Because of the conservation between human and animal genes, the relationships between the genes, proteins, and neural circuits that underlie behavior can be studied in animal models that are experimentally tractable. Two important strategies have been applied with great success in the study of gene function.
In classical genetic analysis organisms are first subjected to mutagenesis with a chemical or irradiation that induces random mutations, and then screened for heritable changes that affect the behavior of interest, say, sleep. This approach does not impose a bias as to the kind of gene involved; it is a random search of all possible mutations that cause detectable changes. Genetic tracing of heritable changes allows the identification of the individual genes that are altered in the mutant organism. Thus the pathway of discovery in classical genetics moves from phenotype to genotype, from organism to gene. In reverse genetics a specific gene of interest is targeted for alteration, a genetically modified animal is produced, and the animals with these altered genes are studied. This strategy is both focused and biased—one begins with a candidate gene—and the pathway of discovery moves from genotype to phenotype, from gene to organism.
These two experimental strategies and their more subtle variations form the basis of experimental genetics. Gene manipulation by classical and reverse genetics is conducted in experimental animals, not in humans.
Circadian Rhythm Is Generated by a Transcriptional Oscillator in Flies, Mice, and Humans
The first large-scale studies of the influence of genes on behavior were initiated by Seymour Benzer and his colleagues around 1970. They used random mutagenesis and classical genetic analysis to identify mutations that affected learned and innate behaviors in the fruit fly Drosophila melanogaster: circadian (daily) rhythms, courtship behavior, movement, visual perception, and memory (Boxes 3–2 and 3–3). These induced mutations have had an immense influence on our understanding of the role of genes in behavior.
Box 3–2 Generating Mutations in Experimental Animals
Random Mutagenesis in Flies
Genetic analysis of behavior in the fruit fly (Drosophila) is carried out on flies in which individual genes have been mutated. Mutations can be made by chemical mutagenesis or by insertional mutagenesis. Chemical mutagenesis, for example with ethyl methanesulfonate (EMS), typically creates random point mutations in genes. Insertional mutagenesis occurs when mobile DNA sequences called transposable elements randomly insert themselves into other genes.
The most widely used transposable elements in Drosophila are the P elements. P elements may be modified to carry genetic markers for eye color, which makes them easy to track in genetic crosses, and they may also be modified to alter expression of the gene into which they are inserted.
To cause P element transposition, Drosophila strains that carry P elements are crossed to those that do not. This genetic cross leads to destabilization and transposition of the P elements in the resulting offspring. The mobilization of the P element causes its transposition into a new location in a random gene.
Chemical mutagenesis and transposable element mutagenesis are random mutagenesis strategies that can affect any gene in the genome. Similar random mutagenesis strategies are used to create mutations in the nematode worm Caenorhabditis elegans, the zebra fish, and mice.
Targeted Mutagenesis in Mice
Advances in molecular manipulation of mammalian genes have permitted precise replacement of a known normal gene with a mutant version. The process of generating a strain of mutant mice involves two separate manipulations. A gene on a chromosome is replaced by homologous recombination in a special cell line known as embryonic stem cells, and the modified cell line is incorporated into the germ cell population of the embryo.
The gene of interest must first be cloned. The gene is mutated and a selectable marker, usually a drug-resistance gene, is then introduced into the mutated fragment. The altered gene is then introduced into embryonic stem cells, and clones of cells that incorporate the altered gene are isolated. DNA samples of each clone are tested to identify a clone in which the mutated gene has been integrated into the homologous (normal) site, rather than some other random site.
When a suitable clone has been identified, the cells are injected into a mouse embryo at the blastocyst stage (3 to 4 days after fertilization), when the embryo consists of approximately 100 cells. These embryos are then reintroduced into a female that has been hormonally prepared for implantation and allowed to come to term. The resulting embryos are chimeric mixtures between the stem cell line and the host embryo.
Embryonic stem cells in the mouse have the capability of participating in all aspects of development, including the germline. Thus injected cells can become germ cells and pass on the altered gene to future generations of mice. This technique has been used to generate mutations in various genes crucial to development or function in the nervous system.
Altering Gene Function by RNA Interference
RNA interference takes advantage of the fact that most double-stranded RNAs in eukaryotic cells are targeted for destruction; the whole RNA is destroyed even if only part of it is double-stranded. By artificially causing a selected mRNA to become double-stranded, researchers can activate this process to reduce the mRNA levels for specific genes.
