frontiers in REVIEW ARTICLE SYSTEMS NEUROSCIENCE published:20 May 2014 dot10.3389/fnsys.2014.00090 Pharmacological enhancement of memory or cognition in normal subjects Gary Lynch12*,Conor D.Cox2 and Christine M.Gall2 Department of Psychiatry and Human Behavior,University of Califomia,Irvine,CA.USA Department of Anatomy and Neurobiology.University of California,Irvine,CA.USA Edited by: The possibility of expanding memory or cognitive capabilities above the levels in high Mikhail Lebedev,Duke University, functioning individuals is a topic of intense discussion among scientists and in society USA at large.The majority of animal studies use behavioral endpoint measures;this has Reviewed by: loan Opris,Wake Forest University, produced valuable information but limited predictability for human outcomes.Accordingly. USA several groups are pursuing a complementary strategy with treatments targeting synaptic Rafael Roesler,Federal University of events associated with memory encoding or forebrain network operations.Transcription Rio Grande do Sul,Brazil and translation figure prominently in substrate work directed at enhancement.Notably. Sam Deadwyler,Wake Forest University Health Sciences,USA the question of why new proteins would be needed for a now-forming memory given Maryam Farahmandfar,Tehran that learning-driven synthesis presumably occurred throughout the immediate past has University of Medical Sciences,Iran been largely ignored.Despite this conceptual problem,and some controversy,recent *Correspondence studies have reinvigorated the idea that selective gene manipulation is a plausible Gary Lynch,Department of route to enhancement.Efforts to improve memory by facilitating synaptic encoding Psychiatry and Human Behavior, Gillespie Neuroscience Research of information have also progressed,in part due of breakthroughs on mechanisms Facility,University of California,837 that stabilize learning-related,long-term potentiation (LTP).These advances point to a Health Science Road,Irvine,CA, reductionistic hypothesis for a diversity of experimental results on enhancement,and 92697.1275,US4 identify under-explored possibilities.Cognitive enhancement remains an elusive goal,in e-mail:glynch@uci.edu part due to the difficulty of defining the target.The popular view of cognition as a collection of definable computations seems to miss the fluid,integrative process experienced by high functioning individuals.The neurobiological approach obviates these psychological issues to directly test the consequences of improving throughput in networks underlying higher order behaviors.The few relevant studies testing drugs that selectively promote excitatory transmission indicate that it is possible to expand cortical networks engaged by complex tasks and that this is accompanied by capabilities not found in normal animals. Keywords:cognitive enhancement,learning,long term potentiation,ampakine,synaptic plasticity,BDNF,F-actin, positive AMPA receptor modulators INTRODUCTION of a system that,while capable of periodically focusing on spe- The present review concerns three topics,two of which involve cific tasks,usually works by integrating a vast amount of disparate terms-enhancement and cognition-that are not sharply material into a product accessible to consciousness.A true cogni- defined.Usage of the former seems straightforward when applied tive enhancer might therefore take the form of a treatment that to memory,although it is often unclear whether accelerated increases the speed or capacity of this assembly process. acquisition or an increase in encoding strength is intended.But Memory enhancement,as suggested,appears to be a much applied to cognition,claims for enhancement face the great prob- more tractable problem.Retention is easily measured as is the lem of how to quantify something for which there is no consensus amount of training needed to produce a given score in a test sub- measurement system.The difficulty can be reduced by focusing sequent to learning.But a curious problem emerges here:few of on cognitive activities of a type that can be described in compu- the many pharmacological agents that produce robust enhance- tational terms.This,however,raises questions about the extent ment of memory in animals are found to have positive effects in to which the sampled process is representative,or a major com- humans.This observation has become the subject of intense pub- ponent,of cognition as the term is typically used.In response,it lic discussion,perhaps with growing skepticism about the utility could reasonably be argued that cognition is a collection of semi- of animal studies on memory enhancement.Some neuroscien- independent operations (e.g.,categorization,value assignment) tists have argued that the"failure to predict"problem reflects the (Sugrue et al.,2005;Tsunada and Sawaguchi,2012)but this seems widespread use of paradigms that have little relevance to human unsatisfactory because the phenomenon is experienced as being, learning.These workers have devised ingenious protocols that can if not unitary,then at least strongly coherent.Electrophysiological be used in rodents and with minor modifications in humans(e.g., and brain imaging results showing coordinated activity across Bari et al.,2008;Demeter et al.,2008;Eichenbaum and Robitsek, broad stretches of neocortex provide some support for the idea 2009;Zeeb et al.,2009;Demeter and Sarter,2013).There is every Frontiers in Systems Neuroscience www.frontiersin.org May 2014 Volume 8 Article 90 1
REVIEW ARTICLE published: 20 May 2014 doi: 10.3389/fnsys.2014.00090 Pharmacological enhancement of memory or cognition in normal subjects Gary Lynch1,2*, Conor D. Cox2 and Christine M. Gall 2 1 Department of Psychiatry and Human Behavior, University of California, Irvine, CA, USA 2 Department of Anatomy and Neurobiology, University of California, Irvine, CA, USA Edited by: Mikhail Lebedev, Duke University, USA Reviewed by: Ioan Opris, Wake Forest University, USA Rafael Roesler, Federal University of Rio Grande do Sul, Brazil Sam Deadwyler, Wake Forest University Health Sciences, USA Maryam Farahmandfar, Tehran University of Medical Sciences, Iran *Correspondence: Gary Lynch, Department of Psychiatry and Human Behavior, Gillespie Neuroscience Research Facility, University of California, 837 Health Science Road, Irvine, CA, 92697-1275, USA e-mail: glynch@uci.edu The possibility of expanding memory or cognitive capabilities above the levels in high functioning individuals is a topic of intense discussion among scientists and in society at large. The majority of animal studies use behavioral endpoint measures; this has produced valuable information but limited predictability for human outcomes. Accordingly, several groups are pursuing a complementary strategy with treatments targeting synaptic events associated with memory encoding or forebrain network operations. Transcription and translation figure prominently in substrate work directed at enhancement. Notably, the question of why new proteins would be needed for a now-forming memory given that learning-driven synthesis presumably occurred throughout the immediate past has been largely ignored. Despite this conceptual problem, and some controversy, recent studies have reinvigorated the idea that selective gene manipulation is a plausible route to enhancement. Efforts to improve memory by facilitating synaptic encoding