New technologies for examining the role of neuronal ensembles in drug addiction and fear

Journal name:
Nature Reviews Neuroscience
Year published:
Published online


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.

At a glance


  1. Neuronal ensembles in the mesocorticolimbic dopamine reward system.
    Figure 1: Neuronal ensembles in the mesocorticolimbic dopamine reward system.

    Hypothetical 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.

  2. The Daun02 inactivation method.
    Figure 2: The Daun02 inactivation method.

    a | The Fos–lacZ transgene contains a Fos promoter that regulates transcription of the lacZ coding sequence. Sufficiently strong and persistent neural activity activates the Fos promoter. As a result, the expression of lacZ mRNA and its protein product, β-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, β-galactosidase catalyses conversion of Daun02 to the active product daunorubicin, which inactivates only those neurons that were activated strongly enough during behaviour to induce β-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 β-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 90 minutes later (the time of maximal β-galactosidase protein induction after neuronal activation). On the test day, 3 days later, the effect of inactivating a specific neuronal ensemble on behaviour in the training context (context A) is assessed.

  3. FACS sorting of activated neurons.
    Figure 3: FACS sorting of activated neurons.

    The fluorescence-activated cell sorting (FACS) 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 90-degree 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.

  4. Electrophysiology of activated neurons using the Fos-GFP rat.
    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.

  5. Manipulating activated fear-encoding neuronal ensembles in the hippocampus and amygdala.
    Figure 5: Manipulating activated fear-encoding neuronal ensembles in the hippocampus and 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 ChR2-expressing neurons and clozapine-N-oxide (CNO) activates the hM3Dq-expressing neurons that were previously activated (red cells), during subsequent behavioural tests. b | A herpes simplex virus (HSV) transgene containing a constitutively active HSV immediate-early 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 GFP–CREB 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 GFP–CREB fusion protein, and the other gene uses a cytomegalovirus (CMV) immediate-early gene promoter to drive expression of the gene that encodes the Drosophila melanogaster Allatostatin receptor (AlstR). Activated neurons (red cells) overexpress GFP–CREB, 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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  1. Intramural Research Program, National Institute on Drug Abuse-National Institutes of Health, 251 Bayview Boulevard, Baltimore, Maryland 21224, USA.

    • Fabio C. Cruz,
    • Eisuke Koya,
    • Danielle H. Guez-Barber,
    • Jennifer M. Bossert,
    • Carl R. Lupica,
    • Yavin Shaham &
    • Bruce T. Hope
  2. Present address: School of Psychology, University of Sussex, Falmer, Brighton, BN1 9QH, UK.

    • Eisuke Koya
  3. Present address: General Pediatrics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.

    • Danielle H. Guez-Barber

Competing interests statement

The authors declare no competing interests.

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Author details

  • Fabio C. Cruz

    Fabio C. Cruz is a postdoctoral visiting fellow at the National Institute on Drug Abuse (NIDA), Intramural Research Program (IRP), Baltimore, Maryland, USA. He received his B.A. in 2002 from the Sao Paulo State University, Araraquara, Brazil. He then received his M.A. in 2004 and his Ph.D. in 2008 from the Federal University of São Carlos, São Carlos, Brazil. His postdoctoral training from 2008 to 2011 was at University Estadual Paulista-UNESP. He joined the NIDA IRP in 2011. His research interest is examining how learned associations related to drug addiction are formed and stored in the brain.

  • Eisuke Koya

    Eisuke Koya is a lecturer and research group leader at the School of Psychology at the University of Sussex, Brighton, UK. He received his B.A. in neurobiology at the University of California at Berkeley, USA, and his Ph.D. at the Vrije Universiteit Amsterdam, in the Netherlands in molecular neurobiology. He then conducted his postdoctoral research at the National Institute on Drug Abuse, Intramural Research Program, Baltimore, Maryland, USA. His group uses molecular biological and electrophysiological approaches to study how learned associations between palatable foods and environmental stimuli are mediated by neuronal ensembles.

  • Danielle H. Guez-Barber

    Danielle H. Guez Barber is currently a resident in General Pediatrics at the Children's Hospital of Philadelphia, Pennsylvania, USA. She received her B.S. in neuroscience from the Massachusetts Institute of Technology, Cambridge, USA in 2003, and her M.D. from Yale University, New Haven, Connecticut, USA, in 2012. She received her Ph.D. in neuroscience from Yale University, USA in 2011, and as part of the Graduate Partnerships Program, she did her thesis work at the National Institute on Drug Abuse, USA. Danielle will begin child neurology training in 2015 and plans to return to basic neuroscience after completing her clinical training.

  • Jennifer M. Bossert

    Jennifer M. Bossert is a staff scientist in the Behavioral Neuroscience branch at National Institute on Drug Abuse (NIDA) Intramural Research Program (IRP), Baltimore, Maryland, USA. She received her B.A. in 1997 from Simon Fraser University in Burnaby, British Columbia, Canada. She then received her Ph.D. in 2003 from McGill University in Montreal, Quebec, Canada. Her postdoctoral training from 2003 to 2006 was at NIDA IRP, and in 2007, she became a staff scientist in the branch. Her research interests are examining the neurobiological mechanisms of relapse to drugs, as assessed in rodent models.

  • Carl R. Lupica

    Carl R. Lupica is Chief of the Electrophysiology Research Section, Cellular Neurobiology Branch, National Institute on Drug Abuse (NIDA) Intramural Research Program (IRP), Baltimore, Maryland, USA. He received his B.A. in 1983 from Ohio University, Athens, USA. He received his M.A. in 1986, and Ph.D. in 1989 from Wayne State University, Detroit, Michigan, USA. His postdoctoral training was in the Department of Pharmacology at the University of Colorado from 1989 to 1992, where he later became an assistant professor. He was Associate Professor in the Department of Pharmacology at the University of Arizona from 2000 to 2002. He joined the NIDA IRP in 2002. He and the members of his laboratory study the mechanisms through which abused drugs affect the functional properties of neurons and neuronal circuits. Carl R. Lupica's homepage.

  • Yavin Shaham

    Yavin Shaham is Chief of the Behavioral Neuroscience Research Branch of the National Institute on Drug Abuse (NIDA) Intramural Research Program (IRP), Baltimore, Maryland, USA. He received his B.S. in 1986 and his M.A. in 1988 from the Hebrew University, Jerusalem, Israel. He then received his Ph.D. in 1992 from the Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA. His postdoctoral training from 1992 to 1995 was at Concordia University, Montreal, Quebec, Canada. From 1996 to 1998, he was an investigator at the Addiction Research Center, Toronto, Canada. He joined the NIDA IRP in 1998. He and the members of his laboratory study the neurobiological mechanisms of relapse to drug and palatable food seeking, as assessed in rodent models. Yavin Shaham's homepage.

  • Bruce T. Hope

    Bruce T. Hope is a tenure-track investigator in the Behavioral Neuroscience Branch of the National Institute on Drug Abuse (NIDA) Intramural Research Program (IRP), Baltimore, Maryland, USA. He received his B.Sc. in 1985 and his Ph.D. in 1991 from the University of British Columbia in Vancouver, British Columbia, Canada. His postdoctoral training from 1990 to 1994 was at Yale University, New Haven, Connecticut, USA. From 1994 to 1997, he was a scientist with New England Medical Center/Tufts University in Boston, Massachusetts, USA. He joined the NIDA IRP in 1998. His laboratory studies the role and mechanisms of neuronal ensembles in learned behaviours related to addiction, as assessed in rodent models. Bruce T. Hope's homepage.

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