To reduce the function of a gene by RNA interference, small-sequence RNA is introduced that will pair with the endogenous mRNA because of complementarity between the small RNA sequence and the desired mRNA. Small RNA is usually 21 or 22 bases in length, and is known as small interfering RNA (siRNA) or small hairpin RNA (shRNA).
Pairing of siRNA or shRNA with mRNA leads to destruction of the endogenous mRNA by ribonucleases within the cell. The siRNA can be introduced by direct transfection of RNA into cells, or transgenes can be introduced into cells that drive expression of siRNA or shRNA.
If the complementarity between the siRNA and mRNA is perfect, the mRNA is usually destroyed. If the complementarity is close but not perfect, the translation of the mRNA is arrested. Although siRNAs and shRNAs are experimental tools, endogenous small miRNAs with imperfect matches to mRNAs are emerging as an important regulator of translation in many contexts.
RNA interference has great potential to increase the power of genetic analysis because it can be used on any species where transgenes or double-stranded RNA can be delivered to cells. It should allow researchers to change the activity of genes in animals that are not now used in classical genetic analysis such as long-lived birds and fish, and even primates.
Box 3–3 Introducing Transgenes in Flies and Mice
Genes can be manipulated in mice by injecting DNA into the nucleus of newly fertilized eggs (Figure 3–8). In some of the injected eggs the new gene, or transgene, is incorporated into a random site on one of the chromosomes. Because the embryo is at the one-cell stage, the incorporated gene is replicated and ends up in all (or nearly all) of the animal's cells, including the germline.
Gene incorporation is illustrated with a coat color marker gene rescued by injecting a gene for pigment production into an egg obtained from an albino strain. Mice with patches of pigmented fur indicate successful expression of DNA. The transgene's presence is confirmed by testing a sample of DNA from the injected animals.
A similar approach is used in flies. The DNA to be injected is cloned into a transposable element (P element). When injected into the embryo, the DNA becomes inserted into the DNA of germ cell nuclei. P elements can be engineered to express genes at specific times and in specific cells. Transgenes may be wild-type genes that restore function to a mutant, or designer genes that activate other genes in new locations or code for a specifically altered protein.
Generating transgenic mice and flies.
Here the gene injected into the mouse causes a change in coat color, while the gene injected into the fly causes a change in eye color. In some transgenic animals of both species the DNA is inserted at different chromosomal sites in different cells (see illustration at bottom). (Reproduced, with permission, from Alberts et al. 2002.)
We have a particularly complete picture of the genetic basis of the circadian control of behavior. An animal's circadian rhythm couples certain behaviors to a 24-hour cycle linked to the rising and setting of the sun. The core of circadian regulation is an intrinsic biological clock that oscillates over a 24-hour cycle. Because of the intrinsic periodicity of the clock, circadian behavior persists even in the absence of light or other environmental influences.
The clock can be reset, so that changes in the day-night cycle eventually result in a matching shift in the intrinsic oscillator, a phenomenon that is familiar to any traveler recovering from jet lag. Light-driven signals are transmitted by the eye to the brain to reset the clock. Finally, the clock drives output pathways for specific behaviors, such as sleep and locomotion.
Benzer's group searched through thousands of mutant flies to look for rare flies that failed to follow circadian rhythms because of mutations in the genes that direct circadian oscillation. From this work emerged the first insight into the molecular machinery of the circadian clock. Mutations in the period, or per, gene affected all circadian behaviors generated by the fly's internal clock.
Interestingly, per mutations could change the circadian clock in several ways (Figure 3–9). Arrhythmic per mutant flies, which exhibited no discernible intrinsic rhythms in any behavior, lacked all function of the per gene, so per is essential for rhythmic behavior. Per mutations that maintained some function of the gene resulted in abnormal rhythms. Long-day alleles produced 28-hour behavioral cycles, whereas short-day alleles produced a 19-hour cycle. Thus per is not just an essential piece of the clock, it is actually a timekeeper whose activity can change the rate at which the clock runs.
A single gene governs the circadian rhythms of behaviors in Drosophila.
Mutations in the period, or per, gene affect all circadian behaviors regulated by the fly's internal clock. (Reproduced, with permission, from Konopka and Benzer 1971).
A. Locomotor rhythms in normal Drosophila and three strains of per mutants: short-day, long-day, and arrhythmic. Files were shifted from a cycle of 12 hours of light and 12 hours of dark into continuous darkness, and activity was then monitored under infrared light. Heavy lines indicate activity.
