NRN 3597

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New technologies for examining the role of neuronal ensembles in drug addiction and fear
Fabio C.Cruz, Eisuke Koya, Danielle H.Guez-Barber, Jennifer M.Bossert, Carl R.Lupica, Yavin Shaham and Bruce T.Hope
roles of neuronal activity in behaviour have manipulated activity in whole brain areas such as permanent excitotoxic lesions, reversible inactivation using GABAergic agonists or the sodium channel blocker tetrodotoxin, intracranial injections of selective receptor antagonists and activation or inhibition using optogenetics or DREADD (designer receptors exclusively activated by designer drugs)23. However, these methods affect neuronal activity of either all neurons or all neurons of a particular cell type, regardless of their activation state during the learned behaviours. In addition, until recently, it was not possible to characterize molecular and electrophysiological alterations within the activated neuronal ensembles that presumably mediate memory formation and learned behaviours. Indeed, the published literature on synaptic plasticity (as assessed by exvivo slice electrophysiology techniques) and molecular mechanisms of learning and memory are based on results obtained from either randomly selected neurons or neurons of a particular cell type independently of their activation state during learned behaviours2427. The goal of this article is to describe several recent technical developments that make it possible to determine causal roles of putative activated neuronal ensembles in learned behaviours, and to characterize molecular and electrophysiological alterations within the activated neurons. We first describe three recently developed tools to study the role of neuronal ensembles in conditioned drug effects and relapse in rats. These include the Daun02 inactivation method28, FACS (flow cytometry and fluorescence-activated cell sorting) of activated FOS-expressing neurons29 and Fos-GFP (green fluorescent protein) rats30, which can be used to selectively inactivate neuronal ensembles and assess molecular and electrophysiological alterations within activated neuronal ensembles. We also describe two other methods that have been used to study the role of neuronal ensembles in conditioned fear: inactivation of CREB (cyclicAMP-responsive elementbinding protein)-overexpressing neurons31,32 and Fos-tTA (tetracycline-off transcriptional activator) mice16,19,33. All of these methods rely on the Fos gene promoter to manipulate the activity of strongly activated neurons
VOLUME 14 | NOVEMBER 2013 | 743 2013 Macmillan Publishers Limited. All rights reserved
Abstract | Correlational data suggest that learned associations are encoded within neuronal ensembles. However, it has been difficult to prove that neuronal ensembles mediate learned behaviours because traditional pharmacological and lesion methods, and even newer cell type-specific methods, affect both activated and non-activated neurons. In addition, previous studies on synaptic and molecular alterations induced by learning did not distinguish between behaviourally activated and non-activated neurons. Here, we describe three new approaches Daun02 inactivation, FACS sorting of activated neurons and Fos-GFP transgenic rats that have been used to selectively target and study activated neuronal ensembles in models of conditioned drug effects and relapse. We also describe two new tools Fos-tTA transgenic mice and inactivation of CREB-overexpressing neurons that have been used to study the role of neuronal ensembles in conditioned fear.
In 1949, Hebb1 proposed that learned associations are encoded within specific patterns of neurons called cell assemblies (now called neuronal ensembles) that were selectively activated by environmental cues. Since then, many electrophysiology and cellular imaging studies have found correlational evidence that supports the idea that learned associations between environmental cues and unconditioned rewards are encoded by neuronal ensembles that are activated by these same cues and rewards2 (FIG.1). The neuronal ensemble hypothesis has had a transforming and long-lasting impact on modern neuroscience research, and has provided the conceptual framework for numerous learning and memory studies28. Since the 1950s9, investigators have primarily used invivo electrophysiology to characterize temporal activity patterns of putative neuronal ensembles in different brain areas in learned behaviours5,1013. Since the late 1990s, investigators have also used double-labelling methods with immediate-early genes (IEGs) as markers of neural activity to characterize the spatial pattern of activated neuronal
NATURE REVIEWS | NEUROSCIENCE
ensembles in the brain1419 (BOX1). More recently, invivo two-photon calcium imaging methods were developed to simultaneously record from hundreds of activated neurons20. These methods, which use calcium-sensitive synthetic dyes and genetically encoded calcium indicator proteins (GCaMPs), have been used to record learning-related alterations in the activity of neuronal ensembles in head-fixed21 or freely moving, awake behavingmice22. An extensive literature, which has been reviewed in the citations above and in many other reviews, supports the notion that neuronal ensemble activity encodes diverse forms of learned associations that mediate learned behaviours. However, until recently, this evidence was based only on correlations between behaviour and invivo electrophysiological neuronal activity or two-photon calcium-imaging patterns during learning and memory tasks or post-mortem activity patterns of different IEGs. Therefore, a causal role of the putative neuronal ensembles in learned behaviours had not been established. Until recently, all methods for assessing causal
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Environmental stimuli Interoceptive stimuli
Neocortex
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Figure 1 | Neuronal ensembles in the mesocorticolimbic dopamine reward system. Hypothetical Nature Reviews | Neuroscience schematic of how drug-associated stimuli activate specific patterns of neurons, or neuronal ensembles, in the mesocorticolimbic dopamine system. Environmental stimuli (for example, tones, lights and odours) and drug-induced interoceptive stimuli (for example, heart rate and blood vessel tone) during drug self-administration activate specific neuronal ensembles in sensory regions of the neocortex and olfactory bulb (OB) that in turn activate specific neuronal ensembles in the prefrontal cortex (PFC), hippocampus, basolateral amygdala (BLA) and thalamus. Activated principal (glutamatergic) neurons in each brain area are indicated by red circles and non-activated principal neurons are indicated by blue circles. Neurons in the nucleus accumbens (NAc) that receive the highest levels of convergent excitatory glutamatergic input (blue lines) from the PFC, BLA and thalamus are selectively activated to form a neuronal ensemble that corresponds to or encodes the specific combination of stimuli and their relationships on the basis of past experience. Depending on the salience and reward value of these stimuli, the ventral tegmental area (VTA) sends dopaminergic input (green lines) to the PFC and NAc that further enhances ongoing activity of the more highly activated neuronal ensembles while attenuating activity in the less activated majority of neurons in the PFC and NAc. Red lines indicate GABAergic outputs from the NAc to the ventral pallidum (VP) and VTA.
and to identify these neurons for subsequent molecular and cellular analysis. The Fos promoter is rapidly induced within strongly activated neurons (see BOX2 for details), and Fos mRNA and FOS protein products have been used in numerous studies as markers of neuronal activation in different neuronal phenotypes in many brain areas3436. Below, we describe the different methods, their application in studying learned behaviours and the limitations of eachmethod.
