with clocks communicate with each other and with those neurons lacking clocks to generate circadian output. This lack of knowledge is a major impediment to understanding how cellular clocks govern circadian behavior. In mammals, these rhythmic and non-rhythmic neurons are organized into anatomical compartments in the SCN, which is the master circadian oscillator. These compartments differ in clock gene expression patterns, as well as in efferent and afferent neuronal connections. The long-term goal of our research is to characterize SCN compartments and their functional significance. Our current goal is to determine the synaptic signaling mechanisms of phenotypically identified neurons in a defined SCN compartment, the calbindin sub-nucleus (CBsn), which is known to be important for generating circadian locomotor behavior. Our hypothesis is that the generation of circadian outputs depends on the micro-circuitry within and between SCN compartments. To test this, we will use a unique combination of electrophysiological recording and immunohistochemical staining techniques to characterize synaptic transmission in the CBsn. Because we are using a defined cell population and a novel combination of techniques, we expect to be able to draw strong conclusions about the micro-circuitry of the CBsn. The specific aims of the proposal are: 1) Determine the circadian phase dependence of responses of CBsn neurons to optic nerve stimulation. 2) Characterize the regulation of action potential firing and synaptic transmission by vasoactive intestinal peptide (VIP) and pituitary adenyl cyclase activating polypeptide (PACAP). 3) Examine the responses of CBsn neurons to Transforming Growth Factor alpha (TGFalpha). 4) Determine whether gastrin releasing peptide (GRP) or VIP immunoreactive neurons fire action potentials in a circadian manner. Completion of these studies will lead to a better understanding of the cellular basis of circadian rhythms as well as the potential to better treat sleep disorders and other disturbances in the circadian clock.

DESCRIPTION (applicant's abstract): To maintain proper temporal coupling with the environment, neural systems have evolved that monitor changing conditions and communicate the information to the circadian clock. This biological clock senses day-night transitions via the retinohypothalamic tract (RHT), a direct glutamatergic projection from retinal ganglion cells to the suprachiasmatic nucleus (SCN). Activation of other afferent neural pathways can modify the light input signal. Presynaptic 5-HT1B, GABA-B, and neuropeptide Y receptors inhibit glutamate release from presynaptic RHT terminals and block light-induced phase shifts of circadian rhythms. The mechanisms of GABA-B, 5-HT1B, and neuropeptide Y receptor inhibition of glutamate release are not known. The inhibition of glutamate release by orphanin-FQ receptors at presynaptic RHT terminals will also be studied. How these multiple afferent inputs interact to regulate RHT activity has not been studied either in vivo or in vitro. Therefore, we have designed experiments using electrophysiological techniques to describe the cellular mechanisms presynaptic pathways use to modulate RHT activity and the environmental light signal. Slices of the hypothalamus containing the SCN with the optic nerve attached will be used to investigate the characteristics of glutamate release by recording evoked and spontaneous excitatory postsynaptic currents. The specific aims of the proposal are: 1) Identify the calcium channel subtypes that regulate the release of glutamate from presynaptic RHT terminals. 2) Determine the mechanisms coupling activation of GABA-B, 5-HT, and neuropeptide Y receptors with the inhibition of glutamate release from presynaptic RHT terminals. 3) Examine whether orphanin FQ receptors produce presynaptic inhibition of RHT terminals. 4) Investigate whether there are co-operative interactions between presynaptic neurotransmitter receptors regulating glutamate release from RHT terminals.

