TRENDS in Neurosciences Vol.25 No.6 June 2002
Giving drugs to knockout mice: can they do that? Paul F. Chapman The past ten years have seen an explosion in the number of genetically modified mice created to aid understanding of the basic mechanisms of learning and memory. There are still significant problems associated with this useful technique, mostly involving the lack of temporal or spatial control over the genetic ‘lesion’. By combining the application of drugs that are sub-threshold in wild-type mice with heterozygosity for gene mutations that do not produce effects alone, it is now possible to avoid many of the problems of both the genetic and the pharmacological approaches.
The basic strategies for understanding the neural basis of learning and memory have been established for some time. Before LTP there was Hebb’s postulate for learning , based on the theoretical work of Cajal and others. This postulate states that some patterns of activity between neurons should cause lasting changes to the synapses between those neurons. If this is how learning occurs, then it should be possible to: (1) map the circuits responsible for a particular form of learning, (2) identify the synapses within those circuits that demonstrate plasticity and (3) determine the mechanisms responsible for the plasticity. Initially, investigations focused on identifying circuits , but with the discovery of LTP there was a significant move towards describing the mechanisms of synaptic plasticity, and their potential contributions to learning and memory, by using pharmacological tools. The discovery that LTP induction could be blocked with the NMDA-receptor antagonist AP5 , for example, prompted a series of investigations into the effects of AP5 on a variety of learning tasks . But, as more of the players in synaptic plasticity were identified, it became clear that many of these components were not readily amenable to pharmacological intervention, because appropriate inhibitors did not exist or were insufficiently specific, or because the target molecules were inaccessible to systemically applied drugs. http://tins.trends.com
Some great successes
The application of targeted gene deletion to the study of synaptic plasticity, learning and memory was therefore as necessary and timely as it was exciting. Two landmark studies reported on the effects of knocking out either αCaMKII (Ca2+–calmodulin-dependent protein kinase IIα, a serine/threonine kinase) [5,6] or FYN (a tyrosine kinase)  on synaptic plasticity and behaviour. These works demonstrated impairments in learning and memory of tasks that are sensitive to lesion of the hippocampus. Moreover, the learning and memory deficits corresponded to impaired LTP, a result that confirmed and extended earlier pharmacological work, and, perhaps more importantly, meant that previously unreachable molecules became accessible for study. The field quickly saw the possibilities offered by the knockout technique, and a large number of mice were created with targeted deletions of genes that encoded molecules thought to be involved in learning and memory . The deleted genes ranged from those encoding neurotransmitter receptors and protein kinases to those encoding nuclear hormone receptors and transcription factors. Although our understanding of which molecules are potentially important for learning, memory and synaptic plasticity has grown (and continues to grow) as a result, some important drawbacks of the knockout technique have also emerged. Some frustrating problems
A major problem with ‘conventional’ knockout technology is that a gene is deleted from the earliest moment of embryonic development. If you are unfortunate, this can mean that animals carrying your deletion do not survive, even to later embryonic stages. If the gene you have deleted is not required for survival, you might still not be out of the woods. The deletion might have an important role in development that is distinct from its role in adult plasticity, and deficits in learning and memory seen in adulthood might reflect those developmental changes
rather than an active role in adult plasticity. Alternatively, a gene deletion that logic and literature say should wreak havoc on learning and memory might have no effect at all. The primary culprit in such cases is likely to be ‘compensation’ – a poorly understood process in which related gene products take over the function of the deleted gene product. In any case, the problems stem not from a lack of specificity or access, as is the case with a pharmacological approach, but from a lack of control over the timing and location of the genetic lesion, and from the long-term response of the nervous system to it. Some attempts to fix the problems
Molecular biologists have devised a truly impressive set of adaptations to standard knockout techniques to address the problems caused by the lack of temporal and spatial control . Space precludes a full description but, basically, these approaches involve combining the transgenic expression of an enzyme (Cre recombinase) with the presence of ‘loxP’ signals within a targeting construct. When Cre recombinase is expressed in cells that also carry a gene flanked by loxP sites (i.e. a ‘floxed’ gene), the recombinase causes selective deletion of the floxed gene from the cells. By creative use of promoters for the Cre recombinase transgene, it is possible to restrict the gene knockout to a particular type of cell (e.g. pyramidal neurons of the CA1 hippocampus) or even to a specific developmental period (if the promoter used to drive Cre recombinase is only active during a particular stage of development). These techniques have provided significant advances, but significant problems remain. It should be possible to generate inducible knockouts, by placing the Cre recombinase gene under the control of an inducible promoter, but progress on this approach has, to date, been disappointing. Moreover, transgenes themselves can be difficult to work with, and getting appropriate levels of expression restricted to the desired cell types is not straightforward .