Neuronal ensembles in addiction research Neuronal ensembles in the nucleus accumbens and prefrontal cortex. Since the 1960s, many studies in humans and laboratory animals have demonstrated that classical and operant conditioning mechanisms play a major part in drug use and relapse3740. With repeated drug use, addicted individuals learn to associate drug effects with stimuli or cues in the drug environment (for example, drug paraphernalia, places of drug taking and cousers), and over time these cues often promote drug craving and drug seeking 3941.
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Drug-related cues are complex combinations of different stimuli that are recognized with a high degree of resolution. Therefore, any neural mechanism capable of encoding these learned associations must have a comparably high degree of resolution. It has been proposed that neuronal ensembles can provide a mechanistic framework for understanding the behavioural and motivational effects of drug-associated cues15,42,43. Indeed, over the past two decades, several studies combining drug self-administration procedures44 with invivo electrophysiology have provided correlative evidence for a role of neuronal ensembles in several brain areas the nucleus accumbens, medial prefrontal cortex (mPFC), ventral pallidum and basolateral amygdala in cue-induced drug seeking 4550. There is also limited correlative evidence from studies that compared context-specific locomotor sensitization behaviour with double-labelling of Fos mRNA and FOSB protein (BOX1) in contextspecific selection of neuronal ensembles in the nucleus accumbens15.
The neuronal ensemble hypothesis has had some impact on addiction research, but studies based on this hypothesis still form only a small proportion of neurobiological research on drug addiction. The vast majority of studies on molecular and synaptic plasticity mechanisms of drug reward, relapse and conditioned drug effects assess drug- or cue-induced molecular and cellular alterations in randomly selected neurons or in neurons of a particular cell type independently of their activation state during behaviour in different animal models of drug addiction5157. Therefore, the alterations assessed in these studies were induced largely in the non-activated majority of neurons and not specifically in the neuronal ensembles that were selectively activated during the behaviour. The unique drug- or cue-induced molecular and cellular alterations in the activated minority of neurons (or neuronal ensembles), which presumably mediate drug-seeking behaviour and conditioned drug effects, were probably missed or masked by drug-induced alterations in the non-activated majority of neurons. On the basis of the above considerations, during the past several years, we have developed a set of pharmacogenetic, molecular biological and genetic tools to selectively inhibit neuronal ensembles and assess their unique molecular and synaptic alterations. We developed these tools in rats because long-term studies using intravenous drug self-administration procedures44 and animal models of drug relapse and craving 5860 are technically very challenging inmice. The Daun02 inactivation method. The Daun02 method28 was developed to manipulate only those sparsely distributed neurons that are activated by specific stimuli or events without affecting either the surrounding non-activated neurons or neurons that are activated by other stimuli or events. We applied the Daun02 inactivation procedure to selectively inactivate neurons that were previously activated by drug-associated cues or contexts28 (FIG.2). Below, we describe the method and its application in studying drugrelated behaviours, as well as its limitations. We used FoslacZ transgenic rats, which have a transgene that contains a Fos promoter to induce transcription of the lacZ coding sequence and translation of the protein product -galactosidase. This induction only occurs in strongly activated (FOS-positive) neurons but not in the surrounding non-activated or weakly activated (FOS-negative) neurons6163. Once -galactosidase has been induced in neurons
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2013 Macmillan Publishers Limited. All rights reserved
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Box 1 | Immediate-early gene-based methods
Over the years, several immediate-early gene (IEG)-based methods have been used to identify putative neuronal ensembles in the brain34,130,131. The general principle has been to use one neuronal activity marker to label neurons activated during the initial learning session or sessions and a different neuronal marker to label neurons that are activated during a subsequent session (which is typically used to assess the expression of the learned behaviour). A high level of double-labelling of the two activity markers would suggest the recruitment of neuronal ensembles that encode the learned behaviours. In the late 1990s, Guzowski and colleagues14 introduced the cellular compartment analysis of temporal activity by fluorescence insitu hybridization (catFISH) method. This method was based on the temporal characteristics of the IEG Arc (activity-regulated cytoskeleton-associated protein) after neuronal activation: a nuclear Arc RNA signal emerges 2min after neuronal activation and persists for up to 16 min, whereas a cytoplasmic Arc RNA signal emerges 2045min after activation17. Accordingly, insitu hybridization can reveal neurons with cytoplasmic Arc mRNA, which are neurons that were active earlier (for example, during the first learning session), and neurons with nuclear Arc mRNA, which are neurons that were active more recently (for example, during the second learning session)17. Along with a variation on the procedure in which the IEG Homer1 is used to label initial neuronal activation and nuclear Arc is used to label subsequent neuronal activation112,132, this method has been used to identify putative neuronal ensembles that encode specific cues or contexts. The method is useful for identifying neuronal ensembles that are activated during short (about 30 min) learning tasks or have short time intervals between presentations of the same or different stimuli14,17. The main limitation of the catFISH method is that it cannot be used in learning tasks in which the learning and the expression of the learned behaviour are separated by hours or days. Another IEG-based method is double-labelling of the IEG Fos (using insitu hybridization) and FOSB (using immunohistochemistry), which is a product of the IEG Fosb. FOSB immunoreactivity labels neurons that were repeatedly activated during the first training or learning sessions, whereas Fos insitu hybridization labels neurons that were activated during the second session. This method is based on the accumulation of long-lasting protein isoforms from a truncated Fosb splice variant called FOSB in repeatedly activated neurons133. This method has been used to identify putative neuronal ensembles in the nucleus accumbens in context-specific locomotor sensitization to cocaine15. In a recently developed approach, a transgenic Fos-tTA (tetracycline (tet)-off transcriptional activator) mouse is used to identify neuronal ensembles16,114. The Fos-tTA transgene uses the Fos promoter to induce the expression of the tTA protein in neurons that are activated during the first learning session. The tTA protein can then bind to a tet operator in the promoter of a second transgene to induce expression of a marker gene. tTA can be induced in activated neurons before and after training, but doxycycline, which binds to and represses tTA, can be added to the mouse diet to block expression of the marker gene. If doxycycline is removed from the diet before the mouse undergoes the first learning session, the marker gene (for example, lacZ or histone2Bgreen fluorescent protein (GFP)) can be expressed in neurons that were activated during a selected time window. Immunohistochemical labelling of the protein products of early growth response 1 (also known as Zif268)16 or Fos116 can be used to label neurons that were activated during the second session. The Fos-tTA tool has been used to identify neuronal ensembles that control fear conditioning16,19,33,116.
