Analysis of circadian mechanisms in the suprachiasmatic nucleus by transgenesis and biolistic transfection

Analysis of the cellular and molecular mechanisms that underlie the circadian pacemaker of the suprachiasmatic nuclei (SCN) requires in vitro preparations amenable to genetic manipulation that can provide dynamic measures of circadian activity in real time over multiple circadian cycles. This article focuses on the value of the SCN organotypic slice for such studies. Specifically, it describes the use of tissues from genetically modified mice in which the circadian promoter of the mPer1 gene is used to drive the expression of either firefly luciferase or destabilized green fluorescent protein optical reporters. Furthermore, we describe a procedure for biolistic (particle-mediated) transfection of SCN organotypic slices with fluorescent reporters that can be used to explore the cis-acting elements and trans-acting factors that control circadian patterning, and also the interactions between subpopulations of neuronal oscillators within the SCN assemblage. Suprachiasmatic nuclei (SCN) of the hypothalamus are the principal circadian pacemaker driving the sleep/wake cycle and the myriad behavioral and neurophysiological rhythms consequential to it (Reppert and Weaver, 2002). Moreover, SCN orchestrate the activity of local circadian oscillators in peripheral tissues and thereby maintain the internal temporal order that underpins physiological adaptation to the solar cycle (Hastings et al., 2003). Current molecular and cellular models of the circadian mechanism of the SCN are based heavily on inferences drawn from biochemical analysis of circadian factors in peripheral tissues and the properties of recombinant proteins expressed in cell lines. While very successful, this approach alone will not lead to an understanding of how the neurons of the SCN intrinsically generate a circadian signal, then synchronize as a population, and finally relay that signal to targets in the brain that ultimately maintain the sleep/wake cycle and control circadian activity in other brain regions. Therefore, more direct analysis of cireadian molecular events in SCN is required. Until recently, most studies of the SCN oscillator used electrophysiological measures to make inferences about the clockwork, usually in acute slice preparations. Long-term organotypic slice cultures (House et al., 1998) offer considerable advantages over these acute preparations and, in several cases, have yielded important insights into cellular events underlying the circadian cycle of electrical firing (Herzog et al., 1997; Ikeda et al., 2003; Nakamura et al., 2001). Moreover, advances in real-time optical imaging techniques now make it possible to observe not only electrical functions in the SCN, which are downstream of the core clockwork, but also key molecular events at the heart of the circadian oscillator. Critical to the success of these approaches has been the development of genetically modified mice and rats bearing optical reporter genes (Kuhlman et al., 2000; Yamaguchi et al., 2001; Yamazaki et al., 2000; Yoo et al., 2004). This approach will be extended further by the refinement of biolistic transfection techniques to introduce novel reporter constructs into SCN neurons in slice culture to achieve acute genetic modification of such neurons (Ikeda et al., 2003; O'Brien et al., 2001).