Regional differences in striatal neuronal ensemble excitability following cocaine and extinction memory retrieval in Fos-GFP mice

Learned associations between drugs of abuse and the drug administration environment play an important role in addiction. In rodents, exposure to a drug-associated environment elicits conditioned psychomotor activation, which may be weakened following extinction learning. While widespread drug-induced changes in neuronal excitability have been observed, little is known about specific changes within neuronal ensembles activated during the recall of drugenvironment associations. Using a cocaine conditioned locomotion procedure, the present study assessed the excitability of neuronal ensembles in the nucleus accumbens core and shell (NAccore and NAcshell), and dorsal striatum (DS) following cocaine conditioning and extinction in Fos-GFP mice that express green fluorescent protein (GFP) in activated neurons (GFP+). During conditioning, mice received repeated cocaine injections (20 mg/kg) paired with a locomotor activity chamber (Paired) or home cage (Unpaired). 7-13 days later both groups were re-exposed to the activity chamber under drug-free conditions, and Paired, but not Unpaired, mice exhibited conditioned locomotion. In a separate group of mice, conditioned locomotion was extinguished by repeatedly exposing mice to the activity chamber under drugfree conditions. Following the expression and extinction of conditioned locomotion, GFP+ neurons in the NAccore (but not NAcshell and DS) displayed greater firing capacity compared to surrounding GFP– neurons. This difference in excitability was due to a generalized decrease in GFP– excitability following conditioned locomotion, and a selective increase in GFP+ excitability following its extinction. These results suggest a role for both widespread and ensemble-specific changes in neuronal excitability following recall of drug-environment associations

Changes in appetitive associative strength modulates nucleus accumbens, but not orbitofrontal cortex neuronal ensemble excitability

Cues that predict the availability of food rewards influence motivational states and elicit food-seeking behaviors. If a cue no longer predicts food availability, animals may adapt accordingly by inhibiting food seeking responses. Sparsely activated sets of neurons, coined neuronal ensembles, have been shown to encode the strength of reward-cue associations. While alterations in intrinsic excitability have been shown to underlie many learning and memory processes, little is known about these properties specifically on cue-activated neuronal ensembles. We examined the activation patterns of cue-activated orbitofrontal cortex (OFC) and nucleus accumbens (NAc) shell ensembles using wild-type and Fos-GFP mice following appetitive conditioning with sucrose and extinction learning. We also investigated the neuronal excitability of recently activated, GFP+ neurons in these brain areas using whole-cell electrophysiology in brain slices. Exposure to a sucrose cue elicited activation of neurons in both the NAc shell and OFC. In the NAc shell, but not the OFC, these activated GFP+ neurons were more excitable than surrounding GFP– neurons. Following extinction, the number of neurons activated in both areas was reduced and activated ensembles in neither area exhibited altered excitability. These data suggest that learning-induced alterations in the intrinsic excitability of neuronal ensembles is regulated dynamically across different brain areas. Furthermore, we show that changes in associative strength modulate the excitability profile of activated ensembles in the NAc shell

Extinction of cue-evoked food seeking recruits a GABAergic interneuron ensemble in the dorsal medial prefrontal cortex of mice

Animals must quickly adapt food-seeking strategies to locate nutrient sources in dynamically changing environments. Learned associations between food and environmental cues that predict its availability promote food-seeking behaviors. However, when such cues cease to predict food availability, animals undergo 'extinction' learning, resulting in the inhibition of food-seeking responses. Repeatedly activated sets of neurons, or 'neuronal ensembles', in the dorsal medial prefrontal cortex (dmPFC) are recruited following appetitive conditioning and undergo physiological adaptations thought to encode cue-reward associations. However, little is known about how the recruitment and intrinsic excitability of such dmPFC ensembles are modulated by extinction learning. Here, we used in vivo 2-Photon imaging in male Fos-GFP mice that express green fluorescent protein (GFP) in recently behaviorally-activated neurons to determine the recruitment of activated pyramidal and GABAergic interneuron mPFC ensembles during extinction. During extinction, we revealed a persistent activation of a subset of interneurons which emerged from a wider population of interneurons activated during the initial extinction session. This activation pattern was not observed in pyramidal cells, and extinction learning did not modulate the excitability properties of activated neurons. Moreover, extinction learning reduced the likelihood of reactivation of pyramidal cells activated during the initial extinction session. Our findings illuminate novel neuronal activation patterns in the dmPFC underlying extinction of food-seeking, and in particular, highlight an important role for interneuron ensembles in this inhibitory form of learning

Acute, but not longer-term, exposure to environmental enrichment attenuates Pavlovian cue-evoked conditioned approach and Fos expression in the prefrontal cortex in mice.

