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 4 d of ad libitum 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.

Sieburg MC ，Ziminski JJ ，Margetts-Smith G ，Reeve HM ，Brebner LS ，Crombag HS ，Koya E ... -  《eNeuro》

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, then 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. Although 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, which express green fluorescent protein (GFP) in activated neurons, after 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. After 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.SIGNIFICANCE STATEMENT Sparsely distributed sets of neurons called "neuronal ensembles" encode learned associations about food and cues predictive of its availability. Widespread changes in neuronal excitability have been observed in limbic brain areas after associative learning, but little is known about the excitability changes that occur specifically on neuronal ensembles that encode appetitive associations. Here, we reveal that sucrose cue exposure recruited a more excitable ensemble in the nucleus accumbens, but not orbitofrontal cortex, compared with their surrounding neurons. This excitability difference was not observed when the cue's salience was diminished after extinction learning. These novel data provide evidence that the intrinsic excitability of appetitive memory-encoding ensembles is regulated differentially across brain areas and adapts dynamically to changes in associative strength.

Ziminski JJ ，Hessler S ，Margetts-Smith G ，Sieburg MC ，Crombag HS ，Koya E ... -  《-》

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 conditioned stimulus-unconditioned stimulus (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 used male Fos-GFP mice that express 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 hyperexcitability. Chemogenetically enhancing the excitability of these neurons throughout subsequent conditioning sessions interfered with the development of reliable cue-selective food seeking, indicated by persistent, nondiscriminated 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 hyperexcitable neurons activated during the initial conditioning session.SIGNIFICANCE STATEMENT Appetitive conditioning endows cues associated with food with the ability to guide food-seeking, through the formation of a food-cue association. Neuronal ensembles in the mPFC control established cue-evoked food-seeking. However, how neurons undergo physiological modifications and become part of an ensemble during conditioning remain unclear. We found that only a minority of dorsal mPFC neurons activated on the initial conditioning session became consistently activated during conditioning and memory recall. These initially activated neurons were also transiently hyperexcitable. We demonstrate the following: (1) how stable neuronal ensemble formation in the dorsal mPFC underlies appetitive conditioning; and (2) how this ensemble may arise from hyperexcitable neurons activated before the establishment of cue-evoked food seeking.

Brebner LS ，Ziminski JJ ，Margetts-Smith G ，Sieburg MC ，Reeve HM ，Nowotny T ，Hirrlinger J ，Heintz TG ，Lagnado L ，Kato S ，Kobayashi K ，Ramsey LA ，Hall CN ，Crombag HS ，Koya E ... -  《-》

Nucleus Accumbens Core and Shell Differentially Encode Reward-Associated Cues after Reinforcer Devaluation.

Nucleus accumbens (NAc) neurons encode features of stimulus learning and action selection associated with rewards. The NAc is necessary for using information about expected outcome values to guide behavior after reinforcer devaluation. Evidence suggests that core and shell subregions may play dissociable roles in guiding motivated behavior. Here, we recorded neural activity in the NAc core and shell during training and performance of a reinforcer devaluation task. Long-Evans male rats were trained that presses on a lever under an illuminated cue light delivered a flavored sucrose reward. On subsequent test days, each rat was given free access to one of two distinctly flavored foods to consume to satiation and were then immediately tested on the lever pressing task under extinction conditions. Rats decreased pressing on the test day when the reinforcer earned during training was the sated flavor (devalued) compared with the test day when the reinforcer was not the sated flavor (nondevalued), demonstrating evidence of outcome-selective devaluation. Cue-selective encoding during training by NAc core (but not shell) neurons reliably predicted subsequent behavioral performance; that is, the greater the percentage of neurons that responded to the cue, the better the rats suppressed responding after devaluation. In contrast, NAc shell (but not core) neurons significantly decreased cue-selective encoding in the devalued condition compared with the nondevalued condition. These data reveal that NAc core and shell neurons encode information differentially about outcome-specific cues after reinforcer devaluation that are related to behavioral performance and outcome value, respectively. Many neuropsychiatric disorders are marked by impairments in behavioral flexibility. Although the nucleus accumbens (NAc) is required for behavioral flexibility, it is not known how NAc neurons encode this information. Here, we recorded NAc neurons during a training session in which rats learned that a cue predicted a specific reward and during a test session when that reward value was changed. Although encoding in the core during training predicted the ability of rats to change behavior after the reward value was altered, the NAc shell encoded information about the change in reward value during the test session. These findings suggest differential roles of the core and shell in behavioral flexibility.

West EA ，Carelli RM 《-》

Bidirectional Modulation of Intrinsic Excitability in Rat Prelimbic Cortex Neuronal Ensembles and Non-Ensembles after Operant Learning.

Learned associations between environmental stimuli and rewards drive goal-directed learning and motivated behavior. These memories are thought to be encoded by alterations within specific patterns of sparsely distributed neurons called neuronal ensembles that are activated selectively by reward-predictive stimuli. Here, we use the Fos promoter to identify strongly activated neuronal ensembles in rat prelimbic cortex (PLC) and assess altered intrinsic excitability after 10 d of operant food self-administration training (1 h/d). First, we used the Daun02 inactivation procedure in male FosLacZ-transgenic rats to ablate selectively Fos-expressing PLC neurons that were active during operant food self-administration. Selective ablation of these neurons decreased food seeking. We then used male FosGFP-transgenic rats to assess selective alterations of intrinsic excitability in Fos-expressing neuronal ensembles (FosGFP+) that were activated during food self-administration and compared these with alterations in less activated non-ensemble neurons (FosGFP-). Using whole-cell recordings of layer V pyramidal neurons in an ex vivo brain slice preparation, we found that operant self-administration increased excitability of FosGFP+ neurons and decreased excitability of FosGFP- neurons. Increased excitability of FosGFP+ neurons was driven by increased steady-state input resistance. Decreased excitability of FosGFP- neurons was driven by increased contribution of small-conductance calcium-activated potassium (SK) channels. Injections of the specific SK channel antagonist apamin into PLC increased Fos expression but had no effect on food seeking. Overall, operant learning increased intrinsic excitability of PLC Fos-expressing neuronal ensembles that play a role in food seeking but decreased intrinsic excitability of Fos- non-ensembles.SIGNIFICANCE STATEMENT Prefrontal cortex activity plays a critical role in operant learning, but the underlying cellular mechanisms are unknown. Using the chemogenetic Daun02 inactivation procedure, we found that a small number of strongly activated Fos-expressing neuronal ensembles in rat PLC play an important role in learned operant food seeking. Using GFP expression to identify Fos-expressing layer V pyramidal neurons in prelimbic cortex (PLC) of FosGFP-transgenic rats, we found that operant food self-administration led to increased intrinsic excitability in the behaviorally relevant Fos-expressing neuronal ensembles, but decreased intrinsic excitability in Fos- neurons using distinct cellular mechanisms.

Whitaker LR ，Warren BL ，Venniro M ，Harte TC ，McPherson KB ，Beidel J ，Bossert JM ，Shaham Y ，Bonci A ，Hope BT ... -  《-》