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TRENDS in Neurosciences Vol.25 No.6 June 2002
Receptor tyrosine kinases
NMDA receptors Ca2+
Substrates involved in synaptic plasticity TRENDS in Neurosciences
Fig. 1. Signalling pathways involved in neuronal plasticity. It is likely that a limited set of common substrates is responsible for synaptic plasticity, learning and memory, and that numerous pathways lead to activation or modification of these substrates. Two examples are the Ras–MAPK (mitogen-activated protein kinase) and NMDA–CaMKII (Ca2+–calmodulin-dependent kinase II) pathways. Disruption of either pathway by genetic or pharmacological manipulations that eliminate a single element [e.g. the homozygous knockout of αCaMKII, the application of MEK (MAPK kinase) inhibitors that would block activation of both ERKI and ERKII MAP kinases, or elimination of the Ras activator Ras-GRF] can prevent LTP and learning. Merely reducing the function of a single element (e.g. the heterozygous knockout of K-Ras or application of low concentrations of NMDA-receptor antagonists) does not significantly affect plasticity or learning alone, but combining the reduction of two elements from the same pathway does. Thus, partial reduction of levels of K-Ras combined with partial reduction of MEK activity blocks learning and LTP effectively, as does partial reduction of αCaMKII combined with low doses of the NMDA-receptor antagonist CPP [(+ –)-(2-carboxypiperazine-4-yl)propyl-1-phosphoric acid]. The independence of the Ras–MAPK and NMDA–CaMKII pathways is indicated by the observation that reducing function of a single element from each of the pathways (e.g. low doses of CPP in K-Ras heterozygous animals) does not affect learning or LTP.
Combining genetic and pharmacological approaches
The difficulties with the knockout approach are largely technical, and have the potential to be solved. As attempts to address these problems become increasingly complex, a simple alternative presents itself. No protein can function without a network with upstream activators and modulators and downstream effectors. By combining a knockout that does not itself produce a significant phenotype with a sub-threshold concentration of a drug that acts either upstream or downstream of the knockedout gene, it is possible to create clean, inducible manipulations with interesting and interpretable consequences. A recent paper by Silva and colleagues  beautifully demonstrates the potential of this approach. The application of drugs to knockout mice is not new. Knockouts created to test http://tins.trends.com
the mechanisms of reward and addiction as models of drug abuse have been given a variety of psychoactive substances . In a particularly elegant study, McKernan and colleagues  used a combination of point mutations in genes encoding GABAA-receptor subunits and application of a newly synthesized compound to dissociate the mechanisms underlying the motor and anxiolytic effects of benzodiazepines. In these studies, however, the knockouts were designed with the explicit goal of understanding drug effects. Silva and colleagues have now, for the first time, used drugs and knockouts as equal and interacting tools in an effort to describe basic mechanisms of learning and synaptic plasticity. In their recent paper, Ohno et al. set out to examine the behavioural and physiological effects of a mutation affecting the Ras–MAPK (mitogenactivated peptide kinase) signalling
pathway . This pathway (Fig. 1) is known to be important for a variety of functions in a variety of cell types, and a significant body of evidence suggests that it is a key element of the pathway responsible for long-term plasticity (and thus, potentially, for long-term memory) [14,15]. This hypothesis has been difficult to prove, because of redundancy in the Ras pathway (the H-, N-, and K- forms of Ras have similar activators and substrates) and the fact that homozygous mutations in genes encoding key elements, such as K-Ras, are lethal . However, heterozygosity for mutation of the gene encoding K-Ras produces a phenotype that is apparently normal. Ohno et al. showed that, by reducing the amount of K-Ras available in the heterozygous mutants, they could lower the concentration of MEK (MAPK kinase) inhibitors required for blockage of the Ras–MAPK pathway. Thus, a mutation that does not affect behaviour or synaptic physiology can, when combined with a concentration of MEK inhibitor that is itself without effect, produce severe deficits. The beauty of these experiments is not only in the demonstration that drugs and genetics can produce a disruption, but also in the indication of how the various elements of this pathway might interact. By showing that reduction of K-Ras and inhibition of MAPK combine to produce dramatic effects on learning and plasticity, Ohno et al. demonstrated that these two molecules belong to a common functional cascade. Moreover, they showed that the synergistic effect of Ras reduction and MAPK inhibition is specific (Fig. 1). Low doses of NMDA-receptor antagonists, which do not interact directly with the Ras–MAPK pathway, do not inhibit learning or plasticity in K-Ras knockout mice but do so when given to heterozygous αCaMKII mutants. Future implications
The concept of giving drugs to genetically modified animals is an appealingly simple one, and its implications are far reaching. First, it will make many genetic and pharmacological studies ‘cleaner’: it will allow the detrimental affects of some knockout events on general health to be avoided and will also permit the use of drugs at concentrations low enough to reduce the risk of affecting unintended targets. Second, it involves animals heterozygous for a mutation, often the only