DESCRIPTION (provided by applicant): Disturbances in circadian rhythms contribute to a variety of diseases and impair mental and physical performance. Circadian rhythms in physiological and behavioral processes are generated by a molecular clock located in the suprachiasmatic nucleus. This clock receives environmental information from cues such as light and subsequently creates timing information that is sent to the rest of the organism. While the signaling pathways involved in this chain of information are poorly understood, evidence suggests a possible role for Ca2+ as a signaling molecule for both input to and output from the circadian clock. But the source of this Ca2+ is unknown. One model proposes that glutamate released from terminals of the retinohypothalamic tract activates NMDA receptors, which generate nitric oxide and release Ca2+ from ryanodine-sensitive stores. However, the release of Ca2+ from ryanodine-sensitive stores by glutamate or nitric oxide has not been directly demonstrated. NMDA receptor activation increases the nuclear Ca2+ concentration of SCN neurons. Nuclear Ca2+ regulates gene expression, including possibly clock gene expression. The long-term goal of this work is to characterize the functional properties of SCN neurons and how the circadian clock regulates these properties. This proposal will determine the roles that cytoplasmic and nuclear Ca2+ play as circadian input and output signals. Our central hypothesis is that changes in cytoplasmic and nuclear Ca2+ concentration are a critical step in light's regulation of the circadian clock. This proposal uses an innovative combination of fluorescent imaging techniques, cell culture, and electrophysiological recording techniques to study the regulation of Ca2+ in SCN neurons during different portions of the circadian day. This research will identify the early steps in the light-signaling pathway. The Specific Aims of the proposal are: 1) Identify the mechanisms and circadian regulation of the increase of the cytoplasmic Ca2+ concentration produced by activating NMDA and AMPA receptors. 2) Determine the mechanisms and circadian phase dependence of the increase in the nuclear Ca2+ concentration produced by NMDA receptor activation. 3) Determine the role that PACAP plays in regulating changes in the cytoplasmic and nuclear Ca2+ concentration induced by NMDA and AMPA receptor activation. 4) Investigate whether the peak of the cytoplasmic Ca2+ rhythm precedes the peak of action potential firing frequency rhythm in SCN neurons. Together, these experiments will identify the early steps in the light-signaling pathway, thus contributing to a better understanding of the cellular basis of circadian rhythms.

Summary A significant number of Americans work non-traditional schedules and suffer the adverse health effects of a disrupted circadian timing system. The master mammalian circadian clock, located in the suprachiasmatic nucleus (SCN) maintains the proper phase relationship between circadian clocks located in tissues throughout the body and entrains the circadian system to the environment. The SCN is composed of individual neuronal oscillators coupled by intercellular communication into a neural network that generates a robust and precise rhythm. The long-term goal of our research is to understand the intercellular signaling mechanisms that couple SCN neurons into a neural network that generates circadian rhythms. GABAergic neurotransmission is a fundamental component of the SCN neural network and changing the strength and polarity of postsynaptic GABA responses modifies the activity of the SCN, and ultimately circadian rhythmicity. GABA serves as a desynchronizing signal under equilibrium conditions and a synchronizing signal when the SCN neural network has been modified by environmental light. GABA acts on synaptic GABA(A) receptors to mediate fast signaling between SCN neurons and on extrasynaptic GABA(A) receptors to activate a tonic GABA(A) current that modulates the activity of individual SCN neurons and communicates the level of network activity to adjacent synapses. We hypothesize that two membrane transporter families play critical roles in the regulation of the circadian activity of GABA neurotransmission in the SCN. The GABA transporters GAT-1 and GAT-3 regulate the amount and duration of neurotransmitter GABA in the extrasynaptic space and the magnitude of the tonic current. In the SCN, the GABA transporters are only expressed in astrocytes suggesting that astrocytes play a vital, but as of yet undetermined role in regulating the physiological actions of GABA in the SCN network. In the adult SCN, GABA serves as both an inhibitory and excitatory neurotransmitter although the physiological significance of this change in the polarity of GABA neurotransmission remains unknown. The chloride cotransporters of the sodium-potassium-chloride (NKCC) and potassium-chloride (KCC) families control the intracellular Cl- concentration and the polarity and magnitude of the GABA(A) receptor-mediated currents. We propose that the circadian clock uses the intracellular second messenger systems WNK-SPAK kinases, Ca2+- activated kinases, and cyclic AMP-activated kinases to regulate the activity of the Cl- transporters. The goal of this application is to understand better the mechanisms regulating GABAergic signaling and how GABA- mediated signaling contributes to the generation of circadian timing signals in the SCN. To accomplish this goal, we will use single cell electrophysiological and imaging techniques together with transgenic mouse models to study GABAergic neurotransmission in identified SCN neurons. Enhanced knowledge of the intercellular signaling mechanisms mediated by GABA will increase our ability to manipulate the circadian clock and reduce the symptoms experienced by patients with circadian-based disorders.