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

Abstract

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.

Keywords: Fos; electrophysiology; intrinsic plasticity; motivated behavior; reward learning; transgenic rat.

Figure 1.

Fos-expressing neuronal ensembles play a causal role in the expression of food-seeking behavior. A, B, Experimental timeline (A) and self-administration data (B) in wild-type rats. C, Representative images (scale bar is 20 μm) and quantification of Fos⁺ PLC neurons (*p < 0.05; no test–day 1: n = 6, no test–day 10: n = 6, test–day 1: n = 10, test–day 10: n = 10). Scale bar, 20 μm. D–F, Experimental timeline (D) and food self-administration (E). F, Cannula placement for Daun02 inactivation experiment. H, I, Lever-pressing behavior in FosLacZ rats on test day (*p < 0.05; vehicle: n = 19, Daun02: n = 18). Shown are representative images of B-gal staining (H) and B-gal quantification (I) after Daun02 inactivation (*p < 0.05; vehicle: n = 16, Daun02: n = 16). Scale bar, 20 μm.

Figure 2.

Bidirectional modulation of PLC FosGFP⁺ and FosGFP⁻ neuronal excitability after food self-administration training. A, Experimental configuration and representative image of FosGFP⁺ neuron in PLC. DIC, Differential interference contrast). B, Example traces from FosGFP⁻ and FosGFP⁺ neurons in test and no test groups after somatic current injection. C, Input–output curves for FosGFP⁻ and FosGFP⁺ neurons in test and no test groups (different from day 1, *p < 0.05; no test–day 1 FosGFP⁻: n = 16 cells/11 rats; no test–day 1 FosGFP⁺: n = 12 cells/8 rats; no test–day 10 FosGFP⁻: n = 7 cells/4 rats; no test–day 10 FosGFP⁺: n = 8 cells/5 rats; test–day 1 FosGFP⁻: n = 11 cells/9 rats; test–day 1 FosGFP⁺: 10 cells/7 rats; test–day 10 FosGFP⁻: n = 15 cells/11 rats; test–day 10 FosGFP⁺: n = 16 cells/11 rats).

Figure 3.

Subthreshold membrane properties of PLC FosGFP⁺ and FosGFP⁻ neurons after food self-administration training. A, Representative traces showing measurement of input resistance using the slope of the I–V curve after injections of hyperpolarizing current. B, Input resistance is significantly greater in FosGFP⁺ neurons on day 10 than on day 1. Input resistance in day 1 FosGFP⁺ neurons was lower than FosGFP⁻ neurons on day 1. C, Resting membrane potential remains consistent in both no test and test groups after operant training. D, Example traces showing measurement of sag current ratio in FosGFP⁻ and FosGFP⁺ neurons in test and no test groups and summary graphs showing percentage sag ratio in no test and test groups. Sag current is reduced in the Day 10 FosGFP⁺ neurons in the no test group but remains unchanged in the test group (*p < 0.05, no test–day 1 FosGFP⁻: n = 16 cells/11 rats; no test–day 1 FosGFP⁺: n = 12 cells/8 rats; no test–day 10 FosGFP⁻: n = 7 cells/4 rats; no test–day 10 FosGFP⁺: n = 8 cells/5 rats; test–day 1 FosGFP⁻: n = 11 cells/9 rats; test–day 1 FosGFP⁺: 10 cells/7 rats; test–day 10 FosGFP⁻: n = 15 cells/11 rats; test–day 10 FosGFP⁺: n = 16 cells/11 rats).

Figure 4.

mAHP is increased in FosGFP⁻ neurons on day 10 in both test and no test groups. A, Representative AP trace from FosGFP⁺ neuron on two different time scales. Red dotted line shows the portion of the trace analyzed to determine the magnitude of the medium AHP. B, Example traces of mAHP currents (AP is truncated) in FosGFP⁻ and FosGFP⁺ neurons in test and no test groups. C, mAHP amplitude is enhanced exclusively in FosGFP⁻ Day 10 neurons in both the test and no test groups (*p < 0.05; no test–day 1 FosGFP⁻: n = 16 cells/11 rats; no test–day 1 FosGFP⁺: n = 12 cells/8 rats; no test–day 10 FosGFP⁻: n = 7 cells/4 rats; no test–day 10 FosGFP⁺: n = 8 cells/5 rats; test–day 1 FosGFP⁻: n = 11 cells/9 rats; test–day 1 FosGFP⁺: 10 cells/7 rats; test–day 10 FosGFP⁻: n = 15 cells/11 rats; test–day 10 FosGFP⁺: n = 16 cells/11 rats). D, AP parameters remain unchanged in no test and test groups (p > 0.05 for all groups and comparisons, no test–day 1 FosGFP⁻: n = 16 cells/11 rats; no test–day 1 FosGFP⁺: n = 12 cells/8 rats; no test–day 10 FosGFP⁻: n = 7 cells/4 rats; no test–day 10 FosGFP⁺: n = 8 cells/5 rats; test–day 1 FosGFP⁻: n = 11 cells/9 rats; test–day 1 FosGFP⁺: 10 cells/7 rats; test–day 10 FosGFP⁻: n = 15 cells/11 rats; test–day 10 FosGFP⁺: n = 16 cells/11 rats).

Figure 5.

Enhanced contribution of SK channels in nonactivated population of neurons after operant learning. A, B, Example traces showing the effect of SK channel antagonism on mAHP currents in FosGFP⁻ neurons in the test group. Summary graph shows larger SK contribution to the mAHP in day 10 FosGFP⁻ neurons (*p < 0.05, day 1 FosGFP⁻: n = 10 cells/6 rats; day 10 FosGFP⁻: n = 9 cells/6 rats). C, Example traces showing larger effect of SK channel antagonism on repetitive firing in FosGFP⁻ day 10 neurons from the test group. D, Summary graph showing the percentage increase in firing after SK channel antagonism in FosGFP⁻ neurons (*p < 0.05, day 1 FosGFP⁻: n = 10 cells/6 rats; day 10 FosGFP⁻: n = 9 cells/6 rats).

Figure 6.

Intra-PLC administration of the SK channel antagonist apamin has no effect on the expression of operant learning. A, B, Experimental timeline and food self-administration behavior. C, Cannula placement for in vivo apamin experiment. D, Lever-pressing behavior on test day (vehicle: n = 9, Apamin n = 9; E). Representative images of Fos expression and Fos quantification after in vivo treatment with apamin are shown (*p < 0.05; vehicle: n = 9, Apamin n = 9).