Chapter Four - Neurophysiological Analysis of the Suprachiasmatic Nucleus: A Challenge at Multiple Levels

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Abstract

Understanding the neurophysiology of the circadian timing system requires investigation at multiple levels of organization. Neurons of the suprachiasmatic nucleus (SCN) function as autonomous single-cell oscillators, which warrant studies at the single-cell level. Combining patch-clamp recordings of ion channels with imaging techniques to measure clock gene expression and intracellular calcium has proven extremely valuable to study cellular properties. To achieve and maintain rhythmic activity, SCN neurons require sufficient stimulation (i.e., input) from surrounding cells. At the network level, SCN rhythms are robust and can be measured in vitro, for example, in brain slices that contain the SCN. These recordings revealed that the collective behavior of the SCN neuronal network is strongly determined by the phase dispersal of the neurons. This phase dispersal is plastic, with high synchronization in short photoperiod, desynchronization in long photoperiod, and antiphase oscillations in aging and/or continuous light. In vivo recordings are needed in order to study the SCN as part of a larger network (i.e., interacting with other brain centers) and to study the SCN's response to light. Interestingly, superimposed on the circadian waveform are higher frequency fluctuations that are present in vivo but not in vitro. These fluctuations are attributed to input from other brain centers and computational analyses suggest that these fluctuations are beneficial to the system. Hence, the SCN's properties arise from several organizational levels, and a combination of approaches is needed in order to fully understand the circadian system.

Introduction

Circadian rhythms are a fundamental property of living organisms. They have evolved early in evolution and are the consequence of an endogenous pacemaker or clock. Clocks evolved in response to the cyclic 24-h changes in the environment due to Earth's rotation around its axis. The evolutionary advantages of having an endogenous clock are clear, as they help the organism anticipate cyclic changes in the environment. In addition, many organisms also have seasonal rhythms in addition to a circadian rhythm. Both circadian and seasonal rhythms are under the control of overlapping biological mechanisms. In mammals, the central clock is located at the base of the brain in a structure called the suprachiasmatic nucleus (SCN), which sits directly above the optic chiasm. The SCN generates circadian rhythms and perceives external light information, thus enabling it to synchronize with both daily and seasonal cyclic changes. In this chapter, we will discuss and review the neurophysiological studies of the SCN.

The generation of biological rhythms is rooted at the molecular level—a negative feedback loop between clock genes and their protein products provides cell-autonomous oscillators. However, although these oscillators are cell-autonomous, they still require input in order to function properly. In the case of the SCN, it seems that most SCN neurons are part of a network and must be excited (i.e., activated) sufficiently to maintain their rhythm-generating capacity (Herzog et al., 2004, Welsh et al., 2010). Thus, the rhythm-generating molecular machinery is itself part of a larger network or loop. At the individual cell level, interactions between molecular machinery—as well as input from other cells—are required to obtain a functional single-cell oscillator. Additional properties of the circadian system arise at the SCN network level and these properties can be measured in vitro.

The SCN network interacts with other brain regions. Based on these interactions, which involve both afferent and efferent pathways, the SCN (i) becomes entrained to the external light cycle, (ii) is responsive to the animal's behavioral activity and sleep stages, and (iii) can function both as a circadian pacemaker and as a mediator of seasonal rhythms. To investigate the SCN in the context of a larger network, including the relevant afferent and efferent pathways, several in vivo recording methods have been developed.

In this review, we will discuss the analysis of the mammalian circadian system at each of these levels, and we will evaluate the currently available physiological approaches that have been applied at each level. In addition, we will use examples to discuss the insights that have been gained from studies performed at each level.

Section snippets

Part I: Clock Mechanisms at the Cellular Level

Following the initial discovery of clock genes and clock cells in both invertebrates and mammals, the study of the molecular mechanisms within circadian pacemaker cells became the new focus of circadian clock researchers in the 1990s. Within a relatively short time span of 10 years, the core molecular players in the cellular clock were identified, and—more importantly—the dynamics of feedback loops were discovered, explaining the underlying basis of the 24-h rhythm in clock gene expression.

Part II: The SCN as a Multi-Oscillator

The SCN is a heterogeneous structure containing many cell-autonomous oscillators (Antle and Silver, 2005, Welsh et al., 2010). Functional coupling between these neurons is essential for obtaining a reliable timing signal. It is perhaps extraordinary that this heterogeneous network can generate rhythms with such high precision, with day-to-day deviations on the order of only a few minutes. This high precision is achieved only at the level of the neuronal network and is not present at the level

Part III: In Vivo Electrophysiology Recordings from the SCN in Anesthetized and Freely Moving Animals

The properties of a circadian system are critically dependent upon interactions between the SCN and other structures. A clear example of this dependence is the phenomenon that photic entrainment—one of the circadian system's primary attributes—is entirely dependent upon the flow of information from intrinsically photosensitive retinal ganglion cells (ipRGCs) to the SCN. Thus, in vivo recording is currently the best system for studying the SCN as a component of a larger network with intact

Conclusions

In this chapter, we described several methodologies that are suitable for performing a neurophysiological analysis of the circadian system at multiple levels. It is important to take all levels into account as higher levels of organization contribute substantially to the basic properties of the SCN. While rudimentary oscillators are obtained at the single-cell level, robustness, precision, entrainment, and adaptations to the changing seasons are all examples of properties that arise at higher

Acknowledgments

We are grateful to Hester van Diepen, Jos Rohling, and Changgui Gu for their helpful comments. This work was supported by NWO/ZonMW grant (TOPGo 91210064) to J. H. M., NWO Complexity grant (645.000.010) to J. H. M., STW perspectief program ONTIME (12190) to J. H. M., and STW perspectief program ONTIME (12191) to S. M.

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