that are activated, these neurons can then be inactivated through injection of the inactive prodrug Daun02 (REFS28,6468). -galactosidase catalyses the conversion of Daun02 into daunorubicin, which inactivates the previously activated neurons through two potential mechanisms: apoptotic cell death65,66 or blockade of voltagedependent calcium channels69. Thus, in our experiments, Daun02 injections disable those neurons that were activated by the cue, context or drug and thus presumably the neuronal ensemble that encodes the association between the cue or context and the drug 24,62,63. On the test day, typically 3days after Daun02 injections, we assess whether these injections decrease the ability of the
drug-associated cues or contexts, or the ability of the drug itself, to reactivate the same drug-related neuronal ensemble and induce a conditioned response or drug seeking. A critical control condition that is required to show a causal role of neuronal ensembles in the drug-related behaviour is that a Daun02 injection after exposure to a nondrug-associated cue or context or to a novel context inactivates neurons distinct from those in the drug-related neuronal ensemble and should, therefore, have no effect on the drug-related behaviour on the testday. We first used the Daun02 inactivation method28 to demonstrate a causal involvement of neuronal ensembles in the nucleus accumbens in context-specific sensitization
of cocaine-induced locomotion15,70,71. We first demonstrated context-specific activation of accumbens neurons by training a group of rats to associate cocaine (7 daily injections) with one context (A) and another group of rats to associate cocaine with a different context (B). After 7 withdrawal days, we injected rats with cocaine or saline in context A and perfused them 90min later to assess FOS expression in the nucleus accumbens. Cocaine injections in test context A enhanced (sensitized) cocaine-induced locomotion and accumbens FOS expression in rats that were previously injected with cocaine in the same context A but not in rats that were previously injected with cocaine in the different context B. Double-labelling immunohistochemistry for FOS and the neuronal marker NeuN showed FOS expression in ~3% of accumbens neurons72. To assess a causal role for this activated FOS-expressing minority of neurons in context-specific cocaine sensitization, we trained FoslacZ transgenic rats to associate context A with cocaine (7 daily injections). After 7 withdrawal days, we injected separate groups of rats with cocaine in context A or in a novel context B, and then injected Daun02 or vehicle into the nucleus accumbens 90min later. Three days later, on the test day, we found that prior Daun02 inactivation of accumbens neurons attenuated cocaine-induced locomotor sensitization and neuronal activation (assessed by Fos promoter-induced galactosidase expression) when Daun02 was injected after cocaine administration in context A but not when it was injected after cocaine administration in the novel context B28. Together, these results indicate that context-specific locomotor sensitization to cocaine is mediated by context-specific selection of accumbens neuronal ensembles that are comprised of a small proportion of sparsely distributed neurons. In our second application67 of the Daun02 inactivation method, we demonstrated a causal role for neuronal ensembles in the ventral mPFC in context-induced reinstatement of drug seeking, an animal model of relapse induced by exposure to the drugassociated environment 73. We first trained rats to self-administer (by lever pressing) heroin in context A and extinguished lever pressing in context B. On the test day, 14 or more days later, reexposure to the heroin context (A), but not to the extinction context (B), increased heroin seeking and increased FOS expression in ~6% of the ventral mPFC neurons. To assess a causal role for this activated FOS-expressing minority of neurons in context-induced reinstatement, we trained
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FoslacZ transgenic rats to self-administer heroin in context A and extinguished lever pressing in context B. On induction day, 14 or more days later, separate groups of rats were exposed to either the heroin context (A) or the extinction context (B) for 30min, and Daun02 or vehicle was injected into the ventral mPFC 90min after the beginning of context exposure. On the test day, 3days later, we found that prior Daun02induced inactivation of the ventral mPFC decreased context-induced reinstatement and neuronal activation when Daun02 was injected after exposure to the heroin-associated context (A) but not when it was injected after exposure to the extinction context (B). Of note, the magnitude of inhibition of contextinduced reinstatement by Daun02 injections was similar to that observed after ventral mPFC injections of a mixture of GABA typeA (GABAA) and GABAB agonists (muscimol and baclofen, respectively) to reversibly inactivate this brain area 510min before the reinstatement tests67. Together, these results indicate that a small subset of ventral mPFC neurons form neuronal ensembles that encode the learned association between heroin reward and the context in which the drug is self-administered. In our most recent application68 of the Daun02 inactivation method, we demonstrated a causal role for neuronal ensembles in the orbitofrontal cortex (OFC) in the incubation of drug craving (as indicated by time-dependent increases in cue-induced drug seeking after withdrawal from the drug)7476. We trained rats to self-administer heroin (6hours per day for 10days; drug infusions were paired with a discrete light cue) and assessed cue-induced heroin seeking in extinction tests after 1 or 14days of withdrawal. Cue-induced heroin seeking increased from 1day to 14days (incubation of heroin craving) and was accompanied by increased FOS expression in ~12% of OFC neurons on withdrawal day 14. To assess a causal role for this activated FOS-expressing minority of OFC neurons in heroin craving, we trained FoslacZ transgenic rats to self-administer heroin. On induction day, 11days later, we reexposed these rats to the light cue in the heroin-associated context or to a novel context without the light cue for 15min and injected Daun02 or vehicle into the OFC 90min after the beginning of context exposure. On the test day, 3days later, we found that prior Daun02 inactivation of OFC neurons decreased cue-induced heroin seeking and OFC neuronal activation when Daun02 was injected after reexposure to the heroin-associated cues but not when Daun02 was injected after exposure to the novel context 68. Non-selective inactivation of OFC neurons with muscimol and baclofen also decreased cue-induced heroin-seeking on withdrawal day 14 (but not on day 1)68. These results indicate that heroin-cue-activated OFC neuronal ensembles have a causal role in persistent responding to heroin cues after withdrawal and incubation of heroin craving.
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Box 2 | The Fos promoter as a marker of neuronal activity during behaviour