viable offspring following the mutation or deletion of a gene. Finally, it should allow more rapid investigations of the interactions between neuronal signalling pathways, by testing for synergistic effects between knockouts and drugs. References 1 Hebb, D.O. (1949) The Organization of Behavior, John Wiley & Sons 2 Thompson, R.F. (1986) The neurobiology of learning and memory. Science 233, 941–947 3 Collingridge, G.L. et al. (1983) The antagonism of amino acid-induced excitations of rat hippocampal CA1 neurones in vitro. J. Physiol. 334, 19–31 4 Morris, R.G.M. et al. (1986) Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP5. Nature 319, 774–776
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5 Silva, A.J. et al. (1992) Deficient hippocampal long-term potentiation in α-calcium–calmodulin kinase II mutant mice. Science 257, 201–206 6 Silva, A.J. et al. (1992) Impaired spatial learning in α-calcium–calmodulin kinase II mutant mice. Science 257, 206–211 7 Grant, S.G.N. et al. (1992) Impaired long-term potentiation, spatial learning, and hippocampal development in fyn mutant mice. Science 258, 1903–1910 8 Mayford, M. and Kandel, E.R. (1999) Genetic approaches to memory storage. Trends Genet. 15, 463–470 9 Tsien, J.Z. et al. (1996) Subregion and cell type restricted gene knockout in mouse brain. Cell 87, 1317–1326 10 Garrick, D. et al. (1998) Repeat-induced gene silencing in mammals. Nat. Genet. 18, 56–59 11 Ohno, M. et al. (2001) Inducible, pharmacogenetic approaches to the study of learning and memory. Nat. Neurosci. 4, 1238–1243
12 Picciotto, M.R. (1999) Knock-out mouse models used to study neurobiological systems. Crit. Rev. Neurobiol. 13, 103–149 13 McKernan, R.M. et al. (2000) Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor α1 subtype. Nat. Neurosci. 3, 587–592 14 Orban, P.C. et al. (1999) Is RAS-dependent signalling necessary for long-term plasticity? Trends Neurosci. 22, 38–44 15 Sweatt, J.D. (2001) The neuronal MAP kinase cascade: a biochemical signal integration system subserving synaptic plasticity and memory. J. Neurochem. 76, 1–10
Paul F. Chapman Cardiff School of Biosciences, Cardiff University, PO Box 911, Cardiff, UK CF10 3US. e-mail: [email protected]
The birth of a memory Leun J. Otten and Michael D. Rugg Laying down new memories has long been thought to involve interactions between the hippocampus and multiple regions of the neocortex. Functional neuroimaging studies performed over the past four years provide evidence for this proposal. A recent electrophysiological study offers a possible mechanism by which interactions between brain regions take place during memory formation.
Since the description of the famous patient H.M. in the late 1950s , it has been recognized that the medial temporal lobe (MTL) plays a crucial role in human memory. Following bilateral resection of the MTL to alleviate persistent epilepsy, H.M. became unable to remember new facts and events for more than a few minutes. Neuropsychological findings such as these indicate that an intact MTL is necessary for normal long-term memory functioning, although it is difficult to discern from such data the extent to which the MTL contributes to memory formation, as opposed to memory storage and retrieval. Studying the neural correlates of memory formation
The question of which neural structures are associated with the formation of lasting human memories can be addressed by recording neural activity while items are initially encoded into memory. A particularly powerful method makes use of the ‘subsequent memory procedure’. http://tins.trends.com
In this approach, neural activity is recorded while volunteers study a sequence of items, after which memory for the items is tested. The activity elicited by items at the time of study is then sorted according to whether the items are remembered or forgotten in the subsequent memory test. Differences between these two types of activity (‘subsequent memory effects’) are taken as candidate neural correlates of encoding. Thus, the procedure provides information about neural structures in which activity is correlated with successful encoding. However, it is important to note that it does not identify which structures are necessary for encoding to occur. The subsequent memory approach has been used with scalp-recorded electrophysiological activity (event-related potentials, or ERPs) for >25 years [2,3]. These studies have provided valuable
information about temporal aspects of the formation of memory traces, but they have been less helpful in identifying the associated brain regions. This is because of both the difficulties of localizing sources of scalp electrical activity and the relative insensitivity of scalp recordings to neural activity in deep brain structures such as the MTL. Non-invasive detection of neural activity with high spatial resolution became possible with the introduction of functional neuroimaging techniques. Memory encoding has been studied since the earliest days of the application of these techniques to cognition , but it was with the advent of ‘event-related’functional MRI (fMRI)  that their full potential was realized. A key development came in 1998, when two papers using the subsequent memory approach with event-related fMRI were published back-to-back in Science [6,7]. These papers described studies of the encoding of visually presented words and pictures, respectively. They showed that relatively greater fMRI signals were elicited by subsequently remembered items in the MTL, among other regions, highlighting a role for the MTL in successful memory encoding. Evidence has now accumulated to suggest that the MTL (both cortex and hippocampus proper) plays a role in encoding across a wide range of task conditions and stimulus materials [3,8–10]. Notwithstanding the puzzling failure to identify encodingrelated MTL activity in some studies
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