The post-genomic era offers behavioural scientists unrivalled opportunities but also many pitfalls. The study of circadian biology is an example of where molecular and behavioural techniques have been effectively integrated to further our understanding of the underlying systems. Behavioural/lesion studies dating back 30 years first identified the suprachiasmatic nucleus (SCN) as the site of the principal mammalian ‘clock’. The identification of this ‘model’ system in turn led to the identification of six genes that form the basis of the molecular ‘clock’ and an understanding of the biochemical mechanisms that allow them to interact to form a biological oscillator.
However, the nature of the control mechanisms that act upon the SCN is poorly understood. Recently, we have demonstrated that the neuropeptide receptor VPAC2, when over-expressed in the mouse, leads to a distinctive behavioural phenotype. This was characterised by a rapid re-entrainment to shifts in the 12 h/12 h light/dark (LD) cycle and a short free running rhythm in constant darkness (DD). By contrast, VPAC2 receptor knock-out (Vipr2-/-) mice exhibited an increase in the variance of activity onset under a 12 h/12 h LD cycle (Wt 0.116 ± 0.013, Vipr2-/- 0.303 ± 0.059 h; P = 0.002) (data reported as means ± S.E.M. derived from ANOVA, n = 10 per group), were virtually arrhythmic in DD (Wt t 23.9 ± 0.04 h, Vipr2-/- undeterminable), and responded with a rapid onset of activity to short periods of darkness interpolated into the light phase unlike control animals (lag in activity onset: Wt 0.75 ± 0.09, Vipr2-/- 0.28 ± 0.06 h, P < 0.001). Parallel studies assessing the molecular rhythmicity of the SCN confirmed that the ‘clock’ was silent.
It is also equally important to confirm which behaviours have been left unaffected following genetic modification. For example, we confirmed that Vipr2-/- mice were able to learn a complex operant, visually guided task with which it was then possible to probe their visual competence. The performance of the knock-outs was found to be indistinguishable from wild-type littermates. Thus a general CNS depression and impairments in the primary visual system can be ruled out as explanations for the deficit in circadian control.
Within the series studies conducted with the over-expressing and knock-out mice, a number of issues arose. These illustrate some of the potential pitfalls in application of molecular techniques to the study of biological function. In this case the knock-out studies were performed using 129P2 stem cells as the basis for the construct. As should be the case the circadian phenotype of the 129P2 strain was also determined. This was found to be abnormal. Although the salient features of the behavioural phenotype were observed in F2 hybrids on a mixed 129P2/OlaHsd X C57BL/6J background and were not seen in littermate controls, or in 129P2/OlaHsd mice, the KO strain was taken to isogenicity prior to final phenotyping.
These data illustrate that with careful hypothesis-driven experimental design, subtle phenotypes can be used to further elucidate the underlying mechanisms in particular functions. In this case peptidergic intercellular signalling, probably by VIP through the VPAC2 receptor, appears to be implicitly involved in normal circadian control.
I would like to acknowledge the contributions of Professor A.J. Harmar and Dr M.J. Hastings to this body of this work.
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