The widespread use of Fos mRNA and its FOS protein products to identify neuronal activation in the brain3436 has led to many studies that have examined the detailed molecular and cellular mechanisms of Fos promoter activation. The relevant literature is immense and is summarized here with only a few selected citations. By necessity, the early studies of Fos and immediate-early gene (IEG) induction were performed under very artificial conditions using cell or slice cultures. These studies were important for identifying candidate signalling mechanisms for IEG induction34,134,135. Many of these mechanisms were also shown in the brain during development, following chemical or mechanical damage or following various manipulations that produced non-physiological activation of signal transduction pathways. However, only a few of these mechanisms have a significant role in Fos promoter activation in the brains of intact behaving rats or mice. In the striatum and hippocampus, Fos expression is mediated by extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)-dependent phosphorylation of ELK1serum response factor (SRF) and phosphorylation, via ribosomal S6 kinase (RSK), of cyclic AMP (cAMP)-responsive element-binding protein (CREB) on the Fos promoter (see the figure)35,136,137 and not by the cAMP pathway (not shown)138,139. Neuronal activation of the ERK/MAPK pathway requires consistent (not sporadic) high levels of calcium influx through NMDA receptors (NMDARs) and voltage-sensitive calcium channels (VSCCs)35,137,140,141. Thus, we hypothesize that Fos expression in behaving animals reflects a summation or integration of neuronal activity-dependent calcium influx over seconds to minutes, and only strong, consistent activity over this time frame will increase calcium levels enough to induce Fos. Glutamatergic excitatory input (along with modulation by GABA inhibitory input) plays a major part in inducing strong neuronal activation35. Drug-induced dopamine release in the striatum is often thought to have a direct pharmacological role in inducing neuronal activity and FOS expression, but this isunlikely because dopamine and dopaminergic agonists tend to hyperpolarize striatal neurons in the absence of ongoing glutamatergic input142. Instead, drug-induced dopamine release is thought more likely to enhance the postsynaptic effect of ongoing glutamatergic input on the most strongly activated neurons, which increases their neural activity even further142 to a level sufficient for Fos promoter activation35. By contrast, drug-induced dopamine attenuates the effect of ongoing glutamatergic input and neural activity of the less activated majority of neurons142. Previous attempts to directly examine the association between electrophysiological activity and FOS expression in the striatum and hippocampus have shown that the level of FOS expression correlates with the level of synaptic activity and not with the number of action potentials131,143. However, results from these studies are difficult to interpret in the context of Glutamatergic input FOS-related neuronal ensemble activity, because electrophysiological recordings of + Dopamine randomly selected striatal or hippocampal Neuronal activity dentate gyrus neurons are almost certainly recordings from the majority of (less activated) neurons and not from the neurons Ca2+ NMDAR that were activated strongly enough to or VSCC express FOS. Invivo calcium imaging has recently been used to demonstrate a low correlation between spontaneous neuronal P activity and FOS expression in single auditory ERK/MAPK 121 cortex neurons in anaesthetized mice . However, these negative data are also difficult to interpret in the context of FOS-related RSK neuronal ensemble activity, because ELK1 P anaesthesia has repeatedly been shown to P block the more behaviourally relevant FOS CREB Fos SRF expression induced in awake behaving rodents144. In the future, it will be important to Fos promoter Transcription repeat similar studies with invivo calcium Fos mRNA imaging to appropriately compare neuronal Translation activity with induced Fos promoter activation FOS protein Nucleus in awake behaving rats or mice. Nature Reviews | Neuroscience
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Neuronal activity Fos promoter lacZ coding sequence Activated cells express -galactosidase lacZ mRNA -galactosidase
that were activated by non-paired contexts (B) or novel contexts did not decrease lever pressing or neuronal activity in the training context (A)28,67,68. In addition, prior Daun02 inactivation of the cocaine-activated nucleus accumbens ensemble in paired context A had no collateral effect on the ability of intraaccumbens injections of a cocktail of AMPA plus picrotoxin to activate all accumbens neurons28. FACS sorting of activated FOS-expressing neurons. The studies described above indicate that selectively activated neuronal ensembles in the nucleus accumbens and cortical areas have a causal role in contextspecific sensitization of cocaine-induced locomotion, context-induced reinstatement of heroin seeking and incubation of heroin craving. Thus, molecular alterations within these neuronal ensembles are likely to have unique and important roles in these drugrelated learned behaviours. FACS can be used to analyse and purify FOS-expressing neurons for molecular analysis. In the flow cytometry component of FACS, brain tissue is enzymatically and mechanically dissociated into single cells, which are then fluorescently labelled with antibodies and forced to pass single-file through a narrow flow cell in a flow cytometer. In the cell-sorting component of FACS, cells are sorted as they leave the flow cell according to their light-scattering and immunofluorescent characteristics7780. We recently developed a FACS-based method to assess gene expression in activated FOS-expressing neurons29,81 (FIG.3). In this method, neurons are identified by labelling with NeuN (a marker of neurons) antibodies, and activated versus non-activated neurons are identified according to their labelling with FOS or galactosidase antibodies. Below, we describe the method and its application in studying drug-related behaviours, as well as its limitations. We used FACS to purify activated neurons from different brain areas in two drug-induced behavioural models: context-dependent cocaine sensitization and incubation of heroin craving 29,68. In the first study, we assessed unique alterations in cocaine-induced gene expression in activated versus non-activated striatal neurons29. We used FACS to purify activated (galactosidase-expressing) neurons 90min after injections of cocaine in naive and cocaine-sensitized FoslacZ transgenic rats. We then compared gene expression in these cell populations with gene expression in all neurons from control rats that had received saline injections29. Microarray and quantitative PCR analyses indicated several unique
Inject Daun02 Daun02 Daunorubicin
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Figure 2 | The Daun02 inactivation method. a | The FoslacZ transgene contains a Fos promoter that regulates transcription of the lacZ coding sequence. Sufficiently strong and persistent neural activity Nature Reviews | Neuroscience activates the Fos promoter. As a result, the expression of lacZ mRNA and its protein product, galactosidase, is increased in these strongly activated neurons (red cells) but not in the surrounding majority of neurons (blue cells). The prodrug Daun02 is injected into the brain area of interest and is initially inactive. However, galactosidase catalyses conversion of Daun02 to the active product daunorubicin, which inactivates only those neurons that were activated strongly enough during behaviour to induce galactosidase. b | The general experimental procedure requires repeated exposures in one context (context A) during the training phase, followed by withdrawal, abstinence or extinction in a different context (context B). On the induction day, specific neuronal ensembles can be (re)activated and -galactosidase induced by exposure to cues and/or the drug in the training context (context A), the extinction context (context B) or a novel context (context C). Vehicle or Daun02 is injected 90minutes later (the time of maximal galactosidase protein induction after neuronal activation). On the test day, 3days later, the effect of inactivating a specific neuronal ensemble on behaviour in the training context (context A) is assessed.
Taken together, the Daun02 inactivation procedure can be used to study the role of neuronal ensembles in the motivational effects of drug cues and contexts. However, the method has some limitations and unresolved issues. The Daun02 method, like all Fos promoter-based methods, cannot manipulate behaviours that are dependent on a lower level of neuronal ensemble activity than that required to activate the Fos promoter. In addition, the method is limited to brain areas in which FOS and galactosidase are highly coexpressed (such as the striatum and the mPFC) and cannot be used to assess neuronal
