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There is a long history documenting the usefulness of genetic approaches in studies of brain function, including learning and memory. These approaches fall into two general categories: forward and reverse genetics. Forward genetics is concerned with the identiﬁcation of genes involved in biological processes such as learning and memory. The starting point of these studies is usually the identiﬁcation of mutant organisms with interesting phenotypic changes, and their goal is to identify the mutations underlying these changes. In reverse genetic studies, the gene is already at hand, and the goal is to deﬁne its role in biological processes of interest. This normally involves the derivation and study of organisms with deﬁned genetic changes. Although the principal purpose of genetic approaches is to study how genetic information determines biological function, recently animals with genetically engineered mutations have been used to develop and test multidisciplinary theories of learning and memory that go well beyond gene function.
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1. The Role of Genetics in Biology
To explore the role of genetics, it is important to place it in the large context of biological investigations. The ultimate goal of biological research is to develop and test explanations of complex phenomena such as learning and memory. At the heart of this process is the establishment of causal connections between phenomena of interest, such as changes in synaptic function and learning. There are four complementary general strategies that science uses to make causal connections between phenomena of interest. One of these strategies is the Lesion strategy. Thus, pharmacological and genetic lesions of calmodulin-induced kinase II (CaMKII) are known to result in deﬁcient long-term potentiation (LTP) of synaptic function, suggesting a connection between the activation of this synaptic kinase and LTP (Silva et al. 1997). It is noteworthy that genetics and pharmacology are the only two approaches to interfere with molecular function in biology. The second strategy that science uses to make causal connections between phenomena of interest is the direct observation of these phenomena in their natural context. For example, the induction of LTP is accompanied by observable increases in CaMKII activity. The third strategy involves the induction of one phenomenon by the other. For example, injection of activated CaMKII into pyramidal neurons in hippocampal slices induces an LTP-like phenomenon. Finally, modeling plays a critical role in making causal connections between phenomena of interest. To assert that two natural phenomena are connected, it is essential to understand something about the mechanism that links them. Thus, CaMKII activation is thought to trigger LTP by phosphorylating and thus enhancing the function of synaptic glutamate receptors. Each of the strategies mentioned above is insuﬃcient to connect two phenomena of interest. Instead, convergent evidence from all four strategies is needed. Therefore, since genetics is one of only two general molecular-lesion approaches available, it is easy to see why it has played a key role in biology. Besides its key role in testing hypotheses (i.e., CaMKII is required for LTP induction), it can be argued that the principal role that genetics has played in biology has been to suggest possible hypotheses or explanations of natural phenomena. Indeed, forward genetic screens have allowed biologists to make major discoveries, even in the absence of a well-delineated hypothesis.
2. Forward Genetics
Long before we had the ability to directly manipulate genes in animals such as ﬂies and mice, geneticists were busy using chemical mutagens to alter genetic information in living systems (forward genetics). The goal of classical or forward genetics, which continues to be used extensively to this day, is to identify the genes critical for biological processes of interest. The idea is that study of those genes is often a critical ﬁrst hint for unraveling underlying biological processes. In forward genetic screens, animals are ﬁrst exposed to a mutagen, for example, the DNA-altering compound ethyl-nitroso-urea, mated, and the progeny are screened for phenotypic changes of interest. The phenotype of a mutant is the sum total of observed biological changes caused by a genetic manipulation. Recent application of this approach in the study of mammalian circadian rhythms resulted in the identiﬁcation of clock, a crucial link in the cascade of transcriptional events that marks molecular time in organisms as diverse as Drosophila and mice (Wilsbacher and Takahashi 1998).
Other molecular components of this pathway, such as per, were isolated in mutagenesis screens in Drosophila. By identifying novel and unexpected molecular components of biological processes of interest, forward genetics has often reshaped entire ﬁelds of research. At times, science can go in circles, obsessively chasing its own tail of half-truths, incapable of escaping the gravitational pull of its worn out paradigms. Forward genetics, in the hands of masters such as Edward Lewis (developmental mutants) and Seymor Benzer (learning mutants), has the ability to turn paradigms upside down, and initiate new lines of scientiﬁc inquiry. The key to the success of forward genetics is the design of biological screens with which the randomly mutagenized animals are tested. If the screens are too stringent, or if the fundamental biological insights underlying the screen’s design are oﬀ mark, one runs the risk of ending up with empty hands, or even worse, with a number of misleading mutants. In contrast, nonstringent designs lead to overwhelming numbers of nonspeciﬁc mutants that are essentially useless.