B. Normal adult fly populations emerge from their pupal cases in cyclic fashion, even in constant darkness. The plots show the number of flies (in each of four populations) emerging per hour over a 4-day period of constant darkness. The arrhythmic mutant population emerges without any discernible rhythm.
The per mutant has no major adverse effects other than the change in circadian behavior. This observation is important because, prior to the discovery of per, many had questioned whether there could be true "behavior genes" that were not required for the other physiological needs of an animal. Per does seem to be such a "behavior gene."
How does per keep time? The protein product PER is a transcriptional regulator that affects the expression of other genes. Levels of PER are regulated throughout the day. Early in the morning PER and its mRNA are low. Over the course of the day the PER mRNA and protein accumulate, reaching peak levels after dusk and during the night. The levels then decrease, falling before the next dawn. These observations provide an answer to the circadian rhythm puzzle—a central regulator appears and disappears throughout the day. But they are also unsatisfying because they only push the question back one step—what makes PER cycle? The answer to this question required the discovery of additional clock genes, which were discovered in flies and also in mice.
Emboldened by the success of the fly circadian rhythm screens, Joseph Takahashi began similar but far more labor-intensive genetic screens in mice in the 1990s. He screened hundreds of mutant mice for the rare mice with alterations in their circadian locomotion period, and found a single gene mutation that he called clock (Figure 3–10). When mice homozygous for the clock mutation are transferred to darkness, they initially experience extremely long circadian periods and later a complete loss of circadian rhythmicity. The clock gene therefore appears to regulate two fundamental properties of the circadian rhythm: the length of the circadian period and the persistence of rhythmicity in the absence of sensory input. These properties are conceptually identical to the properties of the per gene in flies.
Rhythmicity in clock mutant mice.
The records show periods of locomotor activity by three animals: wild-type, heterozygous, and homozygous. All animals were kept on a light-dark (L/D) cycle of 12 hours for the first 7 days, then transferred to constant darkness (D). They later were exposed to a 6-hour light period (LP) to reset the rhythm. The circadian rhythm for the wild-type mouse has a period of 23.1 hours. The period for the heterozygous clock/+ mouse is 24.9 hours. The homozygous clock/clock mice experience a complete loss of circadian rhythmicity on transfer to constant darkness and transiently express a rhythm of 28.4 hours after the light period. (Reproduced, with permission, from Takahashi et al. 1994.)
The mouse clock gene, like the per gene in flies, encodes a transcriptional regulator whose activity oscillates through the day. The mouse CLOCK and fly PER proteins also shared a domain called a PAS domain, characteristic of a subset of transcriptional regulators. This observation suggests that the same molecular mechanism—oscillation of PAS-domain transcriptional regulation—controls circadian rhythm in flies and mice.
More significantly, parallel studies of flies and mice showed that similar groups of transcriptional regulators affected the circadian clock in both animals. After the mouse clock gene was cloned, a fly circadian rhythm gene was cloned and found to be closely related to mouse clock, even more so than per. In a different study a mouse gene similar to fly per was identified and inactivated by reverse genetics. The mutant mouse had a circadian rhythm defect, like fly per mutants. In other words, both flies and mice use both clock and per genes to control their circadian rhythms. A group of genes, not one gene, are conserved regulators of the circadian clock.
Characterization of these genes has led to an understanding of the molecular mechanisms of circadian rhythm, and a dramatic demonstration of the similarity of these mechanisms in flies and mice. In both flies and mice the CLOCK protein is a transcriptional activator. Together with a partner protein, it controls the transcription of genes that determine output behaviors such as locomotor activity levels. CLOCK and its partner also stimulate the transcription of the per gene. However, PER protein represses CLOCK's ability to stimulate per expression, so as PER protein accumulates, per transcription falls (Figure 3–11). The 24-hour cycle comes about because the accumulation and activation of PER protein is delayed by many hours after the transcription of per, a result of PER phosphorylation, PER instability, and interactions with other cycling proteins.
Molecular events that drive circadian rhythm.
The genes that control the circadian clock are regulated by two nuclear proteins, PER and TIM, that slowly accumulate and then bind to one another to form dimers. When PER and TIM accumulate enough to dimerize, they enter the nucleus and shut off the expression of circadian genes including themselves. They do so by inhibiting CLOCK and CYCLE, which stimulate the transcription of per and tim genes. PER is highly unstable; most of the protein is degraded so quickly that it never has a chance to repress CLOCK-dependent per transcription. The degradation of PER is regulated by at least two different phosphorylation events by different protein kinases. When PER binds to TIM, PER is protected from degradation. As CLOCK drives more and more per and tim expression, enough PER and TIM eventually accumulate that the two can bind and stabilize each other, at which point they enter the nucleus to repress their own transcription. As a result, per and tim mRNA levels fall; thereafter, PER and TIM protein levels fall and CLOCK can (once again) drive expression of per and tim mRNA. During daylight TIM protein is degraded by signaling pathways that are regulated by light (including cryptochrome), so PER/TIM complexes form only at night. The CLOCK protein induces PER and TIM expression but is inhibited by PER and TIM proteins.