ensemble activity in brain areas in which coexpression is moderate or low (for example, the thalamus (E.K. and B.T.H., unpublished observations)). Furthermore, we have yet to assess a time course for Daun02 inactivation beyond 3days and do not know the detailed molecular and cellular mechanisms involved in Daun02 inactivation. If daunorubicin in activated neurons ablates these neurons through apoptosis, then some collateral effects of this apoptosis might be expected on the surrounding neurons. However, evidence suggests that this is unlikely: as shown above, Daun02 inactivation of neuronal ensembles
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Figure 3 | FACS sorting of activated neurons. The fluorescence-activated cell sorting (FACS) Nature Reviews | Neuroscience method is used for assessing unique molecular alterations within activated versus non-activated neurons. a | In flow cytometry, including FACS, single cells are enzymatically dissociated from brain tissue and fluorescently labelled with different antibodies. Labelled samples are then forced to pass single file through a narrow flow cell. Absorbance of transmitted laser light for each particle is called forward scatter (FSC) light, whereas light scattered at a 90degree angle is called side scatter (SSC) light. Each particle (cell or non-cell) is called an event. b | Each event is indicated by a dot in the scattergram. The cluster of events in the lower part of the scattergram corresponds to neurons that were subsequently selected (or gated) by the indicated triangle for further analyses of their fluorescence characteristics. Positively labelled events (for example, FOS-positive cells) have high fluorescence levels, whereas negatively labelled events (for example, FOS-negative cells) have low fluorescence levels. c | These events are displayed in a fluorescence scattergram. Rectangular gates are used to select positive events (for example, FOS-positive, neuronal marker NeuN-positive cells) and negative events (for example, FOS-negative, NeuN-positive cells) for collection using FACS. d | Droplets containing gated events can be programmed to receive an electric charge as they leave the flow cell. Magnetic plates direct the charged droplets and sort them into separate positive (red circles) or negative (blue circles) sample tubes for further molecular analysis.
alterations in gene expression levels of IEGs (markers of activity) and other genes within activated neurons. Expression of the IEGs Arc (activity-regulated cytoskeleton-associated protein), Fosb and Nr4a3 (nuclear receptor subfamily 4, group A, member3) was higher in activated neurons from cocaineinjected rats than in non-activated neurons from the same cocaine-injected rats or in all neurons from saline-injected rats. Notably, gene expression was similar in the two
control conditions: non-activated neurons from cocaine-injected rats and all neurons from saline-injectedrats29. In the second study, we used the method to assess unique alterations in heroin-cueinduced gene expression in activated versus non-activated neurons after FACS purification of activated FOS-expressing neurons from the mPFC and OFC82. Rats were trained to self-administer heroin as above and then remained in their home cages for 1430days.
On the test day, we tested half of the rats for cue-induced heroin seeking in an extinction test (a test for incubation of craving after prolonged withdrawal) while the other half remained in their home cage (notest rats). Quantitative PCR analyses indicated several unique alterations in the expression levels of IEGs and other genes within activated neurons. Cue-induced heroin seeking increased the expression of the IEGs Arc, Fosb, Egr1 (early growth response 1) and Egr2 in activated neurons relative to levels in the nonactivated neurons from the same test rats or in all neurons from the no test rats82. Taken together, in both studies, IEGs were induced in activated neurons but not in nonactivated neurons. Together with our immunohistochemical findings29,82, this finding supports the idea of sparse coding, in which only a small proportion of sparsely distributed neurons undergo the molecular and cellular alterations needed to encode conditioned drug effects, whereas the surrounding, larger proportion of neurons presumably have a much smaller role. As many of these IEGs are also transcription factors, it is likely that they can induce further alterations in gene expression within activated neurons that may have uniquely important roles in learned behaviours mediated by activated neuronal ensembles. The FACS-based method has several limitations. We can only identify the relevant neuronal ensembles after they have been activated by acute drug or cue exposure on the test day. Thus, we cannot assess molecular alterations that were induced in these neurons during self-administration training before acute drug or cue exposure on the test day. In addition, it is not possible to manipulate genes selectively in these activated neurons to assess any potential causal roles for these genes in behaviour. We are currently developing methods to overcome these issues. In addition, until recently, our FACSbased method required pooling the relevant brain areas, such as the striatum and PFC, from 610 rats. This makes the method less useful for time- and labour-intensive studies that require intravenous surgery and many weeks of behavioural training (including studies on mechanisms of drug reward and relapse). Furthermore, different subregions of the striatum and frontal cortex are known to have different roles in the behavioural effects of drugs and non-drug rewards, and the cues and contexts associated with them55,8385. To address this issue, we recently combined our existing FACS method81 with the Arcturus PicoPure RNA Kit and pre-amplification of the target genes to assess gene expression
Side scatter (SSC)
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from as few as five FOS-positive neurons. These modifications enable us to reliably measure gene expression changes in a limited number of FOS-expressing neurons from a single dorsal striatum of rats injected with saline or methamphetamine86. We are currently using this improved FACS-method to study unique molecular alterations in activated FOS-expressing accumbens and dorsal striatum neurons using the context-induced reinstatement of drug-seekingmodel. Fos-GFP transgenic mice and rats. Alterations in synaptic efficacy, particularly within excitatory synapses, are regarded as the main cellular mechanism underlying learning and memory processes87,88, including those involved in drug addiction51,89. However, as discussed above, previous studies examined drug-induced or drug-cueinduced global alterations in synaptic efficacy in randomly selected neurons regardless of their activationstate during behaviour. Fos-GFP transgenic mice were developed to assess unique electrophysiological characteristics of activated FOS-expressing cortical neurons during different behavioural states9094. These mice can also be used to study unique synaptic alterations in activated FOS-expressing neuronal ensembles during learned behaviours; we describe this use below, including its application in studying drug-related behaviours and its limitations. The transgene of these mice contains a Fos promoter that induces expression of GFP to identify activated neurons in brain slice preparations. In our experience, confocal microscopy is necessary to visualize GFPlabelled neurons, because regular epifluorescence microscopy does not provide adequate sensitivity. Once a GFP-labelled neuron is identified, infrared differential interference contrast microscopy is used to perform whole-cell patch electrophysiology. We have used these Fos-GFP transgenic mice to assess unique synaptic alterations within activated neurons in the nucleus accumbens following context-specific cocaine sensitization95. We previously found that activated FOS-expressing neuronal ensembles in the nucleus accumbens mediate context-specific sensitization of cocaineinduced locomotion28. The main finding in our study was that cocaine sensitization, but not acute cocaine, produced higher levels of silent synapses (synapses that contain functional NMDA receptors but no functional AMPA receptors96) in activated (GFPpositive) neurons and not in non-activated or weakly activated (GFP-negative) neurons. Interestingly, the silent synapses induced in