of information have also progressed, in part due of breakthroughs on mechanisms that stabilize learning-related, long-term potentiation (LTP). These advances point to a reductionistic hypothesis for a diversity of experimental results on enhancement, and identify under-explored possibilities. Cognitive enhancement remains an elusive goal, in part due to the difficulty of defining the target. The popular view of cognition as a collection of definable computations seems to miss the fluid, integrative process experienced by high functioning individuals. The neurobiological approach obviates these psychological issues to directly test the consequences of improving throughput in networks underlying higher order behaviors. The few relevant studies testing drugs that selectively promote excitatory transmission indicate that it is possible to expand cortical networks engaged by complex tasks and that this is accompanied by capabilities not found in normal animals. Keywords: cognitive enhancement, learning, long term potentiation, ampakine, synaptic plasticity, BDNF, F-actin, positive AMPA receptor modulators INTRODUCTION The present review concerns three topics, two of which involve terms—enhancement and cognition—that are not sharply defined. Usage of the former seems straightforward when applied to memory, although it is often unclear whether accelerated acquisition or an increase in encoding strength is intended. But applied to cognition, claims for enhancement face the great problem of how to quantify something for which there is no consensus measurement system. The difficulty can be reduced by focusing on cognitive activities of a type that can be described in computational terms. This, however, raises questions about the extent to which the sampled process is representative, or a major component, of cognition as the term is typically used. In response, it could reasonably be argued that cognition is a collection of semiindependent operations (e.g., categorization, value assignment) (Sugrue et al., 2005; Tsunada and Sawaguchi, 2012) but this seems unsatisfactory because the phenomenon is experienced as being, if not unitary, then at least strongly coherent. Electrophysiological and brain imaging results showing coordinated activity across broad stretches of neocortex provide some support for the idea of a system that, while capable of periodically focusing on specific tasks, usually works by integrating a vast amount of disparate material into a product accessible to consciousness. A true cognitive enhancer might therefore take the form of a treatment that increases the speed or capacity of this assembly process. Memory enhancement, as suggested, appears to be a much more tractable problem. Retention is easily measured as is the amount of training needed to produce a given score in a test subsequent to learning. But a curious problem emerges here: few of the many pharmacological agents that produce robust enhancement of memory in animals are found to have positive effects in humans. This observation has become the subject of intense public discussion, perhaps with growing skepticism about the utility of animal studies on memory enhancement. Some neuroscientists have argued that the “failure to predict” problem reflects the widespread use of paradigms that have little relevance to human learning. These workers have devised ingenious protocols that can be used in rodents and with minor modifications in humans (e.g., Bari et al., 2008; Demeter et al., 2008; Eichenbaum and Robitsek, 2009; Zeeb et al., 2009; Demeter and Sarter, 2013). There is every Frontiers in Systems Neuroscience www.frontiersin.org May 2014 | Volume 8 | Article 90 | 1 SYSTEMS NEUROSCIENCE
Lynch et al. Cognition enhancement in normal subjects reason to assume that these efforts will ultimately narrow the et al.,2001;Plath et al.,2006;Katche et al.,2010,2012).However gap in cross-species comparisons.But there is a more funda- the basic idea that new protein synthesis is critical to memory for- mental issue from comparative biology that could underlie the mation has been controversial since its introduction more than failure-to-predict problem:humans are enormously encephalized 50 years ago (Abraham and Williams,2008;Gold,2008).Much of animals and rodents aren't (neocortex makes up at least 77%of the dispute revolves around the necessary prediction that protein brain volume in human and just 31%in rat;Stephan et al.,1981;synthesis inhibitors will selectively block recently acquired mem- Swanson,1995).Encephalization is hypothesized to result in a ory;most papers report this result but others do not,or argue that shift of functions from lower brain to cortex;from this perspec- observed disruptions to encoding are due to factors unrelated to tive,humans may be using networks of a very different kind than synthesis(Routtenberg,2008;Gold and Wrenn,2012). those employed by rodents to solve similar problems. Beyond this,the protein synthesis argument faces certain con- An alternative to behaviorally based approaches to developing ceptual problems.Learning is a continuous process in humans, enhancers would be to focus on the neurobiological substrates of and likely other mammals,with new encoding occurring many memory and cognition.This seems feasible in the case of mem- times a minute,as is evident with episodic memory.People recog- ory because of the tremendous progress that has been made in nize or recall a remarkable number of serial events when queried identifying synaptic mechanisms that encode information.There after a 90 min movie.Unless we make the very unlikely assump- is no good reason to think that these processes differ significantly tion that each item of information is encoded on a different between mammalian species and indeed comparative studies sug- neuron,it is difficult to see why,after hours of producing pro- gest that certain essential elements are evolutionarily ancient teins needed for consolidation,a given cell would need further (Crystal and Glanzman,2013).It follows from this that treat- synthesis to stabilize a now forming memory.Along this line,it ments acting on memory substrates in rodents are likely to have has been argued that animals exposed to an enriched environ- similar actions in human brain.Cognition again represents a ment which would entail constitutively elevated basal activity,and much more challenging problem.However,the universally held thus activity-driven protein synthesis,may not require additional assumption that cognitive operations arise from the transient synthesis to support LTP(Abraham and Williams,2008)and the formation of telencephalic networks points to a relatively sim- related encoding of hippocampus-dependent memories.There is, ple idea for enhancement.Communication within and between however,a special case in which transcription and/or broadly cortical regions is mediated by glutamatergic transmission;if distributed translation could be required to securely encode a so,then agents that augment the release of glutamate,or the specific memory;namely,a circumstance in which continuous post-synaptic response to it,should facilitate the formation of learning of similar material does not precede the new instance. cognition's substrates. Under these conditions,consolidation could depend upon pro- The following sections consider attempts to develop enhancers teins generated by the