The molecular properties of per, clock, and related genes generate all properties essential for circadian rhythm. The key elements of the regulatory process are:
The transcription of circadian rhythm genes varies with the 24-hour cycle: PER activity is high at night, CLOCK activity is high during the day.
The circadian rhythm genes are transcription factors that affect each other's mRNA level, generating the oscillations. CLOCK activates per transcription and PER represses CLOCK function.
The circadian rhythm genes also control the transcription of output genes that affect many downstream responses. For example, in flies the neuropeptide gene pdf controls locomotor activity levels.
The oscillation of these genes can be reset by light.
Recent work has shown that the same genetic network controls circadian rhythm in humans. People with advanced sleep-phase syndrome have short 20-day cycles and an extreme early-to-bed, early-to-rise "morning lark" phenotype. Louis Ptáček and Ying-hui Fu found that these individuals have mutations in a human per gene. These results demonstrate that genes for behavior are conserved from insects to humans. Advance sleep-phase syndrome is discussed in detail in the chapter on sleep (see Chapter 51).
Natural Variation in a Protein Kinase Regulates Activity in Flies and Honeybees
In the genetic studies of circadian rhythm described above, random mutagenesis was used to identify genes of interest in a biological process. All normal individuals have functional copies of per, clock, and the related genes; only after mutagenesis were different alleles generated. Another, more subtle question about the role of genes in behavior is to ask which genetic changes make normal individuals behave differently from each other. Work by Marla Sokolowski and her colleagues led to the identification of the first gene associated with variation in behavior among normal individuals in a species.
Larvae of Drosophila vary in activity level and locomotion. Some larvae, called rovers, move over long distances (Figure 3–12). Others, called sitters, are relatively stationary. Drosophila larvae isolated from the wild can be either rovers or sitters, indicating that these are natural variations and not laboratory-induced mutations. These traits are heritable; rover parents have rover offspring and sitter parents have sitter offspring.
Foraging behavior of Drosophila melanogaster larvae.
The larvae feast on patches of yeast. Rover-type larvae move from patch to patch, whereas sitter-type larvae stay put on a single patch for a long time. When foraging within a single patch, rover larvae move about more than sitter larvae. On agar alone, rover and sitter larvae move about equally. This difference in foraging behavior maps to a single protein kinase gene, for, that varies in activity in different fly larvae. (Reproduced, with permission, from Sokolowski 2001.)
Sokolowski used crosses between different wild flies to investigate the genetic differences between rover and sitter larvae. These crosses showed that the difference between rover and sitter larvae lies in a single major gene, the for (forager) locus. The for gene encodes a signal transduction enzyme, a protein kinase activated by the cellular metabolite cyclic guanosine 3′-5′monophosphate (cGMP). Thus this natural variation in behavior arises from altered regulation of signal transduction pathways. Many neuronal functions are regulated by protein kinases such as the cGMP-dependent kinase encoded by the for gene. Molecules such as protein kinases are particularly significant at transforming short-term neural signals into long-term changes in the property of a neuron or circuit.
Why would variability in signaling enzymes be preserved in wild populations of Drosophila, which typically include both rovers and sitters? The answer is that variations in the environment favor alternative genetic forms: There is balanced selection for both behaviors. Crowded environments favor the rover larva, which is more effective at moving to new, unexploited food sources in advance of competitors, whereas sparse environments favor the sitter larva, which exploits the current source more thoroughly.
The for gene also affects honeybee behavior. Honeybees exhibit different behaviors at different stages of their life; in general, young bees are nurses, while older bees become foragers that leave the hive. The for gene is expressed at high levels in the brains of active foraging honeybees, and at low levels in the younger and more stationary nurse bees. Activation of cGMP signaling in young bees can cause them to enter the forager stage prematurely. Presumably, this change is normally programmed by an environmental stimulus or the bee's advancing age. Thus the same gene controls variation in a behavior in two different insects, the fruit fly and the bee. However, in the fruit fly the variation is expressed in different individuals, whereas in the honeybee it is expressed in one individual at different ages. The difference illustrates how an important regulatory gene can be recruited to different behavioral strategies in different species.
