Using c-fos to study neuronal ensembles in corticostriatal circuitry of addiction

Abstract

Learned associations between drugs and environment play an important role in addiction and are thought to be encoded within specific patterns of sparsely distributed neurons called neuronal ensembles. This hypothesis is supported by correlational data from in vivo electrophysiology and cellular imaging studies in relapse models in rodents. In particular, cellular imaging with the immediate early gene c-fos and its protein product Fos has been used to identify sparsely distributed neurons that were strongly activated during conditioned drug behaviors such as drug self-administration and context- and cue-induced reinstatement of drug seeking. Here we review how Fos and the c-fos promoter have been employed to demonstrate causal roles for Fos-expressing neuronal ensembles in prefrontal cortex and nucleus accumbens in conditioned drug behaviors. This work has allowed identification of unique molecular and electrophysiological alterations within Fos-expressing neuronal ensembles that may contribute to the development and expression of learned associations in addiction.

Keywords: Conditioned cues; Daun02 inactivation; Drug environment; Extinction; Nucleus accumbens; Prefrontal cortex; Self-administration.

Figure 1

Schematic drawing to illustrate how different activity patterns of stimulatory afferent input to a brain area can select different patterns of neurons called ‘neuronal ensembles’ that mediate distinct learned associations. The longer red arrows symbolize different patterns of afferent input activated by Drug and Drug-related cues while the longer blue arrows symbolize different patterns of afferent input activated by Non-drug rewards and Non-drug-related cues. Those neurons that receive the highest and most persistent levels of integrated stimulatory input will be part of the neuronal ensemble. The red ovals symbolize neurons that receive the highest levels of excitatory drug-related afferent inputs and encode drug-related learned associations that mediate drug-related behaviors while the blue ovals symbolize neurons that receive the highest levels of excitatory non-drug-related afferent inputs and encode non-drug-related learned associations that mediate non-drug-related behaviors. In the real brain, each of these neuronal ensembles is comprised of thousands to millions of neurons in many different brain areas.

Figure 2

Schematic drawing to illustrate the neurochemistry and molecular mechanisms of c-fos and Fos expression in strongly activated neurons in prefrontal cortex and nucleus accumbens of awake behaving animals. Glutamate is the main excitatory neurotransmitter that increases neural activity. Dopamine enhances (red arrow and + sign) glutamate-mediated neural activation of the small proportion of neurons that have the highest levels of neural activity while inhibiting neural activation of the majority of neurons that have lower levels of neural activity. The detailed electrophysiological mechanisms underlying this glutamate-dopamine interaction are described in (Surmeier et al., 2007). Strong persistent neural activity induces calcium (Ca²⁺) influx through NMDA-type glutamate receptors and voltage-sensitive calcium channels (VSCCs) to levels that are sufficient for phosphorylating and activating ERK/MAPK via the Ras-Raf-MEKK pathway. ERK/MAPK activation leads to phosphorylation of Elk-1 that is associated with serum response factor (SRF) as well as phosphorylation of CREB via ribosomal S6 kinase (RSK). Elk-1/SRF and CREB are transcription factors that, when phosphorylated, can induce transcription of the coding sequence for c-fos. Transcribed c-fos mRNA and the translated protein product Fos can be used as markers of strongly activated neurons.

Figure 3

Daun02 inactivation of Fos-expressing neuronal ensembles in rat nucleus accumbens attenuated sensitized cocaine-induced locomotion and β-galactosidase-labeled neural activity (shown on the y-axes) on Test day only when Daun02 was injected into the nucleus accumbens following cocaine injections to rats in the Cocaine-paired locomotor activity chamber on Induction day. The x-axes indicate conditions used on Induction day three days prior to Test day. On induction day, Vehicle or Daun02 was injected into the nucleus accumbens 90 min following systemic injections of Saline or Cocaine to rats. Daun02 did not attenuate cocaine-induced locomotion or neural activity when non-drug-associated neurons were activated in the Saline-injected rats. The column on the left indicates cocaine-induced locomotion on the first day of repeated injections during sensitization training, and is provided only to show that Daun02 attenuated cocaine-induced locomotion on test day in sensitized rats to approximately the same level as that seen in non-sensitized rats. * indicates a significant difference (p<0.05) between Vehicle and Daun02 groups following cocaine injections three days earlier on induction day. Modified figure from (Koya et al., 2009).

Figure 4

Daun02 inactivation of Fos-expressing neuronal ensembles in different drug relapse models in rats. Lever-pressing behavior on test day is indicated on the y-axes of graphs in the left-hand column. Neural activity, indicated by number of β-galactosidase (β-gal)-labeled neurons, on test day is indicated on the y-axes of graphs in the right-hand column. The x-axes indicate conditions used on Induction day three days prior to Test day. Panel A: Daun02 (but not Vehicle) injections into ventromedial prefrontal cortex (vmPFC) following exposure to the Heroin context (but not the Extinction context) on Induction day attenuated context-induced reinstatement of heroin seeking (lever pressing) and neural activity three days later on Test day. Modified figure from (Bossert et al., 2011). Panel B: Daun02 (but not Vehicle) injections into nucleus accumbens shell following exposure to the Cocaine context (but not to a Novel context) on Induction day attenuated context-induced reinstatement of cocaine seeking (lever pressing) and neural activity three days later on Test day. Modified figure from (Cruz et al., 2014). Panel C: Daun02 (but not Vehicle) injections into orbitofrontal cortex (OFC) following exposure to the Heroin context (but not to a Novel context) on Induction day attenuated cue-induced heroin seeking (lever pressing) and neural activity three days later on Test day. Modified figure from (Fanous et al., 2012). * indicates a significant difference (p<0.05) between Vehicle and Daun02 groups following exposure to the drug context three days earlier on induction day.