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activated, GFP-expressing neurons appear to be different from those previously observed in randomly selected nucleus accumbens neurons following repeated cocaine injections97,98. Specifically, NMDA receptors in silent synapses from randomly selected neurons were characterized by high levels of the NR2B subunit98, whereas this was not the case for silent synapses in our activated, GFPpositive neurons95. We hypothesize that silent synapses in GFP-positive neurons may result from AMPA receptor endocytosis following strong activation of these neurons. The data from this study 95, together with the finding described above28, suggest that distinct synaptic alterations are induced in the activated nucleus accumbens neurons that mediate context-specific cocaine sensitization. The Fos-GFP transgenic mouse is an excellent tool for studying unique synaptic alterations in activated neuronal ensembles following relatively simple behavioural tests used in the addiction field, such as locomotor sensitization and conditioned place preference. However, transgenic mice are not ideal subjects for complex studies of drug reward and relapse that are based on intravenous drug self-administration. For this reason, we developed a Fos-GFP transgenic rat using the genetic construct described earlier 30 (FIG.4). In the initial neurobiological study with these transgenic rats, we adapted the classic reinstatement model of drug relapse58 to study reinstatement of food seeking as an animal model of relapse during dieting 99. We assessed whether stress-induced reinstatement of palatable food seeking 100, which is dependent on dorsal mPFC activity 101,102, is associated with unique synaptic alterations in this brain area.
a Coronal slice
We found that reinstatement of food seeking induced by the pharmacological stressor yohimbine103 was associated with reduced AMPAR/NMDAR current ratios (indicating reduced glutamatergic synaptic efficacy 104) and increased paired-pulse facilitation (indicating decreased synaptic glutamate release105) in activated GFP-positive neurons but not non-activated or weakly activated GFP-negative neurons30. Taken together, these studies in Fos-GFP transgenic rats and mice as well as earlier studies92,93 demonstrate that these transgenic rodents are suitable for studying unique synaptic alterations in the activated minority of neurons (neuronal ensembles) that presumably control learned behaviours. There are, however, several limitations of this approach. One limitation is that, as with the FACS procedure, we cannot assess synaptic alterations that were induced in these neurons during training before acute drug or cue exposure on test day. Nor can we manipulate these altered synaptic mechanisms selectively in activated neurons to assess their causal roles in learned behaviours. Another limitation is that combining behavioural studies with synaptic physiology of activated neurons is technically challenging, because of the difficulties associated with identifying a sufficient number of GFP-positive neurons in a slice preparation. This difficulty arises from the fact that only a minority of neurons express GFP, and this expression is transient, lasting only a few hours. In addition, to date, we have only used this approach after pharmacological activation (using cocaine or yohimbine) that may induce stronger neuronal activation than that induced by
c Fill patched cell with
Alexa 568 Alexa 568
b Electrode attached to
GFP-positive neuron GFP
Nature Reviews Neuroscience Figure 4 | Electrophysiology of activated neurons using the Fos-GFP rat.| Assessing unique electrophysiological alterations within activated versus non-activated neurons. The Fos-GFP (green fluorescent protein) transgene in transgenic rats (or mice) contains a Fos promoter that regulates transcription of the coding sequence for GFP. Sufficiently strong and persistent neural activity activates the Fos promoter, which induces GFP in these strongly activated neurons but not in the surrounding majority of neurons. a | Coronal slices are obtained for electrophysiological analysis. b | GFP expression (induced by drug or cue exposure) can be used to guide the electrode to GFP-positive or GFP-negative neurons, and then use differential interference contrast optics to patch the cell. The arrow indicates a GFP-positive neuron with the shadow of the attached electrode to the right. c | The fluorescent dye Alexa 568 in the electrode can diffuse into the attached cell to confirm that the recorded cell was GFP-positive.
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exposure to drug or food cues. As is the case with FoslacZ rats, electrophysiological studies with Fos-GFP rats and mice are limited to behaviours that increase neural activity enough to activate the Fos promoter and induce a high level of coexpression of FOS and GFP in the brain areas of interest.
Neuronal ensembles in fear conditioning The neuronal ensembles hypothesis has been the inspiration for many studies on neuronal mechanisms of fear conditioning and extinction13,106. As in other neuroscience disciplines, most published work on this topic was derived from correlational studies between behaviour and invivo electrophysiology 107,108 or cellular imaging 109112 (BOX1) methods. Recently, investigators have developed two methods to manipulate putative neuronal ensembles and examine their causal role in conditioned fear 18,113. We describe these methods below (FIG.5). a
Mouse transgene Fos promoter Neuronal activity tTA cds Activation by light (ChR2) or CNO (hM3Dq)
Second transgene
tTA Protein expression in activated neurons tTA ChR2 or hM3Dq cds
Doxycycline
Tet operator
GFPCREB fusion protein
HSV transgene HSV IE4/5 promoter GFP cds CREB cds IoxP Dtr
IRES Cre recombinase cds Diphtheria toxin
Mouse transgene IoxP ROSA26 promoter Stop
Manipulation of neuronal ensembles in FostTA transgenic mice. In two recent studies, Fos-tTA transgenic mice16,114 were used in combination with optogenetic or DREADD methods to examine causal roles of neuronal ensembles in fear conditioning 19,33. As described in BOX1, the Fos promoter induces expression of the tTA protein in strongly activated neurons. Doxycycline provided in the drinking water prevents tTA protein binding to the tet operator prior to the learning task. Doxycycline is then removed before the first learning session, allowing the tTA activator protein to bind to a tet operator in the promoter of a second transgene and drive the expression of this gene only in neurons that were activated (FOS-positive) during the learning task. Investigators have used optogenetic or DREADD genetic constructs as the second transgenes to selectively reactivate these neurons during tests for the expression of fear learning 19,33 (FIG.5a). Liu etal.19 used Fos-tTA transgenic mice to determine causal involvement of hippocampal neuronal ensembles in Pavlovian fear conditioning. They tested whether reactivation of neuronal ensembles in the dentate gyrus that were activated during learning was sufficient for fear memory recall, which was operationally defined as increased freezing. The experimental group consisted of Fos-tTA mice that were injected with an adeno-associated virus (AAV) expressing channelrhodopsin2 (ChR2)enhanced yellow fluorescent protein and implanted with an optical fibre in the dentate gyrus. Mice were kept on doxycycline during habituation days, so that their basal freezing levels in
DTR protein
GFPCREB fusion protein
AlstR Allatostatin
HSV transgene HSV IE4/5 promoter GFP cds CREB cds CMV promoter AlstR
Figure 5 | Manipulating activated fear-encoding neuronal ensembles in the hippocampus and Nature Reviews | Neuroscience amygdala. a | The Fos-tTA transgene contains a Fos promoter that regulates RNA transcription from the coding DNA sequence (cds) for the tetracycline (tet)-off transcriptional activator (tTA) protein. Sufficiently strong and persistent neural activity during a particular learned behaviour induces tTA in these strongly activated neurons but not in the surrounding majority of neurons. Doxycycline provided to the mice (commonly through the diet) inactivates tTA transcriptional activity. When doxycycline is removed from the diet, tTA can bind to the tet operator and activate a second transgene (viral or genomic) that expresses the optogenetic activating protein channelrhodopsin 2 (ChR2) or the pharmacogenetic activating DREADD (designer receptors exclusively activated by designer drugs) receptor hM3Dq in those neurons that were previously activated during the behaviour. Blue light activates and manipulates the ChR2expressing neurons and clozapine-Noxide (CNO) activates the hM3Dqexpressing neurons that were previously activated (red cells), during subsequent behavioural tests. b | A herpes simplex virus (HSV) transgene containing a constitutively active HSV immediateearly 4/5 (IE4/5) promoter that regulates RNA transcription from the two DNA sequences encoding the green fluorescent protein (GFP)cyclic AMP-responsive element-binding protein (CREB) fusion protein and Cre recombinase, which are separated by an internal ribosome entry site (IRES). Overexpression of GFPCREB increases the sensitivity of neurons to synaptic input (red cells). Cre recombinase in the same neurons recognizes loxP DNA sequences in the diphtheria toxin receptor (Dtr) transgene of transgenic mice to cut out the stop DNA sequence; this permits constitutive ROSA26 promoter-induced expression of DTR protein. Subsequent injections of diphtheria toxin ablate DTR-expressing neurons. c | An HSV transgene containing two separate genes; one gene uses a constitutively active HSV IE4/5 promoter that regulates RNA transcription from the DNA sequence encoding the GFPCREB fusion protein, and the other gene uses a cytomegalovirus (CMV) immediateearly gene promoter to drive expression of the gene that encodes the Drosophila melanogaster Allatostatin receptor (AlstR). Activated neurons (red cells) overexpress GFPCREB, which increases both the sensitivity of neurons and the expression of the AlstR. Subsequent site-specific injections of the allatostatin peptide can inactivate these neurons during a behavioural test.
