Photoperiodic information is stored in the SCN by an alteration of the synchrony in the timing of (subpopulations of) neurons (Schaap et al., 2003; VanderLeest et al., 2007; Rohling et al., 2006a; Brown and Piggins, 2009). Although it is now clear that heterogeneity in phases exists within the SCN, the mechanism by which the multitude of cellular clock phases are synchronized to produce rhythms that are well adapted to the environmental light-dark cycles are yet unknown. Several neurotransmitters that are involved in synchronization are known and may play a role in the capability of the SCN to code for day length.
VIP
Vasoactive intestinal polypeptide (VIP) is thought to synchronize neighboring cells, acting upon the VPAC2 receptor (Aton et al., 2005;
Vosko et al., 2007; Brown et al., 2007). Cellular electrical activity rhythms are dampened when VIP signaling is absent but compensatory mechanisms (signaling through Gastrin Releasing peptide) may reduce the severity of the absence of this key signaling pathway. To investigate the possible mechanisms that enable the SCN to adapt to different photoperiods, it would be interesting to block synchronizing pathways such as VIP signaling in slices from different day lengths. In short days it can be hypothesized that absence of VPAC2 receptor signaling will result in a loss of synchrony
between SCN neurons. This will result in a wider MUA peak over
time, while different neurons have a slightly different τ, phase
differences will accumulate over time and cells become less synchronized after application of the VPAC2 receptor blocker.
We performed pilot experiments in VIP/PHI deficient mice, where there is no functional VIP signaling, but VPAC2 receptors are
cellular rhythms, enabling some of the animals to remain rhythmic in their behavior (Brown et al., 2007). During entrainment to a short day light-dark cycle the wheel running rhythms of VIP deficient mice showed adaptation to the day length. These wheel running rhythms could however be masked by the light-dark cycle. We therefore exposed the animals to constant darkness to detect the endogenous circadian behavior, but behavioral rhythms were hard to interpret because of an extremely positive phase angle and seemingly arrhythmic behavior. It was shown that slices from behaviorally arrhythmic animals also are arrhythmic in electrical multiunit activity in vitro (Brown et al., 2007). In our experiments, electrical activity in slices from VIP/PHI -/- was severely distorted and multiunit
recordings yielded only a single MUA peak, out of six slices, which was of very low amplitude. Based on these pilot experiments we are unable to determine the involvement of VIP signaling in day length encoding by the SCN. In an LD cycle, animals may seem rhythmic due to masking effects of the LD cycle, but entrainment is hard to asses. Furthermore, because of a high number of arrhythmic slices, the effect of VIP on the MUA peak cannot be determined by these methods. As an alternative strategy, we propose would perform in vitro experiments in slices from WT mice, entrained to a short day length and apply a blocker of the VPAC2
GABA
receptor to inhibit VIP signaling.
Another known synchronizing agent in the SCN is γ- aminobutyric acid (GABA) of which it is shown that it synchronizes the electrical activity between regions (Albus et al., 2005; Liu and Reppert, 2000; Belenky et al., 2008; Choi et al., 2008). In long days, the in vitro multiunit electrical activity patterns are broad, because of a broad phase distribution of single units. If GABA is synchronizing SCN neurons, application of a GABA agonist, which increases GABA signaling, will increase the level of synchronization when applied chronically. In behavioral experiments, the GABA agonist midazolam
was shown to acutely increase alpha, consistent with a short day activity profile (Vansteensel et al., 2003a). If GABAergic signaling on the other hand is blocked, a loss of synchrony will occur, leading to an increase in phase dispersion in the timing of neurons, which would lead to an increase in peak width. We entrained mice to a long and short day photoperiod and prepared hypothalamic slices containing the SCN and recorded electrical activity in vitro. We chronically applied 20 µM bicuculline in slices from short days and 100 nM midazolam in slices from long days to assess the effects of GABA signaling in the SCN on synchronization between neurons.
Our preliminary results show that chronic application of midazolam did not affect the peak width in slices from long days, indicating that chronic GABA activation does not induce an increased synchronization among the SCN neurons. In slices from short day in which we chronically blocked GABAergic signaling by application of bicuculline, we did not observe an increment in the duration of elevated electrical activity. We furthermore performed experiments in which we increased the concentration of potassium ions in the ACSF, which increases neuronal spiking activity and increases GABA signaling. No differences were found in the width of the multiunit peak. If GABA is essential for day length encoding, we would expect that at least in the slices from long day length a decrease in MUA peak width would be observed when a GABA agonist was applied. However, in all these experiments, we did not observe any changes in MUA activity relative to the control situation. This suggests that GABAergic activity is not involved in the temporal synchronization between neurons needed for photoperiodic encoding, although more experiments are needed to strengthen this conclusion. These results are in agreement with the finding that GABA signaling increases electrical rhythm amplitude and single cell rhythm precision, but does not synchronize between neurons (Aton et al., 2006). However it should be noted that those results were obtained in dispersed cell cultures rather than acute SCN slices. Furthermore, regional synchronization, perhaps by GABA, may play a role in photoperiodic
encoding in a manner we are not able to elucidate by means of stationary extracellular electrodes. The observed change in alpha after application of midazolam (Vansteensel et al., 2003a), mimicking the short day activity profile, may have been induced by GABAergic connections downstream of the SCN (Kalsbeek et al., 2008).
Taken together, our preliminary results do not yet indicate what mechanism in the SCN is responsible for tighter or loser synchronization, by which the SCN is able to code for day length. It is possible that a loss of synchronizing signaling does not lead to an increase in peak width, measurable in about 48h in vitro, since the starting point is a tightly synchronized system, and a lack of a synchronizing signal does not necessarily lead to an acutely desynchronized situation, but may take several cycles to become visible