3. The First Screens for Learning and Memory Mutants
Seymor Benzer and colleagues working with Drosophila at the California Institute of Technology designed the ﬁrst successful screen for learning and memory mutants in the 1970s (Dudai 1988). Benzer and colleagues developed a behavioral procedure with operant and Pavlovian components. During training the ﬂies were allowed to enter two chambers, each with a diﬀerent odorant, but they only got shocked in one of the chambers. During testing approximately twothirds of the trained ﬂies avoided the chamber with the odorant that previously had been paired with shock. With this procedure, Benzer and colleagues tested a number of Drosophila lines derived from ﬂies treated with ethylmethane sulfonate (EMS). The ﬁrst mutant line isolated from this screen was dunce (Dudai 1988).
Remarkably, three out of the four learning and memory mutations, ﬁrst discovered in genetic screens in Drosophila, code for members of the cAMP-signaling pathway. For example, dunce lacks a phosphodiesterase that degrades cAMP. Importantly, these ﬁndings have recently been extended into vertebrates, where electrophysiological and behavioral studies have conﬁrmed the critical importance of cAMP signaling to learning and memory (Silva et al. 1998). Remarkably, in the early 1970s Eric Kandel and his colleagues at Colombia University also found evidence for the importance of cAMP signaling in learning and memory with a completely diﬀerent approach. They used a reduced cellular preparation to study sensitization, a nonassociative form of learning, in the sea snail Aplysia (Byrne and Kandel 1996). They also found that sensitization depends on cAMP signaling. This is a fascinating example of convergent evidence in science, but it also serves to illustrate that genetics, like any other tool in science, is most successful when used in parallel with other approaches. The persuasive power of convergent evidence cannot be overemphasized. Besides identifying new genes, genetics can also be used to test hypotheses about the function of cloned genes (reverse genetics).
4. Reverse Genetics
In classical genetics an interesting phenotype is usually the driving force behind the molecular experiments required to identify the underlying mutant gene(s). In contrast, in reverse genetics, the interesting molecular properties of a gene usually drive the generation and study of the mutant animal (hence, the word reverse). It is now possible to delete and add genes to many species, ranging from bacteria to mice. For example, mice can be derived with the deletion (knockouts) or overexpression (transgenics) of almost any cloned gene. These manipulations can involve whole genes or they can target speciﬁc domains or even single base pairs.
To generate knockout mice, the desired mutation is engineered within the cloned gene, and this mutant DNA is introduced into embryonic stem (ES) cells. Since ES cells are pluripotent, they can be used to derive mice with the genetically engineered lesion. For that purpose, they are injected into blastocysts (early embryos), and the blastocysts are implanted in host mothers. The resulting chimeric (having mutant and normal cells) oﬀspring are then mated to obtain mutant mice. In contrast, transgenic mice are derived by injecting the pronuclei of fertilized eggs with a DNA construct carrying a gene of interest under the regulation of an appropriate promoter. The injected eggs are transplanted into pregnant females and some of the resulting progeny will have the transgenic construct inserted randomly in one of its chromosomes.
With classical knockout and transgenic techniques it is not possible to regulate the time and the regions aﬀected by the mutation transgene. However, recent techniques promise to circumvent these limitations with a variety of techniques. For example, the expression of the gene of interest can be regulated by gene promoters that can be controlled by exogenously provided substances, such as tetracycline derivatives (Mayford et al. 1997). Alternatively, it is also possible to regulate the function of a protein of interest by fusing it with another protein that can be regulated by synthetic ligands such as tamoxifen (Picard 1993). For example, our laboratory has recently showed that a transcriptional repressor called CREB can be activated at will when fused with a ligand-binding domain (LBDm) of a modiﬁed estrogen receptor. Addition of tamoxifen (the ligand of the modiﬁed receptor) activates the CREBr LBDm fusion protein. It is important to note that irrespective of the exact method used, the general idea of reverse genetic studies is that the function of a gene can be deduced from the phenotype of the mutant animal.