Funder: The University of Sussex Strategic Development FundsFunder: Sussex Neuroscience 4‐year PhD programmeExposure to environmental enrichment can modify the impact of motivationally relevant stimuli. For instance, previous studies in rats have found that even a brief, acute (~1 day), but not chronic, exposure to environmentally enriched (EE) housing attenuates instrumental lever pressing for sucrose-associated cues in a conditioned reinforcement setup. Moreover, acute EE reduces corticoaccumbens activity, as measured by decreases in expression of the neuronal activity marker "Fos." Currently, it is not known whether acute EE also reduces sucrose seeking and corticoaccumbens activity elicited by non-contingent or "forced" exposure to sucrose cues, which more closely resembles cue exposure encountered in daily life. We therefore measured the effects of acute/intermittent (1 day or 6 day of EE prior to test day) versus chronic (EE throughout conditioning lasting until test day) EE on the ability of a Pavlovian sucrose cue to elicit sucrose seeking (conditioned approach) and Fos expression in the medial prefrontal cortex (mPFC), orbitofrontal cortex (OFC), and nucleus accumbens (NAc) in mice. One day, but not 6 day or chronic EE , reduced sucrose seeking and Fos in the deep layers of the dorsal mPFC. By contrast, 1 day, 6 day, and chronic EE all reduced Fos in the shallow layers of the OFC. None of the EE manipulations modulated NAc Fos expression. We reveal how EE reduces behavioral reactivity to sucrose cues by reducing activity in select prefrontal cortical brain areas. Our work further demonstrates the robustness of EE in its ability to modulate various forms of reward-seeking across species

The emergence of a stable neuronal ensemble from a wider pool of activated neurons in the dorsal medial prefrontal cortex during appetitive learning in mice

Animals selectively respond to environmental cues associated with food reward to optimize nutrient intake. Such appetitive CS-US associations are thought to be encoded in select, stable neuronal populations or neuronal ensembles, which undergo physiological modifications during appetitive conditioning. These ensembles in the medial prefrontal cortex (mPFC) control well-established, cue-evoked food seeking, but the mechanisms involved in the genesis of these ensembles are unclear. Here, we utilized male Fos-GFP mice that express the green fluorescent protein (GFP) in recently behaviorally-activated neurons, to reveal how dorsal mPFC neurons are recruited and modified to encode CS-US memory representations using an appetitive conditioning task. In the initial conditioning session, animals did not exhibit discriminated, cue-selective food seeking, but did so in later sessions indicating that a CS-US association was established. Using microprism-based in vivo 2-Photon imaging, we revealed that only a minority of neurons activated during the initial session was consistently activated throughout subsequent conditioning sessions and during cue-evoked memory recall. Notably, using ex vivo electrophysiology we found that neurons activated following the initial session exhibited transient hyper-excitability. Chemogenetically enhancing the excitability of these neurons throughout subsequent conditioning sessions interfered with the development of reliable cue-selective food seeking, indicated by persistent, non-discriminated performance. We demonstrate how appetitive learning consistently activates a subset of neurons to form a stable neuronal ensemble during the formation of a CS-US association. This ensemble may arise from a pool of hyper-excitable neurons activated during the initial conditioning session

Reward devaluation attenuates cue-evoked sucrose seeking and is associated with the elimination of excitability differences between ensemble and non-ensemble neurons in the nucleus accumbens