isolated learning episode.Note that this via actions on (i)different aspects of the complex machinery scenario loosely describes the great majority of animal studies underlying learning-related synaptic modifications,or(ii)com- testing for the contributions of protein synthesis.Certain of these munication within and between cortical networks. arguments make relatively straightforward,readily tested predic- tions.For example,animals with a well-developed learning set MEMORY ENHANCEMENT could be given protein synthesis inhibitors after learning a single Most research on memory enhancement deals with psychological problem with or without having dealt with many such problems events that precede the actual encoding of information.There is in the preceding hours.Such a paradigm can be achieved for rats for example a very large literature describing attempts,typically using two-odor discriminations.If continual learning obviates the using chemical agents,to increase the speed of learning by mod- need for problem-specific synthesis,then the blockers should have ulating arousal and attention (Lynch et al.,2011).It has become no effect in a group given many trials prior to being introduced common to refer to resultant improvements as cognitive enhance- to the new test items ment,presumably because key elements of cognition are being There is a variant of the translation hypothesis that addresses manipulated,but there are reasons to question this assumption the problem of why prior synthesis doesn't provide a sufficient (see below).There is a smaller,but rapidly growing,body of work supply of proteins for current learning.This involves the ample directed at the machinery responsible for converting patterns of evidence for dendritic (local)translation from already in place afferent activity into the long lasting increases in synaptic strength mRNAs.One could posit a set of conditions in which new synthe- assumed to encode specific information.This section evaluates sis,even after recent experience,needs to occur post-acquisition the latter material. for transfer into long-term storage;e.g.,(1)translation occurs within very small dendritic compartments;(2)such active regions GENE EXPRESSION AND PROTEIN SYNTHESIS are only found in the immediate vicinity of recently modified Work in this area begins with the hypothesis that learning triggers synapses;and(3)newly formed proteins do not diffuse to any the transcription or local translation of proteins that serve to con- great degree.These circumstances would reduce the probabil- solidate the newly acquired memories,something that can take ity that proteins from earlier learning would be present at the anywhere from many minutes to hours.Compounds that facili-large majority of current sites.But"synaptic tagging"experi- tate production of the pertinent RNAs or proteins could accord- ments,conducted for instances where LTP in hippocampal slices ingly increase the likelihood that recent learning will lead to stable is blocked by protein synthesis inhibitors,describe results that are memory,and there are many reports of such effects (Guzowski not consistent with these postulates.Specifically,LTP induction at Frontiers in Systems Neuroscience www.frontiersin.org May 2014 Volume 8 Article 90 2
Lynch et al. Cognition enhancement in normal subjects reason to assume that these efforts will ultimately narrow the gap in cross-species comparisons. But there is a more fundamental issue from comparative biology that could underlie the failure-to-predict problem: humans are enormously encephalized animals and rodents aren’t (neocortex makes up at least 77% of brain volume in human and just 31% in rat; Stephan et al., 1981; Swanson, 1995). Encephalization is hypothesized to result in a shift of functions from lower brain to cortex; from this perspective, humans may be using networks of a very different kind than those employed by rodents to solve similar problems. An alternative to behaviorally based approaches to developing enhancers would be to focus on the neurobiological substrates of memory and cognition. This seems feasible in the case of memory because of the tremendous progress that has been made in identifying synaptic mechanisms that encode information. There is no good reason to think that these processes differ significantly between mammalian species and indeed comparative studies suggest that certain essential elements are evolutionarily ancient (Crystal and Glanzman, 2013). It follows from this that treatments acting on memory substrates in rodents are likely to have similar actions in human brain. Cognition again represents a much more challenging problem. However, the universally held assumption that cognitive operations arise from the transient formation of telencephalic networks points to a relatively simple idea for enhancement. Communication within and between cortical regions is mediated by glutamatergic transmission; if so, then agents that augment the release of glutamate, or the post-synaptic response to it, should facilitate the formation of cognition’s substrates. The following sections consider attempts to develop enhancers via actions on (i) different aspects of the complex machinery underlying learning-related synaptic modifications, or (ii) communication within and between cortical networks. MEMORY ENHANCEMENT Most research on memory enhancement deals with psychological events that precede the actual encoding of information. There is for example a very large literature describing attempts, typically using chemical agents, to increase the speed of learning by modulating arousal and attention (Lynch et al., 2011). It has become common to refer to resultant improvements as cognitive enhancement, presumably because key elements of cognition are being manipulated, but there are reasons to question this assumption (see below). There is a smaller, but rapidly growing, body of work directed at the machinery responsible for converting patterns of afferent activity into the long lasting increases in synaptic strength assumed to encode specific information. This section evaluates the latter material. GENE EXPRESSION AND PROTEIN SYNTHESIS Work in this area begins with the hypothesis that learning triggers the transcription or local translation of proteins that serve to consolidate the newly acquired memories, something that can take anywhere from many minutes to hours. Compounds that facilitate production of the pertinent RNAs or proteins could accordingly increase the likelihood that recent learning will lead to stable memory, and there are many reports of such effects (Guzowski et al., 2001; Plath et al., 2006; Katche et al., 2010, 2012). However, the basic idea that new protein synthesis is critical to memory formation has been controversial since its introduction more than 50 years ago (Abraham and Williams, 2008; Gold, 2008). Much of the dispute revolves around the necessary prediction that protein synthesis inhibitors will selectively block recently acquired memory; most papers report this result but others do not, or argue that observed disruptions to encoding are due to factors unrelated to synthesis (Routtenberg, 2008; Gold and Wrenn, 2012). Beyond this, the protein synthesis argument faces certain conceptual problems. Learning is a continuous process in humans, and likely other mammals, with new encoding occurring many times a minute, as is evident with