Rhythmic coupling among cells in the suprachiasmatic nucleus.

The circadian timing system regulates many aspects of an organism’s behavior and physiology. In mammals, the part of the nervous system responsible for most circadian behavior can be localized to a pair of structures in the hypothalamus known as the suprachiasmatic nucleus (SCN). Importantly, when SCN cells are removed from the organism and maintained in a brain slice preparation, they continue to generate 24-h rhythms in electrical activity, secretion, and gene expression (reviewed by Gillette, 1997). Previous studies suggest that the basic mechanism responsible for the generation of these rhythms is intrinsic to individual cells in the SCN (Welsh et al., 1995) and perhaps in other cell populations (Balsalobre et al., 1998). The core molecular mechanism driving these cellular oscillations appears to be a negative feedback loop operating at the transcriptional/translational levels (e.g., Reppert, 1998; Sangoram et al., 1998; Wilsbacher and Takahashi, 1998). There is also evidence that the cells within the SCN are coupled to one another and that this coupling is important for the normal functioning of the circadian system. In the brain slice, SCN cells are thought to show only weakly coupled electrical activity (Bouskila and Dudek, 1993). But this coupling is sufficient to maintain the expression of rhythmic electrical activity in the SCN population. In contrast, in dissociated cultures of rat SCN, it appears that gap junctions couple astrocytes but not neurons (Welsh and Reppert, 1996). In these conditions, individual SCN neurons continue to express 24-h rhythms in electrical activity, but the cells drift out of phase with each other (Welsh et al., 1995) so that the population can no longer function as a time-keeping system. One mechanism that would coordinate electrical activity is the presence of direct electrical connections between cells mediated by gap junctions. Gap junction channels form the basis of cell-to-cell electrotonic communication in the developing nervous system. These channels allow the passage of ions and other small molecules (up to 1 kD) between coupled cells and function to connect cells both electrically and metabolically. A gap junction channel is formed by two hemichannels, each composed of 6 connexin proteins. Connexins (Cx) are encoded by a multigene family of which Cx26, 32, 36, and 43 are the major isoforms expressed in the developing brain (e.g., Kumar and Gilula, 1996; Nadarajah et. al., 1997; Srinivas et al., 1999). Previous studies have found that Cx32 is typically expressed in neurons and oligodendrocytes, Cx43 is expressed in astrocytes, whereas Cx26 is restricted to nonneuronal cells in the ependyma and meninges. This distinction is not absolute as a previous study found that Cx43 was also expressed in a subpopulation of cortical neurons (Nadarajah et al., 1996). Nevertheless, Cx32 and perhaps Cx36 appear to be important for the formation of gap junctions in the central nervous system (CNS). Despite the recent progress made by the molecular description of this growing family of gap junction proteins, our understanding of the specific functions of these channels and their regulation is still limited. The present study utilized whole-cell patch-clamp electrophysiology with biocytin-filled electrodes to examine if direct connections exist between developing SCN neurons in a brain slice preparation. The passage of biocytin from one cell to another will be taken as evidence for the presence of these direct connections presumably mediated by gap junctions. Next, experiments were run to determine if the permeability of these connections, that is, the extent of dye coupling, was modulated by activity-dependent mechanisms. The possibility of a circadian rhythm in electrical connections between SCN cells also was examined by making a day—night comparison between dye coupling in SCN slices of animals maintained in a light—dark (LD) cycle and in constant darkness. Finally, immunocytochemical analysis was used to characterize the possible expression of the Cx32 protein in the SCN of postnatal animals.