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

Abstract

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.

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

“…More specifically, among interneurons persistently activated during extinction training, the proportion of those neurons with a previous history of persistent activation in acquisition is decreased. Thus, we suggest that extinction learning recruits a stable interneuron ensemble from a wider pool of neurons activated in the initial extinction session and that this ensemble is partly distinct from those interneurons that were persistently activated in conditioning (Brebner et al, 2020).…”

“…Half-width was measured as the AP width at half-maximal spike following cubic spline interpolation to increase sampling rate by a factor of 4. Post-spike fAHPs and mAHPs were measured ~ 3 and 40 ms following the AP threshold, respectively, similar to (Ishikawa et al, 2009 2.5 | In vivo 2-Photon imaging 2.5.1 | Habituation and imaging sessions 2-Photon imaging experiments were conducted as described in (Brebner et al, 2020). Imaging sessions took place on headfixed, awake mice that were able to freely run on a polystyrene cylinder (Figure 2c).…”

“…Interneuron activation has been associated with multiple forms of associative learning, including appetitive and aversive conditioning (Courtin et al, 2014;Doron & Rosenblum, 2010;Pinto & Dan, 2015;Stefanelli et al, 2016). Furthermore, we and others have shown that such forms of learning recruit persistently activated populations of neurons called neuronal ensembles (Brebner et al, 2020;Cao et al, 2015;Czajkowski et al, 2014;Mattson et al, 2008;Tayler, Tanaka, Reijmers, & Wiltgen, 2013). Our results provide an important new step by demonstrating that (a) extinction learning recruits a stable interneuron ensemble in the dmPFC, and (b) this ensemble was preferentially recruited from interneurons activated during the initial phase of extinction (i.e., extinction session 1), when behavioral responding is generally elevated.…”

“…Similar behavioral experimental procedures and apparatus were utilized as in Brebner et al (2020) and Ziminski et al (2017). Briefly, behavioral experiments were performed in standard mouse conditioning chambers (15.9 × 14 × 12.7 cm; Med Associates), each fitted with a recessed magazine for dispensing boluses of ~15 µl of 10% sucrose solution that served as the unconditioned stimulus (US).…”

Coordinated prefrontal state transition leads extinction of reward-seeking behaviors

“…This sustained responsiveness did not correspond, however, to stability in single-unit coding. While, on the one hand, some single units in mPFC can maintain their response pattern across days (Brebner et al, 2020;Powell and Redish, 2014), on the other hand, we found that the majority of units significantly changed their average firing rate and their stimulus-responsiveness within tens of minutes. It is important to note that our approach captures longer-lasting changes in unit coding, i.e., significant jumps in firing probability between periods of relative stability, and not trial-to-trial variability as observed in many brain regions.…”