episodic memory. People recognize or recall a remarkable number of serial events when queried after a 90 min movie. Unless we make the very unlikely assumption that each item of information is encoded on a different neuron, it is difficult to see why, after hours of producing proteins needed for consolidation, a given cell would need further synthesis to stabilize a now forming memory. Along this line, it has been argued that animals exposed to an enriched environment which would entail constitutively elevated basal activity, and thus activity-driven protein synthesis, may not require additional synthesis to support LTP (Abraham and Williams, 2008) and the related encoding of hippocampus-dependent memories. There is, however, a special case in which transcription and/or broadly distributed translation could be required to securely encode a specific memory; namely, a circumstance in which continuous learning of similar material does not precede the new instance. Under these conditions, consolidation could depend upon proteins generated by the isolated learning episode. Note that this scenario loosely describes the great majority of animal studies testing for the contributions of protein synthesis. Certain of these arguments make relatively straightforward, readily tested predictions. For example, animals with a well-developed learning set could be given protein synthesis inhibitors after learning a single problem with or without having dealt with many such problems in the preceding hours. Such a paradigm can be achieved for rats using two-odor discriminations. If continual learning obviates the need for problem-specific synthesis, then the blockers should have no effect in a group given many trials prior to being introduced to the new test items. There is a variant of the translation hypothesis that addresses the problem of why prior synthesis doesn’t provide a sufficient supply of proteins for current learning. This involves the ample evidence for dendritic (local) translation from already in place mRNAs. One could posit a set of conditions in which new synthesis, even after recent experience, needs to occur post-acquisition for transfer into long-term storage; e.g., (1) translation occurs within very small dendritic compartments; (2) such active regions are only found in the immediate vicinity of recently modified synapses; and (3) newly formed proteins do not diffuse to any great degree. These circumstances would reduce the probability that proteins from earlier learning would be present at the large majority of current sites. But “synaptic tagging” experiments, conducted for instances where LTP in hippocampal slices is blocked by protein synthesis inhibitors, describe results that are not consistent with these postulates. Specifically, LTP induction at Frontiers in Systems Neuroscience www.frontiersin.org May 2014 | Volume 8 | Article 90 | 2
Lynch et al. Cognition enhancement in normal subjects one input protects subsequently induced potentiation at a second 2003),and does not disturb already potentiated contacts as likely input to the same region from the effects of the inhibitor (Frey required for a high capacity memory system.A very large body of and Morris,1997;Shires et al.,2012).Given the small number experimental work has confirmed the tight connection between of synapses that generate EPSPs of conventional amplitudes,it LTP and diverse instances of memory (e.g.,Roman et al.,1987; is extremely likely that connections from the two inputs are,for Rioult-Pedotti et al.,2000;Whitlock et al.,2006).Moreover,LTP the most part,located on different dendritic segments.It follows is intimately related to the theta rhythm,an oscillation long asso- then that proteins from the first episode must have been synthe- ciated with learning (Buzsaki,2005;Vertes,2005;Snider et al., sized,or traveled,throughout much of the dendritic arborization, 2013);i.e.,five brief(30 ms)bursts of high frequency stimulation a point that is reinforced by evidence for tagging in the apical den- pulses(a pattern that mimics"theta bursting"during learning) drites after stimulation of basal afferents (Alarcon et al.,2006).It prove to be near optimal for inducing extremely stable LTP but will be noted that these findings align with the broad idea that only when separated by the period of the theta wave (Larson continual learning maintains relevant proteins at levels sufficient et al.,1986;Capocchi et al.,1992).The reasons for this have been for LTP-related plasticity,obviating the need for synthesis after identified (Figure 1). individual learning events. These observations suggest the possibility of enhancing learn- The above discussion concerns interpretative issues rather than ing with drugs that promote theta activity and correlated bursts the likelihood of achieving enhancement using the transcription/of high frequency discharges.Agents such as physostigmine, translation strategy.It may well be the case that increasing within- that facilitate central cholinergic transmission,promote the theta cell levels of proteins that support consolidation reduces the rhythm (Olpe et al.,1987;Hasselmo,2006)and are reported requirements for encoding persistent memories and/or increases to improve learning scores in certain experimental situations. their stability.Signaling from synapses to the nucleus or to local Notably,drugs of this type are among the few treatments protein synthesis machinery involves many steps and so is likely approved for Alzheimer's Disease (Clarke and Francis,2005; to be a variable and somewhat uncertain process.It would not Noetzli and Eap,2013).However,cholinergic systems perform be surprising,then,if the ongoing production of memory-related varied functions in brain,some of which are homeostatic in elements operates at a less than optimal rate even in high per- nature.This likely explains why drugs targeting cholinergic forming,normal subjects.In line with this,there are multiple mechanisms have not gained widespread acceptance as plausi- demonstrations that treatment with compounds that inhibit par- ble enhancers.Another approach based on theta activity involves ticular histone deacetylases,leading to increased transcription of the large hyperpolarizing potentials triggered within target neu- select gene families,can markedly enhance memory after single rons by the short train of theta bursts used to induce LTP.These training sessions (Stefanko et al.,2009;McQuown et al.,2011).after-hyperpolarizing potentials(AHPs),set in motion by cell dis- Also of interest are the numerous studies showing that selective charges,persist throughout the duration of the theta train and phosphodiesterase-4 inhibitors have potent enhancing effects on serve to counteract the depolarization needed to unblock the memory.Inhibitors of this class(e.g.,Rolipram),drive the protein voltage dependent,synaptic NMDA receptors.Influx of calcium kinase A-CREB transcription pathway implicated in learning through these receptors,followed by release of the cation from in a broad array of animals (including invertebrates),and so intracellular stores,triggers the chain of events leading to poten- is argued to be a very ancient,evolutionarily conserved mem- tiation (Figure 1).AHPs are mediated by a set of voltage-and ory substrate (Tully et al.,2003;Normann and Berger,2008).calcium-sensitive potassium channels,prominent among which Evidence that