An elegant variation on conventional gene-knockout techniques can delete a gene at specific times and locations in mice. The approach shows when and where a serotonin receptor protein is needed during development.
On page 396 of this issue, Gross and colleagues1 look in depth at how serotonin, one of the chemical messengers that nerve cells use to communicate, is involved in anxiety. Perhaps one of the best known of these messengers, or neurotransmitters, serotonin has a role in many different neurobiological processes. For example, it helps to regulate our moods — a fact that has been well established since the 1950s, with the discovery that drugs that deplete serotonin precipitate depression whereas increasing serotonin levels has antidepressant effects. The idea that serotonin might also affect anxiety was first suspected in the 1980s following the serendipitous finding that buspirone, a drug developed to treat psychotic patients, is also useful for treating anxiety disorders, and stimulates a type of serotonin-detecting molecule in the body, the serotonin1A receptor. Later came the discovery that mice that have been genetically engineered to lack this receptor, and so cannot respond normally to serotonin, show increased 'anxiety-like' behaviour2,3,4.
But the underlying mechanisms have been elusive. For instance, the relevant brain regions have not been delineated. Moreover, the findings in receptor-deficient mice appear to contradict observations that compounds that block serotonin1A receptors do not cause anxiety in adult mice. Gross et al.1 substantially clarify these issues. By using mice in which the serotonin1A receptor can be knocked out at will, the authors show that the absence of the receptor in newborns does indeed lead to anxiety-like behaviour, whereas its knockout during adult life has no effect. Gross et al. also discriminate between the role of the receptors in the hindbrain and in forebrain structures such as the hippocampus and cerebral cortex.
Conventional gene-knockout techniques are powerful tools for working out what a protein does. But they have major limitations compared with using drugs (which might, for example, activate or inhibit the protein of interest). Genes tend to be knocked out during embryonic life, generally affecting the whole organism throughout its lifetime. By contrast, a drug can be administered at any time and, in the brain, can be injected into specific areas.
The approach adopted by Gross et al.1 is an ingenious way of addressing the shortcomings of gene knockouts, providing time- and tissue-specific deletion and restoration of serotonin1A receptors. To achieve time-specific knockouts, Gross et al. produced mice in which expression of the serotonin1A-receptor gene was under the control of the antibiotic doxycycline. The gene could be switched off — with a certain time lag — simply by feeding mice the antibiotic.
The authors also engineered mice in which the serotonin1A receptor was expressed only in the forebrain. This was a little more complicated. They first produced a 'transgenic' mouse line in which the serotonin1A-receptor gene could be activated only in the presence of a bacterial gene- transcription factor. In a second mouse line, the gene encoding that transcription factor was joined with a control region from the gene encoding calcium–calmodulin-dependent protein kinase II, which is active specifically in the forebrain. So the transcription factor is likewise expressed selectively in the forebrain. Mating the two lines of mice produced double-transgenic offspring with normal serotonin1A receptors expressed in the hippocampus and cerebral cortex, but no receptors on the serotonin-sensitive neurons in the raphe nuclei of the hindbrain.
Gross et al. found that mice lacking serotonin1A receptors throughout the brain showed pronounced anxiety-like behaviour, as judged in a variety of tasks (Fig. 1), whereas selective restoration of the receptors in the forebrain restored normal behaviour. Thus, serotonin1A receptors in the forebrain regulate anxiety, whereas receptors in the hindbrain do not seem to be directly involved.
The authors also delineated critical periods of development during which the receptors influence anxiety. Feeding dox cycline to juvenile (10–12-week-old) double-transgenic mice eliminated forebrain serotonin1A receptors in adult mice. This did not elicit anxiety-like behaviour. By contrast, administering doxycycline during gestation eliminated the forebrain receptors from newborns, and this did lead to pronounced anxiety-like behaviour. So forebrain serotonin1A receptors are needed during the development of newborns to modulate the predisposition to anxiety-like behaviour, but are no longer critical during adult life.
These findings1, together with earlier evidence, suggest that selected serotonin-sensitive neuronal systems may participate in brain development. Like most neurotransmitter-sensitive neurons, serotonin-responsive neurons first appear during embryonic life and then gradually increase in numbers until adulthood. However, discrete but relatively prominent populations of such neurons are produced transiently at key periods during development and then vanish, suggesting that they may help to trigger particular aspects of brain development5. Moreover, the serotonin1A receptor has been specifically implicated in the development of synapses (neuronal junctions) in the forebrain, as treatment of newborn rodents with drugs that block these receptors decreases the numbers of spines (neuronal extensions) on neurons in the dentate gyrus of the forebrain6.
More speculatively, perhaps variations in serotonin-sensitive neurons and serotonin receptors in early life account for the importance of maternal nurturing in preventing emotional disturbance in adults. Thus, adult rats that were not licked and groomed adequately as pups by their mothers display increased anxiety, and this may reflect serotonin-dependent mechanisms7. Assuming that we can equate developmental stages in mice and humans, these findings might be relevant to brain development and the genesis of anxiety in people, too.
Serotonin appears to be an all-purpose neurotransmitter; it has been implicated in many aspects of brain function and in the effects of many important drugs that are used to treat anxiety, depression, migraine headaches, nausea, pain and irritable bowel syndrome. Gross et al.'s discovery1 — that anxiety is linked to the need for serotonin1A receptors in a specific brain region, at a particular period of development — adds a new layer of understanding of serotonin's function. More generally, the authors' technique lends greater precision and flexibility to gene-knockout approaches for understanding neurotransmitter function, and will hopefully soon be extended to many other neurotransmitters and behaviours.
Gross, C. et al. Nature 416, 396–400 (2002).
Parks, C. L., Robinson, P. S., Sibille, E., Shenk, T. & Toth, M. Proc. Natl Acad. Sci. USA 95, 10734–10739 (1998).
Ramboz, S. et al. Proc. Natl Acad. Sci. USA 95, 14476–14481 (1998).
Heisler, L. K. et al. Proc. Natl Acad. Sci. USA 95, 15049–15054 (1998).
D' Amato, R. J. et al. Proc. Natl Acad. Sci. USA 84, 4322–4326 (1987).
Heim, C. & Nemeroff, C. B. Biol. Psychiatry 49, 1023–1039 (2001).
Yan, W., Wilson, C. C. & Haring, J. H. Brain Res. Dev. Brain Res. 98, 185–190 (1997).
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