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 ... -  《-》

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 ... -  《-》

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 dmPFC 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 pyramidal cells. 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.

Brebner LS ，Ziminski JJ ，Margetts-Smith G ，Sieburg MC ，Hall CN ，Heintz TG ，Lagnado L ，Hirrlinger J ，Crombag HS ，Koya E ... -  《-》

Medial Prefrontal Cortex Neural Plasticity, Orexin Receptor 1 Signaling, and Connectivity with the Lateral Hypothalamus Are Necessary in Cue-Potentiated Feeding.

Cognitive processes contribute to the control of feeding behavior and help organism's survival when they support physiological needs. They can become maladaptive, such as when learned food cues drive feeding in the absence of hunger. Associative learning is the basis for cue-driven food seeking and consumption, and behavioral paradigms with Pavlovian cue-food conditioning are well established. Yet, the neural mechanisms underlying circuit plasticity across cue-food learning, cue memory recall, and subsequent food motivation are unknown. Here, we demonstrated the medial prefrontal cortex (mPFC) is a site of learning-induced plasticity and signaling of the neuropeptide orexin within the mPFC mediates cue potentiated feeding (CPF). First, using a marker of neuronal activation, c-fos, we confirmed that the mPFC is activated during CPF. Next, to assess whether the same mPFC neuronal ensemble is activated during cue-food learning and later CPF, we used the Daun02 chemogenetic inactivation method in c-fos-lacZ transgenic male and female rats. Selective inactivation of the mPFC neurons that were active during the last cue-food training session abolished CPF during test, demonstrating that the mPFC is a site of plasticity. We postulated that integration of food cue memory and feeding motivation requires mPFC communications with lateral hypothalamus and showed that disconnection of that system abolished CPF. Then we showed that lateral hypothalamus orexin-producing neurons project to the mPFC. Finally, we blocked orexin receptor 1 signaling in the mPFC and showed that it is a neuromodulator necessary for the cue-driven consumption. Together, our findings identify a causal function for the mPFC in the cognitive motivation to eat.SIGNIFICANCE STATEMENT Obesity has reached epidemic proportions, and the associated health consequences are serious and costly. The causes of obesity are complex because, in addition to physiological energy and nutrient needs, environmental cues can drive feeding through hedonic and cognitive processes. Learned food cues from the environment can powerfully stimulate appetite and food consumption in the absence of hunger. Using an animal model for cue-potentiated feeding, the current study determined the mPFC neuronal plasticity and neuropeptide orexin signaling are critical circuit and neurotransmitter mechanisms involved in this form of cognitive motivation to eat. These findings identify key targets for potential treatment of excessive appetite and overeating.

Cole S ，Keefer SE ，Anderson LC ，Petrovich GD ... -  《-》

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》