the same results obtain after extensive experience is the SK3 channel (Hosseini et al.,2001).The bee toxin apamin with similar problems in the recent past,and presumably a great blocks this channel with some selectivity and,as predicted,aug- deal of learning-driven transcription,would constitute support ments post-synaptic responses to theta burst trains;this results in for there being less than optimal production of proteins needed a striking increase in the magnitude of LTP (Kramar et al.,2004). for encoding under normal circumstances.This would certainly While a number of studies have found substantial improve- encourage the idea that enhanced protein synthesis is a viable ments in rodent learning with apamin treatment (Ikonen and route to augmented memory. Riekkinen,1999;Brennan et al.,2008;Vick et al.,2010),this is not a likely enhancer because of toxicology issues.But given SYNAPTIC PLASTICITY AND MEMORY ENHANCEMENT increasing interest in applications of channel blockers for diverse Most mechanism-based efforts directed at improving memory clinical problems,the apamin results suggest an intriguing mech- have focused on synaptic plasticity and in particular the long term anistic target for the development of enhancers.It is of note in this potentiation (LTP)effect.Researchers since the late 19th cen- regard that Brain Derived Neurotrophic Factor (BDNF),which tury have argued that the enormous capacity of memory is best appears to be released from terminals by theta bursts(Balkowiec explained by assuming that physical encoding of new information and Katz,2000;Chen et al.,2010b),also reduces AHPs at least occurs at small numbers of connections between neurons.The in rats (Kramar et al,2004).Elevating endogenous levels of discovery of LTP demonstrated that individual synapses in the this neurotrophin,which can be achieved by pharmacological cortical telencephalon do in fact possess the properties expected manipulations described later,thus provides another avenue for for a memory substrate (Bliss and Collingridge,1993;Lynch, enhancement. 1998,2004b;Morris,2003).The increase in transmission strength Identification of the initial triggers for LTP,as schematized in (magnitude of EPSCs)develops quickly,persists for a remark- Figure 1,pointed to NMDA receptor-mediated calcium influxes able period (weeks at least)(Staubli and Lynch,1987;Abraham, as a logical target for enhancement.The existence of multiple Frontiers in Systems Neuroscience www.frontiersin.org May 2014 Volume 8 Article 90 3
Lynch et al. Cognition enhancement in normal subjects one input protects subsequently induced potentiation at a second input to the same region from the effects of the inhibitor (Frey and Morris, 1997; Shires et al., 2012). Given the small number of synapses that generate EPSPs of conventional amplitudes, it is extremely likely that connections from the two inputs are, for the most part, located on different dendritic segments. It follows then that proteins from the first episode must have been synthesized, or traveled, throughout much of the dendritic arborization, a point that is reinforced by evidence for tagging in the apical dendrites after stimulation of basal afferents (Alarcon et al., 2006). It will be noted that these findings align with the broad idea that continual learning maintains relevant proteins at levels sufficient for LTP-related plasticity, obviating the need for synthesis after individual learning events. The above discussion concerns interpretative issues rather than the likelihood of achieving enhancement using the transcription / translation strategy. It may well be the case that increasing withincell levels of proteins that support consolidation reduces the requirements for encoding persistent memories and/or increases their stability. Signaling from synapses to the nucleus or to local protein synthesis machinery involves many steps and so is likely to be a variable and somewhat uncertain process. It would not be surprising, then, if the ongoing production of memory-related elements operates at a less than optimal rate even in high performing, normal subjects. In line with this, there are multiple demonstrations that treatment with compounds that inhibit particular histone deacetylases, leading to increased transcription of select gene families, can markedly enhance memory after single training sessions (Stefanko et al., 2009; McQuown et al., 2011). Also of interest are the numerous studies showing that selective phosphodiesterase-4 inhibitors have potent enhancing effects on memory. Inhibitors of this class (e.g., Rolipram), drive the protein kinase A—CREB transcription pathway implicated in learning in a broad array of animals (including invertebrates), and so is argued to be a very ancient, evolutionarily conserved memory substrate (Tully et al., 2003; Normann and Berger, 2008). Evidence that the same results obtain after extensive experience with similar problems in the recent past, and presumably a great deal of learning-driven transcription, would constitute support for there being less than optimal production of proteins needed for encoding under normal circumstances. This would certainly encourage the idea that enhanced protein synthesis is a viable route to augmented memory. SYNAPTIC PLASTICITY AND MEMORY ENHANCEMENT Most mechanism-based efforts directed at improving memory have focused on synaptic plasticity and in particular the long term potentiation (LTP) effect. Researchers since the late 19th century have argued that the enormous capacity of memory is best explained by assuming that physical encoding of new information occurs at small numbers of connections between neurons. The discovery of LTP demonstrated that individual synapses in the cortical telencephalon do in fact possess the properties expected for a memory substrate (Bliss and Collingridge, 1993; Lynch, 1998, 2004b; Morris, 2003). The increase in transmission strength (magnitude of EPSCs) develops quickly, persists for a remarkable period (weeks at least) (Staubli and Lynch, 1987; Abraham, 2003), and does not disturb already potentiated contacts as likely required for a high capacity memory system. A very large body of experimental work has confirmed the tight connection between LTP and diverse instances of memory (e.g., Roman et al., 1987; Rioult-Pedotti et al., 2000; Whitlock et al., 2006). Moreover, LTP is intimately related to the theta rhythm, an oscillation long associated with learning (Buzsaki, 2005; Vertes, 2005; Snider et al., 2013); i.e., five brief (30 ms) bursts of high frequency stimulation pulses (a pattern that mimics “theta bursting” during learning) prove to be near optimal for inducing extremely stable LTP but only when separated by the period of the theta wave (Larson et al., 1986; Capocchi et al., 1992). The reasons for this have been identified (Figure 1). These observations suggest the possibility of enhancing learning with drugs that promote theta activity and correlated bursts of high frequency discharges. Agents such as physostigmine, that facilitate central cholinergic transmission, promote the theta rhythm (Olpe et al., 1987; Hasselmo, 2006) and are reported to improve learning scores in certain experimental situations. Notably, drugs of this type are among the few treatments approved for Alzheimer’s Disease (Clarke and Francis, 2005; Noetzli and Eap, 2013). However, cholinergic