The Social Behaviors of Several Species Are Regulated by Neuropeptide Receptors
Many aspects of behavior are associated with an animal's social interactions with other animals. Social behaviors are highly variable between species, yet have a large innate component within a species that is controlled genetically. A simple form of social behavior has been analyzed in the roundworm C. elegans. These animals live in soil and eat bacteria. Different wild-type strains exhibit profound differences in feeding behavior. Some strains are solitary, dispersing across a lawn of bacterial food and failing to interact with each other. Other strains have a social feeding pattern, joining large feeding groups of dozens or hundreds of animals (Figure 3–13). The difference between these strains is genetic, as both feeding patterns are stably inherited.
Feeding behavior of the roundworm Caenorhabditis elegans depends on the level of activity of a neuropeptide receptor gene.
In one strain individual worms graze in isolation (left), whereas in another strain individuals mass together to feed. The difference is explained by a single amino acid substitution in the neuropeptide receptor gene. (Reproduced, with permission, from Mario de Bono and Cell Press.)
The difference between social and solitary worms is caused by a single amino acid substitution in a single gene, a member of a large family of genes involved in signaling between neurons. This gene, npr-1, encodes a neuropeptide receptor. Neuropeptides have long been appreciated for their roles in coordinating behaviors across networks of neurons. For example, a neuropeptide hormone of the marine snail Aplysia stimulates a complex set of movements and behavior patterns associated with a single behavior, egg laying. Mammalian neuropeptides have been implicated in feeding behavior, sleep, pain, and many other behaviors and physiological processes. The existence of a mutation in the neuropeptide receptor that alters social behavior suggests that this kind of signaling molecule is important both for generating the behavior and for generating the variation between individuals.
Neuropeptide receptors have also been implicated in the regulation of mammalian social behavior. The neuropeptides oxytocin and vasopressin stimulate mammalian affiliative behaviors such as pair bonding and parental bonding with offspring. Oxytocin is required in mice for social recognition, the ability to identify a familiar individual. Oxytocin and vasopressin have been studied in depth in prairie voles, rodents that form lasting pairs to raise their young. Oxytocin released in the brain of female prairie voles during mating stimulates pair-bond formation. Likewise, vasopressin released in the brain of male prairie voles during mating stimulates pair-bond formation and paternal behavior.
The extent of pair-bonding varies substantially between mammalian species. Male prairie voles form pair-bonds with females and help them raise their offspring, but the closely related male montane voles breed widely and do not engage in paternal behavior. The difference between the behaviors of males in these species correlates with differences in the expression of the V1a class of vasopressin receptors in the brain. In prairie voles V1a vasopressin receptors are expressed at high levels in a specific brain region, the ventral pallidum (Figure 3–14). In montane voles the levels are much lower in this region, although high in other brain regions.
Distribution of vasopressin (V1a) receptor binding in the ventral pallidum of montane voles (nonmonogamous) and prairie voles (monogamous).
(Adapted, with permission, from Young et al. 2001.)
A. V1a receptor expression is high in the lateral septum (LS) but low in the ventral pallidum (VP) in the nonmonogamous montane vole.
B. Expression is high in the ventral pallidum of the monogamous prairie vole. Expression of the receptor in the ventral pallidum allows vasopressin to link the social recognition pathway to the reward pathway.
The importance of oxytocin and vasopressin and their receptors has been confirmed and extended by reverse genetic studies in mice, which are genetically easier to manipulate than voles. For example, introducing the V1a vasopressin receptor gene from prairie voles into male mice, which behave more like montane voles, increases the expression of the V1a vasopressin receptor in the ventral pallidum and increases the affiliative behavior of the male mice toward females. Thus differences between species in the pattern of expression of the vasopressin receptor can contribute to differences in social behaviors.
The analysis of vasopressin receptors in different rodents provides insight into the mechanisms by which genes and behaviors can change during evolution. Genetic changes in evolution alter the pattern of expression of the V1a vasopressin receptor in the ventral forebrain. These changes in turn alter the activity of a neural circuit, so that the function of the ventral forebrain is linked to the function of the vasopressin-secreting neurons that are activated by mating. As a result, social behaviors are altered.
The importance of oxytocin and vasopressin in human social behavior is not known, but the central role of pair-bonding and pup rearing in mammalian species suggests that these molecules might play a role in our species as well.