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context A could be determined during lightoff and lighton epochs of optical stimulation. The mice showed little freezing during these habituation sessions before fear conditioning. Mice were then taken off doxycycline and underwent tone (conditioned stimulus) shock (unconditioned stimulus) pairing (in other words, fear conditioning) in context B to induce ChR2 expression in FOS-positive neurons that were activated during fear conditioning. Mice were given doxycycline again and tested during light-off and lighton epochs in context A. ChR2 activation by optical stimulation induced reactivation of the neurons that had previously been activated during fear conditioning in context B and produced increased freezing behaviour in context A. In an important control condition, after habituation with doxycycline in contextA, the authors removed doxycycline and induced ChR2 in neuronal ensembles activated by exposure to a novel context C in the absence of fear conditioning. Doxycycline was then given to the mice before fear conditioning in context B. ChR2induced activation of context Crelated ensembles in these mice did not produce higher levels of freezing in context A, presumably because this ensemble was not associated with fear conditioning 19. The results of this elegant study 19 suggest a causal role for dentate gyrus neuronal ensembles in the formation of stable fear memories. This conclusion would be strengthened if it could be shown that halorhodopsin-dependent inhibition of the neuronal ensembles that were previously activated during fear conditioning in context B (and that presumably encode the fear memory) prevents tonecueinduced freezing in context B. In other words, although the authors showed that activation of a dentate gyrus neuronal ensemble is sufficient for reactivating a fear memory, it is unknown whether endogenous activity of this putative ensemble is necessary for encoding the fearmemory. In a second study, Garner etal.33 used Fos-tTA transgenic mice with DREADD technology to activate FOS-expressing fearencoding neurons in the brain (FIG.5a). These transgenic mice have two transgenes that are widely expressed in many brain areas. The first transgene is the Fos-promoter-driven tTA (Fos-tTA) transgene described above. The second transgene contains a tet operator that drives tTA-dependent expression of hM3Dq, an artificial Gqcoupled receptor that binds the drug clozapine-Noxide (CNO), which is typically injected systemically to activate neurons expressing this receptor 115. One of the experiments in this study 33 was similar to that used in the earlier study 19.
Specifically, doxycycline was removed for 2days, after which mice underwent fearconditioning training in context B, so that the hM3Dq protein was induced in neurons that were activated during the fear conditioning. On the test day, the mice were placed in a novel context (A) and CNO was systemically injected to activate hM3Dq and thereby reactivate those neurons that were previously activated during fear conditioning in contextB. However, CNO did not induce freezing in context A, suggesting that DREADD-mediated reactivation of neurons paired with fear conditioning was not sufficient to recall the fear memory. The authors also performed several experiments suggesting that coactivation of artificially induced neuronal ensembles can interfere with real cue-activated neuronal ensembles. It is beyond the scope of this article to describe these other experiments, because they did not directly test a causal role for neuronal ensembles in conditionedfear. The reasons for the different results between the two studies19,33 are unknown. Optogenetic activation of neurons may be stronger than DREADD-based activation of neurons. Beyond methodological differences related to the fear-conditioning procedures, a very important difference is that one study 19 reactivated neuronal ensembles only in the dentate gyrus, whereas the other 33 reactivated ensembles in multiple brain areas, which may interfere with or mask the expression of conditioned fear. Finally, it should be noted that in a different study 116 using Fos-tTA mice, the number of tTA-induced GFP-expressing hippocampal neurons was less than the number of FOS-expressing neurons following context-induced reactivation of fear on test day. This finding suggests that tTA activation in other studies using Fos-tTA mice may underestimate the number of neurons that are activated during fear conditioning. Inactivation of CREB-overexpressing neurons. Viral overexpression of CREB combined with diphtheria toxin or activation of allatostatin receptors for subsequent inactivation of CREB-overexpressing neurons has been proposed as a method to study neuronal ensembles in the lateral amygdala in fear conditioning 31,32. Han etal.31 used herpes simplex virus (HSV) to overexpress both CREB and Cre recombinase from the same viral construct in a small number of lateral amygdala neurons in mice carrying a Cre-inducible diphtheria toxin receptor (Dtr; also known as Hbegf) transgene. Subsequent injections of diphtheria toxin (which binds to DTR) can then selectively inactivate
neurons that express DTR31 (FIG.5b). In the first phase of the experiment, test mice that overexpressed CREB and DTR in the same neurons and control mice that overexpressed DTR but not CREB underwent fear conditioning in what was termed weak training (one toneshock pairing) or strong training (two toneshock pairings) in one context. All mice were tested 1day later for expression of fear conditioning (freezing) in a different context. Similar to results in a previous study 117, CREB overexpression in the test mice increased neural responsiveness of the CREB-overexpressing neurons, which led to their preferential activation during fear learning and enhanced fear expression in the weak but not strong training condition (presumably the strong training condition already produced maximal levels of fear learning)31. In the subsequent lesioning phase of the experiment, control and test mice were systemically injected with diphtheria toxin to ablate DTRexpressing neurons and were then tested for expression of fear conditioning. Diphtheria toxin reduced the expression of fear memory in test mice but not in control mice, in which a similar number of lateral amygdala neurons overexpressing DTR (but not CREB) were inactivated by diphtheriatoxin. In a second study, Zhou etal.32 used a similar strategy. They used HSV to overexpress both CREB and the inhibitory Drosophila melanogaster Allatostatin receptor (which is not expressed in rodents) in the same neurons (FIG.5c); the peptide allatostatin binds to this receptor to reversibly inactivate neurons. The authors used experimental methods similar to those described above31 to demonstrate that following auditory fear learning, inactivation of CREB-overexpressing neurons by allatostatin decreased the expression of conditionedfear 32. Although these two studies successfully inactivated CREB-overexpressing neurons and decreased the expression of conditioned fear, the CREB overexpression method is not optimal for studying causal roles for neuronal ensembles in learned behaviours. This is because the neuronal ensembles that are inactivated by this method are artificially selected CREB-sensitized neurons rather than neuronal ensembles that were naturally selected by cue or context exposure during fear-conditioning training. That is, the neurons overexpressing CREB in these experiments are randomly selected during HSV infection before any learning experience. CREB overexpression in these neurons results in the formation of a hypersensitive cell type with synaptic alterations that make them highly responsive to many
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stimuli97,118,119. This leads to the formation of artificial neuronal ensembles that probably have a very different composition than the putative endogenous neuronal ensembles that would be selected during the same learning experience. For comparison, in the Fos promoter-based neuronal ensemble inactivation methods described above, only the neurons that are strongly activated by cues, contexts or drugs during learning are selected for subsequent inactivation. A promising future direction would be to combine the diphtheria toxin or allatostatin inactivation methods with Fos promoter selection of neurons activated during learned behaviours.