systems perform varied functions in brain, some of which are homeostatic in nature. This likely explains why drugs targeting cholinergic mechanisms have not gained widespread acceptance as plausible enhancers. Another approach based on theta activity involves the large hyperpolarizing potentials triggered within target neurons by the short train of theta bursts used to induce LTP. These after-hyperpolarizing potentials (AHPs), set in motion by cell discharges, persist throughout the duration of the theta train and serve to counteract the depolarization needed to unblock the voltage dependent, synaptic NMDA receptors. Influx of calcium through these receptors, followed by release of the cation from intracellular stores, triggers the chain of events leading to potentiation (Figure 1). AHPs are mediated by a set of voltage- and calcium-sensitive potassium channels, prominent among which is the SK3 channel (Hosseini et al., 2001). The bee toxin apamin blocks this channel with some selectivity and, as predicted, augments post-synaptic responses to theta burst trains; this results in a striking increase in the magnitude of LTP (Kramar et al., 2004). While a number of studies have found substantial improvements in rodent learning with apamin treatment (Ikonen and Riekkinen, 1999; Brennan et al., 2008; Vick et al., 2010), this is not a likely enhancer because of toxicology issues. But given increasing interest in applications of channel blockers for diverse clinical problems, the apamin results suggest an intriguing mechanistic target for the development of enhancers. It is of note in this regard that Brain Derived Neurotrophic Factor (BDNF), which appears to be released from terminals by theta bursts (Balkowiec and Katz, 2000; Chen et al., 2010b), also reduces AHPs at least in rats (Kramar et al., 2004). Elevating endogenous levels of this neurotrophin, which can be achieved by pharmacological manipulations described later, thus provides another avenue for enhancement. Identification of the initial triggers for LTP, as schematized in Figure 1, pointed to NMDA receptor-mediated calcium influxes as a logical target for enhancement. The existence of multiple Frontiers in Systems Neuroscience www.frontiersin.org May 2014 | Volume 8 | Article 90 | 3
Lynch et al. Cognition enhancement in normal subjects A AMPA-Rs NMDA-Rs AHP membrane voltage first theta second theta burst burst (+NMDA) first theta burst second theta burst GABA-A GABA-A GABA GABA-B interneuron autoreceptors maximum hyperpolarization at 180 msec FIGURE 1|Why theta burst stimulation (TBS)is so effective at after-hyperpolarization (AHP).The AHP which is largely mediated by inducing LTP.TBS (Larson et al.,1986)mimics a firing pattern found in calcium and voltage dependent potassium channels,tends to counteract cortical neurons during leaming (Otto et al..1991)and elicits a robust, the depolarization produced by the burst,thus capping the magnitude of non-decremental LTP that persists for weeks (at least).(A)(left side).A NMDA receptor responses.(C-E)How the second and subsequent single stimulation pulse releases glutamate (black dots)and a partial bursts generate large depolarization and unblock NMDA receptors.(C)A membrane depolarization via current flux through AMPA receptors (dotted glutamatergic axon innervates a pyramidal cell dendrite (gray)and a line).NMDA receptors do not open because of voltage dependent block feedforward,GABAergic interneuron (orange);note that both contacts use of the ion channel (open circle).(right side)Trains of high frequency AMPA receptors (red).(D)A first theta burst triggers GABA release from stimulation cause a greater depolarization (light blue)that removes the the interneuron onto the pyramidal neuron thereby producing a channel block and thereby allows current flow through calcium permeant di-synaptic (slightly delayed)IPSC via post-synaptic GABA-A receptors NMDA receptors.Calcium is the initial trigger for LTP (B)Intracellular (orange ellipses);this shunts the EPSCs produced at neighboring recording shows that the first theta burst (four pulses at 100 Hz)in a glutamatergic synapses.The released GABA also binds to pre-synaptic, train causes a relatively modest depolarization accompanied by a single metabotrophic GABA-B auto-receptors on the releasing intemeuron spike;NMDA receptors make a very small contribution to this response. terminal (purple).(E)The auto-receptors hyperpolarize the GABAergic A second burst administered after a delay corresponding to the period of terminal and block release,an effect that reaches its maximum at the the theta wave produces a more profound depolarization with multiple period of the theta wave.A theta burst arriving at this time point spikes;this burst response contains a large NMDA receptor mediated generates an excitatory response that is only weakly counteracted by the component.Note that each theta burst in the train is followed by a large opening of post-synaptic GABA-A receptors (see B). modulatory sites (e.g.,for glycine and polyamine)on the recep- cholesterol metabolite that facilitates NMDA receptor currents tors suggested a plausible route for building positive allosteric through a novel oxysterol modulatory site and markedly increases drugs (Monaghan et al,2012).Most of this effort has been the magnitude of LTP(Paul et al,2013).The development of pos- directed toward treatments for neuropathology and psychiatric itive NMDA receptor modulators is clearly a promising area with disorders,most notably schizophrenia and depression(Labrie and regard to enhancement. Roder,2010;Dang et al.,2014),rather than memory enhance- Increasing current flux through AMPA receptors results ment.Perhaps the most widely studied agent of this type is in greater post-synaptic depolarization and thereby promotes D-cycloserine,a compound that targets the glycine binding removal of the voltage block on NMDA receptors.This sug- pocket on the receptor and facilitates channel opening(Sheinin gests that increasing AMPA receptor currents should facilitate the et al.,2001;Dravid et al.,2010).It has been known for some induction of LTP.Tests of this became possible with the inven- time that the site is important for induction of LTP(Oliver et al.,tion of AMPA receptor modulators that freely enter the brain and 1990)and,as expected from this,D-cycloserine enhances var- increase fast glutamatergic transmission(Lynch,2004a).The ini- ious forms of memory in animals (Flood et al,1992;Baxter tial positive modulators were small benzamide compounds but et al.,1994;Tsai et al.,1999;Normann and Berger,2008;Peters subsequent work from many laboratories resulted in diverse fam- and De Vries,2013).There is also evidence that the endogenous ilies of compounds that slow deactivation or desensitization (or neurosteroid pregnenolone sulfate (Wu et al,1991),and other both)of ligand bound AMPA receptors.Here we will refer to steroid-like substances(Madau et al.,2009),promote the open- all agents of this type by the term,"ampakines,"used for the ing of NMDA receptors and facilitate both LTP and memory.Also original compounds.Through a series of electrophysiological and of note,recent work led to discovery of a naturally occurring X-ray crystallography studies,the mechanism of ampakine action Frontiers in Systems Neuroscience www.frontiersin.org May 2014 Volume 8 Article 90 4