5. Knockouts, Transgenics, and Learning
The ﬁrst knockout transgenic studies of learning and memory analyzed mice with a targeted mutation of the α isoform of CaMKII (Grant and Silva 1994). Pharmacological studies had previously shown that this family of calcium calmodulin induced kinases present in synapses were required for LTP, a stable enhancement in synaptic eﬃcacy thought to contribute to learning and memory. Remarkably, deleting αCaMKII resulted in profound deﬁcits in hippocampal LTP and in hippocampal-dependent learning and memory. Additional studies showed that either the overexpression of a constitutively expressed form of the kinase or a mutation that prevented its autophosphorylation also disrupted LTP and learning. Importantly, studies of hippocampal circuits that ﬁre in a place-speciﬁc manner (place ﬁelds) showed that these CaMKII genetic manipulations disrupted the stability of these place representations (but not their induction) in the hippocampus. Altogether, these studies suggested the provocative hypothesis that this kinase is important for the induction of stable synaptic changes, that the stability of synaptic changes is crucial for the stability of hippocampal circuits coding for place, and that these circuits are essential for spatial learning (Elgersma and Silva 1999). Even this very abbreviated summary demonstrates that these studies went well beyond gene function. Instead, they used mutations to test hypotheses that connected molecular, cellular, circuit, and behavioral phenomena. Although it is reasonable to claim that the αCaMKII has a direct role in the regulation of synaptic function (for example, by phosphorylating glutamate receptors), it is more problematic to argue that the kinase is regulating spatial learning. There are many more phenomenological steps between kinase function and the animal’s ability to ﬁnd a hidden platform in a water maze than between the biochemical properties of this kinase and its role in synaptic plasticity. By comparison, it is easier to see how the stability of place ﬁelds in the hippocampus could be an important component of hippocampal-dependent spatial learning.
6. Common Concerns with the Interpretation of Transgenic Knockout Studies
Despite the power of genetics there are a number of concerns that must be kept in mind when using genetic approaches. One of the most commonly discussed is the possibility that developmental eﬀects or any other change caused by the mutation preceding the study could confound its interpretation. Another pertains to the possible eﬀects of genetic compensation. Since proteins do not work alone, but instead function in highly dynamic networks, it is often observed that speciﬁc genetic changes lead to alterations compensations in the function of other related proteins. A related concern pertains to genetic background. Extensive studies have shown that the genetic background of a mutation has a profound eﬀect on its phenotype. The concerns listed above are not limitations of genetics, but simply reﬂect the properties of the biological systems that genetics manipulates. At the heart of many of the concerns described above are two misconceptions concerning the nature and organization of biological systems.
First, genetics is essentially a lesion tool. Like other lesion tools, it cannot be used in isolation. To establish causal connections between any two phenomena (A and B) in science, it is never enough to lesion A and
document the alteration of B. As described above, it is also critical to fulﬁl three other criteria: ﬁrst, A must be observed to precede B; second, triggering A should result in B; ﬁnally, it is essential to have a clear hypothesis of how A triggers B. Fulﬁlling only one or two of those four criteria is simply not enough to establish a causal connection between A and B. Therefore, although studying the eﬀects of a deleted protein is an important component in determining its function, it is by no means suﬃcient.
Second, biological systems are dynamic and adaptive, and, therefore, the lesion of any one component is always followed by changes in several other components. Although it is often a helpful simpliﬁcation to think of biological components as independent functional units, it is important to remember that they are not. Thus, it is hardly surprising that the eﬀect of a mutation is dependent on biological variables such as genetic background.
7. The Future of Genetic Manipulations
In the near future it will be possible to delete or modify any gene, anywhere in most organisms of interest, and at any time of choice. Additionally, more powerful forward genetic strategies will allow the isolation of entire pathways of genes involved in any neurobiological phenomenon of interest, including learning, attention, emotion, addition, etc. In parallel with expected advances in genetics, there will also be advances in the methods used to analyze mutants. These advances are just as critical to genetic studies as advances in genetic methodology. For example, imaging the brain of mutant mice may yield insights into how molecular lesions aﬀect the function of brain systems. Most genetic studies of learning and memory in mice have focused on the relationship between cellular phenomena (i.e., LTP) and behavior. Advances in small animal magnetic resonance imaging (MRI) may enable the kind of functional system analysis in the brains of mutant mice that have so far only been possible in large primates. Similarly, multiple-single unit recording techniques are starting to yield system-wide snapshots of circuit activity in the brains of mutant mice. At a molecular level, small-size positron emission tomography (PET) devices will allow the imaging of molecular function in living animals such as mice. For example, it may be possible to image the activation of a receptor such as the dopamine receptor during learning or memory. Microarray techniques and other molecular cloning approaches will allow the identiﬁcation of gene proﬁles in mutant mice. These molecular proﬁles will be critical to delineating the molecular changes behind the expression of a mutant phenotype. It is important to note that genetics allows us to reprogram the biology of organisms. The ﬁner and more sophisticated the phenotypic and genotypic tools that we have at our disposal, the deeper we may be able to probe the magical natural programs embedded in our genes.
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