Animals must learn relationships between foods and the environmental cues that predict their availability for survival. Such cue-food associations are encoded in sparse sets of neurons or ‘neuronal ensembles’ in the nucleus accumbens (NAc). For these ensemble-encoded, cue-controlled appetitive responses to remain adaptive, they must allow for their dynamic updating depending on acute changes in internal states such as physiological hunger or the perceived desirability of food. However, how these neuronal ensembles are recruited and physiologically modified following the update of such learned associations is unclear. To investigate this, we examined the effects of devaluation on ensemble plasticity at the levels of recruitment, intrinsic excitability, and synaptic physiology in sucrose conditioned Fos-GFP mice that express green fluorescent protein (GFP) in recently activated neurons. Neuronal ensemble activation patterns and their physiology were examined using immunohistochemistry and slice electrophysiology, respectively. Reward-specific devaluation following four days of ad lib sucrose consumption, but not general caloric devaluation, attenuated cue-evoked sucrose seeking. This suggests that changes in the hedonic and/or incentive value of sucrose, and not caloric need drove this behavior. Moreover, devaluation attenuated the size of the neuronal ensemble recruited by the cue in the NAc shell. Finally, it eliminated the relative enhanced excitability of ensemble (GFP+) neurons against non-ensemble (GFP–) neurons observed under Non-devalued conditions, and did not induce any ensemble-specific changes in excitatory synaptic physiology. Our findings provide new insights into neuronal ensemble mechanisms that underlie the changes in the incentive and/or hedonic impact of cues that support adaptive food seeking

Reward devaluation attenuates cue-evoked sucrose seeking and is associated with the elimination of excitability differences between ensemble and non-ensemble neurons in the nucleus accumbens

Animals must learn relationships between foods and the environmental cues that predict their availability for survival. Such cue-food associations are encoded in sparse sets of neurons or ‘neuronal ensembles’ in the nucleus accumbens (NAc). For these ensemble-encoded, cue-controlled appetitive responses to remain adaptive, they must allow for their dynamic updating depending on acute changes in internal states such as physiological hunger or the perceived desirability of food. However, how these neuronal ensembles are recruited and physiologically modified following the update of such learned associations is unclear. To investigate this, we examined the effects of devaluation on ensemble plasticity at the levels of recruitment, intrinsic excitability, and synaptic physiology in sucrose conditioned Fos-GFP mice that express green fluorescent protein (GFP) in recently activated neurons. Neuronal ensemble activation patterns and their physiology were examined using immunohistochemistry and slice electrophysiology, respectively. Reward-specific devaluation following four days of ad lib sucrose consumption, but not general caloric devaluation, attenuated cue-evoked sucrose seeking. This suggests that changes in the hedonic and/or incentive value of sucrose, and not caloric need drove this behavior. Moreover, devaluation attenuated the size of the neuronal ensemble recruited by the cue in the NAc shell. Finally, it eliminated the relative enhanced excitability of ensemble (GFP+) neurons against non-ensemble (GFP–) neurons observed under Non-devalued conditions, and did not induce any ensemble-specific changes in excitatory synaptic physiology. Our findings provide new insights into neuronal ensemble mechanisms that underlie the changes in the incentive and/or hedonic impact of cues that support adaptive food seeking

Reward devaluation attenuates cue-evoked sucrose seeking and is associated with the elimination of excitability differences between ensemble and non-ensemble neurons in the nucleus accumbens

Animals must learn relationships between foods and the environmental cues that predict their availability for survival. Such cue-food associations are encoded in sparse sets of neurons or ‘neuronal ensembles’ in the nucleus accumbens (NAc). For these ensemble-encoded, cue-controlled appetitive responses to remain adaptive, they must allow for their dynamic updating depending on acute changes in internal states such as physiological hunger or the perceived desirability of food. However, how these neuronal ensembles are recruited and physiologically modified following the update of such learned associations is unclear. To investigate this, we examined the effects of devaluation on ensemble plasticity at the levels of recruitment, intrinsic excitability, and synaptic physiology in sucrose conditioned Fos-GFP mice that express green fluorescent protein (GFP) in recently activated neurons. Neuronal ensemble activation patterns and their physiology were examined using immunohistochemistry and slice electrophysiology, respectively. Reward-specific devaluation following four days of ad lib sucrose consumption, but not general caloric devaluation, attenuated cue-evoked sucrose seeking. This suggests that changes in the hedonic and/or incentive value of sucrose, and not caloric need drove this behavior. Moreover, devaluation attenuated the size of the neuronal ensemble recruited by the cue in the NAc shell. Finally, it eliminated the relative enhanced excitability of ensemble (GFP+) neurons against non-ensemble (GFP–) neurons observed under Non-devalued conditions, and did not induce any ensemble-specific changes in excitatory synaptic physiology. Our findings provide new insights into neuronal ensemble mechanisms that underlie the changes in the incentive and/or hedonic impact of cues that support adaptive food seeking