Lynch et al. Cognition enhancement in normal subjects FIGURE 1 | Why theta burst stimulation (TBS) is so effective at inducing LTP. TBS (Larson et al., 1986) mimics a firing pattern found in cortical neurons during learning (Otto et al., 1991) and elicits a robust, non-decremental LTP that persists for weeks (at least). (A) (left side). A single stimulation pulse releases glutamate (black dots) and a partial membrane depolarization via current flux through AMPA receptors (dotted line). NMDA receptors do not open because of voltage dependent block of the ion channel (open circle). (right side) Trains of high frequency stimulation cause a greater depolarization (light blue) that removes the channel block and thereby allows current flow through calcium permeant NMDA receptors. Calcium is the initial trigger for LTP. (B) Intracellular recording shows that the first theta burst (four pulses at 100 Hz) in a train causes a relatively modest depolarization accompanied by a single spike; NMDA receptors make a very small contribution to this response. A second burst administered after a delay corresponding to the period of the theta wave produces a more profound depolarization with multiple spikes; this burst response contains a large NMDA receptor mediated component. Note that each theta burst in the train is followed by a large after-hyperpolarization (AHP). The AHP, which is largely mediated by calcium and voltage dependent potassium channels, tends to counteract the depolarization produced by the burst, thus capping the magnitude of NMDA receptor responses. (C–E) How the second and subsequent bursts generate large depolarization and unblock NMDA receptors. (C) A glutamatergic axon innervates a pyramidal cell dendrite (gray) and a feedforward, GABAergic interneuron (orange); note that both contacts use AMPA receptors (red). (D) A first theta burst triggers GABA release from the interneuron onto the pyramidal neuron thereby producing a di-synaptic (slightly delayed) IPSC via post-synaptic GABA-A receptors (orange ellipses); this shunts the EPSCs produced at neighboring glutamatergic synapses. The released GABA also binds to pre-synaptic, metabotrophic GABA-B auto-receptors on the releasing interneuron terminal (purple). (E) The auto-receptors hyperpolarize the GABAergic terminal and block release, an effect that reaches its maximum at the period of the theta wave. A theta burst arriving at this time point generates an excitatory response that is only weakly counteracted by the opening of post-synaptic GABA-A receptors (see B). modulatory sites (e.g., for glycine and polyamine) on the receptors suggested a plausible route for building positive allosteric drugs (Monaghan et al., 2012). Most of this effort has been directed toward treatments for neuropathology and psychiatric disorders, most notably schizophrenia and depression (Labrie and Roder, 2010; Dang et al., 2014), rather than memory enhancement. Perhaps the most widely studied agent of this type is D-cycloserine, a compound that targets the glycine binding pocket on the receptor and facilitates channel opening (Sheinin et al., 2001; Dravid et al., 2010). It has been known for some time that the site is important for induction of LTP (Oliver et al., 1990) and, as expected from this, D-cycloserine enhances various forms of memory in animals (Flood et al., 1992; Baxter et al., 1994; Tsai et al., 1999; Normann and Berger, 2008; Peters and De Vries, 2013). There is also evidence that the endogenous neurosteroid pregnenolone sulfate (Wu et al., 1991), and other steroid-like substances (Madau et al., 2009), promote the opening of NMDA receptors and facilitate both LTP and memory. Also of note, recent work led to discovery of a naturally occurring cholesterol metabolite that facilitates NMDA receptor currents through a novel oxysterol modulatory site and markedly increases the magnitude of LTP (Paul et al., 2013). The development of positive NMDA receptor modulators is clearly a promising area with regard to enhancement. Increasing current flux through AMPA receptors results in greater post-synaptic depolarization and thereby promotes removal of the voltage block on NMDA receptors. This suggests that increasing AMPA receptor currents should facilitate the induction of LTP. Tests of this became possible with the invention of AMPA receptor modulators that freely enter the brain and increase fast glutamatergic transmission (Lynch, 2004a). The initial positive modulators were small benzamide compounds but subsequent work from many laboratories resulted in diverse families of compounds that slow deactivation or desensitization (or both) of ligand bound AMPA receptors. Here we will refer to all agents of this type by the term, “ampakines,” used for the original compounds. Through a series of electrophysiological and X-ray crystallography studies, the mechanism of ampakine action Frontiers in Systems Neuroscience www.frontiersin.org May 2014 | Volume 8 | Article 90 | 4
Lynch et al. Cognition enhancement in normal subjects is now fairly well understood.As illustrated in Figure 2,each between AMPA and NMDA receptor pharmacology:compounds subunit of the tetrameric AMPA receptor has two large extra- widely used to block the former also exhibit high affinity antag- cellular domains that form a"clamshell"that closes upon gluta- onism of the glycine modulatory site on the latter(Kessler et al., mate binding (Sun et al.,2002).Relaxation to the resting state, 1989).However,the ampakine pocket is distant to the extracel- and transmitter release,terminates current flow;this process is lular domain of AMPA receptor antagonist binding and there referred to as"deactivation."The four subunits form two dimers,is no evidence that these drugs affect NMDA receptor-gated an arrangement that can be disrupted by ligand binding;under currents. these conditions the channel closes but the transmitter is retained. Early work established that ampakines enhance both LIP and This interesting,high affinity (slow dissociation constant)state memory (Granger et al.,1993;Staubli et al.,1994),results that constitutes the desensitized condition of the receptor (Hall et al., have been multiply replicated by different groups(Lynch,2004a; 1993).It was originally thought that desensitization is the normal Lynch and Gall,2013).Versions of the drugs that simply slow route for terminating the EPSC but it now appears that deacti-deactivation lower the threshold for inducing LTP whereas those vation is responsible for the decay rate of the synaptic response. that affect both deactivation and desensitization also raise the The ampakine binding pocket is located at the dimer interface ceiling on the degree of potentiation produced by theta bursts near the hinge of the clamshell(Jin et al,2005);this strategic (Arai et al,2002).By changing rate constants for both recep- position explains how ampakines can affect both deactivation tor inactivation processes,the latter compounds lead to much and desensitization (Arai et al.,1996)(Figure 2).Apparently, longer EPSCs and thus prolonged NMDA receptor-mediated cal- the orientation of the compounds within the pocket determines cium influxes.This presumably explains their greater potency. which of the two processes is most affected.There is overlap Surprisingly,there appear to have been no studies testing for A resting bound c deactivation deactivation rate (1 ms pulse) plus membrane ampakine 的 bound,channel ampakine open binding pocket desensitization hinge'for extracellular domains also,dimer interface glutamate desensitization rate(500 ms plus ampakine pulse) ampakine bound,channel closed FIGURE 2 Mode of action for positive allosteric modulators of AMPA ms pulse of glutamate to an excised patch:delivery of the ligand causes a receptors (ampakines).