Conclusions and future directions We have discussed recent technical developments that make it possible to determine causal roles of putative activated neuronal ensembles in learned behaviours and to characterize the molecular and synaptic physiology of the activated neurons. These methods are unique and largely orthogonal to current mainstream neuroscience research that followed the introduction of optogenetic- and DREADD-based methods to the field23,27. This is because the goal of most optogenetic and DREADD studies is to identify causal roles of specific cell types, receptors or cellular signalling molecules in a given brain area or a particular cell-specific projection in learned behaviour, independently of the activation state of the neurons. The study of the causal role of neuronal ensembles in drug addiction and fear is in its infancy, and as discussed above, each method has its own limitations. The clearest demonstration of necessary causal roles for endogenous neuronal ensembles in learned behaviours has come from studies using the Daun02 inactivation procedure, in which inhibition of a small proportion of activated neurons in the nucleus accumbens or cortex inhibited context-specific cocaine-induced locomotor sensitization, context-induced reinstatement of heroin seeking and incubation of heroin craving 28,67,68. By contrast, as discussed above, studies combining optogenetic or DREADD methods with Fos-tTA mice either demonstrated only the sufficiency (but not the necessity) of neuronal ensembles in conditioned fear 19 or did not provide clear evidence for a role of neuronal ensembles in fear conditioning 33. In addition, although studies using CREB overexpression demonstrated that selective inactivation of a small proportion of activated neurons decreases the expression of fear conditioning 32,117, the neurons to be activated during
752 | NOVEMBER 2013 | VOLUME 14 2013 Macmillan Publishers Limited. All rights reserved
fear learning and subsequently inactivated were pre-selected by the HSV viral manipulation. Thus, the neuronal ensembles identified in these studies may not reflect the composition of the endogenous amygdala neuronal ensembles that are normally selected by the learning experience. There are also important limitations in the methods that have been developed for examining molecular 29,81 and synaptic physiology 30,95 alterations that were induced in neuronal ensembles activated during prior learning or on test day. One main limitation is that we cannot assess the basal conditions of the putative neuronal ensembles before activation, because we must acutely induce GFP, galactosidase or FOS to identify the activated neurons. Thus, we cannot determine whether any observed alterations are due to prior learning during the training phase or due to acute expression of the learned behaviour on the test day. To solve this problem we need to identify neurons that are destined to be activated and participate in an ensemble, before their activation on test day. The second limitation is that although we can potentially demonstrate causal roles of selectively activated neurons in learned behaviours, there are no tools to manipulate the molecular and synaptic alterations induced selectively in the activated neurons to demonstrate how these molecular or synaptic alterations affect learned behaviours or cell function. A third limitation is that the Fos promoterbased techniques described above cannot identify groups of neurons that are inhibited or insufficiently activated by cues for Fos promoter activation to occur. Therefore, an absence of Fos activation in neurons does not necessarily imply that they were not active and have no role in the behaviour. It may be possible to overcome the first limitation regarding basal conditions by using the Fos-tTA mouse system or our recently developed transgenic rat system that expresses tetracycline-activated Fos promoterdriven Cre recombinase (F.C.C., Y.S. and B.T.H., unpublished observations), in which previously activated neurons can be identified with a constitutively expressed molecular marker (for example, GFP) that persists for many days after the last manipulation, when basal conditions are reestablished. A similar strategy for identifying previously activated neurons involves tamoxifen-sensitive Cre recombinase induced by either the Fos or Arc promoter 120. In addition, a recent and promising technology uses transgenic mice with photoactivable GFP that, once activated, can be placed in a long-lasting fluorescent
state that permits neurons to be identified at a later time for more detailed analysis using slice electrophysiology 121. One study 122 has recently achieved selective manipulations within activated neurons by using mice carrying both aFos-tTA transgene and a GFPGluA1 transgene123. The authors used the GFPGluA1 construct to assess structural changes in dendritic spines on hippocampal neurons that were activated at the time of fear learning. The main finding was that spines on active (FOS-positive) neurons but not those on inactive (FOS-negative) neurons of context-fear-conditioned mice were reduced 24h after conditioned fear training, and this reduction did not occur in control mice (exposed to the context alone or to an unpaired shock). To date, however, none of the published techniques described above has been used to define the interactions between neuronal ensembles in different brain areas that are active at the same time during learning and on the test day and that may form the circuitry underlying learned behaviour. It will also be important to expand on the present work using other activity-dependent promoters such as Arc, which has been used for twophoton imaging of integrated neural activity in Arc promoter-driven GFP mice124 as well as for inducing Cre recombinase in activated neurons120. The Arc promoter is similar to the Fos promoter but generally has higher levels of basal activity and a lower threshold for activation125. Finally, it is perhaps too early to speculate about the clinical implications of a better understanding of the unique molecular and synaptic physiology alterations in behaviourally activated neuronal ensembles. Nevertheless, one potential implication is the shift in direction of medication development from strategies that target specific receptor, cell type or signalling mechanisms that are independent of the neurons activity status to strategies that target specific mechanisms that are observed only in activated neuronal ensembles. For example, drugs such as ketamine or memantine that bind preferably to activated NMDA receptors126128 can be used to potentially erase (via interference with memory reconsolidation26,129) or diminish the motivational impact of memories of drug-associated or fear-associated cues. Thus, giving ketamine or memantine immediately after exposing drug users or patients with post-traumatic stress disorder to drug- or trauma-associated cues to reactivate neuronal ensembles that encode the drug- or trauma-associated memory may diminish the motivational impact of these cues and decrease relapse.
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Fabio C.Cruz, Jennifer M.Bossert, Carl R.Lupica, Yavin Shaham and Bruce T.Hope are at the Intramural Research Program, National Institute on Drug AbuseNational Institutes of Health, 251 Bayview Boulevard, Baltimore, Maryland 21224, USA. Eisuke Koya was previously at the Intramural Research Program, National Institute on Drug Abuse-National Institutes of Health, 251 Bayview Boulevard, Baltimore, Maryland 21224, USA. Present address: School of Psychology, University of Sussex, Falmer, Brighton, BN1 9QH, UK. Danielle H. Guez-Barber was previously at the Intramural Research Program, National Institute on Drug Abuse-National Institutes of Health, 251 Bayview Boulevard, Baltimore, Maryland 21224, USA. Present address: General Pediatrics, Childrens Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA. Correspondence to B.T.H. e-mail: [email protected] doi:10.1038/nrn3597 Published online 3 October 2013
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Acknowledgements
The writing of this article was supported by the US National Institute on Drug Abuse, Intramural Research Program. We thank the members of the Hope, Lupica and Shaham laboratories who contributed to the development and implementation of the new technologies described in this article.
Competing interests statement
The authors declare no competing financial interests.
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PROGRESS

Non-activated neurons Activated neurons

Thalamus NAc VTA

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Box 2 | The Fos promoter as a marker of neuronal activity during behaviour

Inject Daun02 Daun02 Daunorubicin

Previously activated cells now inactive

Extinction, withdrawal or abstinence

A Drug and cue Behaviour

B Not applicable Behaviour

A or B (control) or C (control) Drug or cue Behaviour

A Drug or cue Behaviour

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Forward scatter (FSC)

FOS labelling (log)

Side scatter (SSC)

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VOLUME 14 | NOVEMBER 2013 | 749

tTA Protein expression in activated neurons tTA ChR2 or hM3Dq cds

GFPCREB fusion protein

IRES Cre recombinase cds Diphtheria toxin

Mouse transgene IoxP ROSA26 promoter Stop

GFPCREB fusion protein

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VOLUME 14 | NOVEMBER 2013 | 753

Competing interests statement

The authors declare no competing financial interests.

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