DISCUSSION

The first experiment shows that expression of the human insulin transgene in habenular neurons caused a specific increase in the amount of SWS and a decreased rebound for both states of sleep. Before discussing the possible mechanisms of these variations, it is interesting to take into consideration the results of the second experiment.

This experiment was designed to study appropriate control mice, i.e. transgenic mice with a modified 5'-flanking sequence leading either to expression of the transgene only in the pancreas (DELTA-258 mice) or to its total nonexpression (DELTA-58 mice). In these mice, the amount of SWS did not differ from that seen in the transgenic mice (DELTA-168) expressing human insulin in habenular neurons. We then asked how these results could be explained and whether the transgenic process (insertion of the transgene) alters the expression of other genes involved in sleep regulation; such a change has been previously described when the human interferon P gene was fortuitously inserted into the MAO-A gene, resulting in its inactivation by deletion of two exons and a resultant altered phenotype (Cases et al. 1995, 1996). Insertion of the human insulin gene does not cause a dramatic change in phenotype. However, it might possibly cause alterations in some candidate genes for sleep regulation recently localized on the mouse chromosome map (Tafti et al. 1997). Unfortunately, the precise localization and number of the insulin transgene copies in the mouse genome are still unknown.

Another explanation may be the strain into which the transgene was injected. In the original publication concerning these mice, the authors mentioned that the transgene was injected into the male pronucleus before the first division of the egg from an F2 hybrid C57BL/6 x CBA (Fromont-Racine et al. 1990). Thus, mice bearing the transgene have a genetic background that is a recombination of the B6 and CBA genomes. In terms of sleep, these two strains differ markedly. Several years ago, we demonstrated that all CBA sleep parameters were dominantly transmitted when these mice were crossed with C57 mice (Valatx et al. 1972), and it would therefore be possible that the sleep characteristics of the three transgenic lines were inherited from the CBA parent of the injected hybrid embryo. Two arguments support this hypothesis. Firstly, in CBA mice, during SWS, EEG recordings show high amplitude spindles (8-10Hz, 800 uV) that are characteristic of several strains derived from BALB/c mice (e.g. C3H and CBA) and dominantly transmitted to F1 hybrids (Valatx et al. 1974), whereas different sleep spindles are seen in B6 mice (12-15Hz, 400 uV); CBA-type spindles were recorded during SWS in several of our transgenic mice (Fig. 3). Secondly, comparison of the present results with previously obtained sleep data from CBA mice (Valatx et al. 1974) shows that the duration of SWS and PS sleep in CBA mice is very similar to that seen in the transgenic mice (Fig. 1B). However, the sleep rebound in transgenic mice was intermediate between that seen in B6 and CBA mice, suggesting that the regulatory mechanisms for rebound are inherited differently from those for spontaneous sleep.

Because of the gene insertion process, it is difficult to interpret the present results in terms of a specific effect of human brain insulin on sleep regulation. Potential users of transgenic mice should be aware of such difficulties and should carefully examine the entire process of transgenic mouse construction. Many mice containing a knockout gene deletion are constructed by alteration of embryonic stem (ES) cells from the 129 strain, which are then injected into a hybrid embryo or another strain. To obtain a stable genetic background, at least 10 or more backcrosses to the recipient strain are necessary. Unfortunately, strain 129 is not often used in behavioural studies and therefore the heritability of most of its behavioural traits is unknown. In conclusion, transgenic mice are potentially good models for sleep studies, as long as certain rules are followed during their construction and breeding, e.g. using the same mouse strain for the entire process of transgenesis. Recent progress has made it possible to perform conditional gene deletion or activation, which becomes effective only in the adult animal, and each animal can therefore be used as its own control.

Figure 3

Examples of 30-s EEG recordings during SWS in a control mouse (A) and in a transgenic mouse (B). Note the high amplitude spindles in the transgenic mouse (vertical bars: 100 uV). Record derived from the fronto-parietal derivation: 11 min frontal to bregma and lambda, and 1 mm lateral to midline.

ACKNOWLEDGEMENTS

This work was supported by INSERM U52 and U257. We would like to thank Tom Barkas for proof-reading the manuscript.