(A)Schematic shows two of the four subunits that sharp influx of current that decays after rapid washout.Bound ampakines comprise the AMPA receptor tetramer in the resting state;the C-tails are not slow reopening,resulting in a significant retardation of deactivation (bottom included.Each subunit has two large extracellular domains that form a trace).(D)Prolonged stimulation of the receptor can disrupt the dimer "clamshell"containing the glutamate binding site.The hinge of the structure configuration,leading to a condition in which transmitter remains bound but is indicated by the double circle.The subunits dimerize at a zone close to the the ion channel returns to the closed state (desensitization).The upper trace hinge.The ampakine pocket (star)is strategically located at the dimer to the right describes an instance of this in which glutamate was applied for interface adjacent to the two hinges.There are thus four neurotransmitter, 500 msec.An initial influx of current was followed by decay,despite and two ampakine,sites on the full AMPA receptor.(B)Glutamate binding is continuing presence of the transmitter,to a steady state value about 1/10 of accompanied by a closing of each subunits'clamshell,resulting in opening of the peak flux.Ampakines stabilize the dimer configuration and,as predicted, the ion channel and inward flux of current.The receptor then shifts into one greatly slow desensitization-current flow continues throughout the 500 ms of two configurations;the gray arrows denote the time required for the application of glutamate.The receptor structural dynamics,including transitions in the presence (dotted)and absence (solid line)of an ampakine. interactions with an ampakine,illustrated here are based on X-ray (C)Normally,single transmission events are followed by opening of the crystallography studies (Sun et al.,2002:Jin et al.,2005);physiological data extracellular domains and release of the transmitter,a process referred to as are from patches taken from hippocampal slices (Arai et al..1996.2002:Arai 'deactivation."The upper trace to the right describes deactivation after a one and Lynch,1998). Frontiers in Systems Neuroscience www.frontiersin.org May 2014 Volume 8 Article 905
Lynch et al. Cognition enhancement in normal subjects is now fairly well understood. As illustrated in Figure 2, each subunit of the tetrameric AMPA receptor has two large extracellular domains that form a “clamshell” that closes upon glutamate binding (Sun et al., 2002). Relaxation to the resting state, and transmitter release, terminates current flow; this process is referred to as “deactivation.” The four subunits form two dimers, an arrangement that can be disrupted by ligand binding; under these conditions the channel closes but the transmitter is retained. This interesting, high affinity (slow dissociation constant) state constitutes the desensitized condition of the receptor (Hall et al., 1993). It was originally thought that desensitization is the normal route for terminating the EPSC but it now appears that deactivation is responsible for the decay rate of the synaptic response. The ampakine binding pocket is located at the dimer interface near the hinge of the clamshell (Jin et al., 2005); this strategic position explains how ampakines can affect both deactivation and desensitization (Arai et al., 1996) (Figure 2). Apparently, the orientation of the compounds within the pocket determines which of the two processes is most affected. There is overlap between AMPA and NMDA receptor pharmacology: compounds widely used to block the former also exhibit high affinity antagonism of the glycine modulatory site on the latter (Kessler et al., 1989). However, the ampakine pocket is distant to the extracellular domain of AMPA receptor antagonist binding and there is no evidence that these drugs affect NMDA receptor-gated currents. Early work established that ampakines enhance both LTP and memory (Granger et al., 1993; Staubli et al., 1994), results that have been multiply replicated by different groups (Lynch, 2004a; Lynch and Gall, 2013). Versions of the drugs that simply slow deactivation lower the threshold for inducing LTP whereas those that affect both deactivation and desensitization also raise the ceiling on the degree of potentiation produced by theta bursts (Arai et al., 2002). By changing rate constants for both receptor inactivation processes, the latter compounds lead to much longer EPSCs and thus prolonged NMDA receptor-mediated calcium influxes. This presumably explains their greater potency. Surprisingly, there appear to have been no studies testing for FIGURE 2 | Mode of action for positive allosteric modulators of AMPA receptors (ampakines). (A) Schematic shows two of the four subunits that comprise the AMPA receptor tetramer in the resting state; the C-tails are not included. Each subunit has two large extracellular domains that form a “clamshell” containing the glutamate binding site. The hinge of the structure is indicated by the double circle. The subunits dimerize at a zone close to the hinge. The ampakine pocket (star) is strategically located at the dimer interface adjacent to the two hinges. There are thus four neurotransmitter, and two ampakine, sites on the full AMPA receptor. (B) Glutamate binding is accompanied by a closing of each subunits’ clamshell, resulting in opening of the ion channel and inward flux of current. The receptor then shifts into one of two configurations; the gray arrows denote the time required for the transitions in the presence (dotted) and absence (solid line) of an ampakine. (C) Normally, single transmission events are followed by opening of the extracellular domains and release of the transmitter, a process referred to as “deactivation.” The upper trace to the right describes deactivation after a one ms pulse of glutamate to an excised patch: delivery of the ligand causes a sharp influx of current that decays after rapid washout. Bound ampakines slow reopening, resulting in a significant retardation of deactivation (bottom trace). (D) Prolonged stimulation of the receptor can disrupt the dimer configuration, leading to a condition in which transmitter remains bound but the ion channel returns to the closed state (desensitization). The upper trace to the right describes an instance of this in which glutamate was applied for 500 msec. An initial influx of current was followed by decay, despite continuing presence of the transmitter, to a steady state value about 1/10 of the peak flux. Ampakines stabilize the dimer configuration and, as predicted, greatly slow desensitization—current flow continues throughout the 500 ms application of glutamate. The receptor structural dynamics, including interactions with an ampakine, illustrated here are based on X-ray crystallography studies (Sun et al., 2002; Jin et al., 2005); physiological data are from patches taken from hippocampal slices (Arai et al., 1996, 2002; Arai and Lynch, 1998). Frontiers in Systems Neuroscience www.frontiersin.org May 2014 | Volume 8 | Article 90 | 5