Multi-channel unit recordings were performed rats undergoing auditory fear conditioning and extinction (see ). Freezing behavior served as a measure of fear. As shown in , rats acquired significant levels of freezing to the tone as conditioning proceeded (repeated measures ANOVA: F(4,64) = 59.2, p < 0.001), and diminished freezing with extinction training (F(19,304) = 23.5, p < 0.001). Rats showed substantial recall of extinction 24 hr later (freezing levels at the beginning of extinction vs. at the beginning of recall, F(1,16) = 23.9, p < 0.001).
Multi-channel unit recordings were performed in the prelimbic subregion of the medial prefrontal cortex of rats undergoing auditory fear conditioning and extinction
Fear conditioning induces sustained conditioned unit responses in a subpopulation of prelimbic neurons
Tone responses were examined for 81 PL neurons recorded from 17 rats that were maintained throughout habituation, conditioning and extinction training. Fear conditioning dramatically increased the spiking rate of a number of PL neurons, throughout the presentation of tones. These sustained excitatory responses can be observed in , which shows spike trains of two representative neurons prior to fear conditioning (during habituation trials) and after fear conditioning (during early extinction trials). Peri-event time histograms for these neurons revealed that tone-elicited responses were typical of high-fear states (conditioning and early extinction), but not of low-fear states (habituation and late extinction).
Prelimbic neurons showed sustained excitatory responses to conditioned tones
The percentage of neurons showing excitatory tone responses (z > 2.58 in two or more tone bins) was calculated at different time points of the experiment. As illustrated in , fear conditioning increased the percentage of neurons showing excitatory responses from 11% to 26% (black-filled area in pie charts; χ2 = 4.98, p = 0.026). This conditioning effect was reversed by extinction training, which reduced the percentage of responsive neurons from 23% to 10% (χ2 = 4.95, p = 0.026). Only a small proportion of neurons showed inhibitory tone responses (gray-filled area in pie charts), which did not change significantly with conditioning or extinction (p’s > 0.27).
The time course of conditioned responses in the prelimbic cortex is highly correlated with expression of freezing
Combined with prior inactivation studies, the sustained nature of PL conditioned responses suggests that they contribute to the generation of sustained freezing responses. To examine this hypothesis, we compared the activity of PL neurons showing significant excitatory tone responses with freezing on a second-to-second timescale. We observed that conditioned responses in PL were correlated with the time course of freezing. The top panel on shows the time course of freezing prior to conditioning (habituation), after conditioning (early extinction), and after extinction (late extinction). Second-to-second freezing significantly differed among the training phases, according to repeated measures ANOVA (main effect of training phase, F(2,48) = 35.2, p < 0.001; seconds, F(21,1008) = 8.48, p < 0.001; phase vs. seconds interaction, F(42,1008) = 8.23, p < 0.001). PL activity tended to parallel freezing responses in all phases. The lower panel in illustrates the average activity of those PL neurons showing excitatory tone responses during early extinction (19/81 neurons, 23%). Similar to freezing during early extinction, the activity of these neurons significantly increased at tone onset, remained high throughout the tone presentation, outlasted the tone, and gradually came back to pretone levels. Furthermore, as with freezing, the response of these neurons returned to habituation levels by the end of extinction training. An ANOVA comparing tone responses across phases showed significantly higher tone-elicited PL activity during early extinction (training phase, F(2,54) = 13.9, p < 0.001; seconds, F(21,1134) = 2.67, p < 0.001; interaction, F(42,1134) = 3.38, p < 0.001). A bin-by-bin comparison of PL activity (, lower panel) and freezing (, top panel) showed that the two measures were significantly correlated during early extinction (r = 0.83, df = 20, p < 0.001). No significant correlations were observed during habituation (r = 0.41, p = 0.06) and late extinction (r = 0.07, p = 0.76).
Conditioned responses in prelimbic neurons were highly correlated with the time course of conditioned freezing
A significant correlation between PL activity and freezing during early extinction supports the hypothesis that PL activity contributes to the generation of freezing. An alternative hypothesis is that freezing triggers PL activity. To distinguish between these two alternatives, we examined the response latency of freezing and PL activity during early extinction, in 1-sec bins. As shown in , the onset of PL tone responses (blue line plot) preceded the onset of freezing responses (gray bar graph). Significant tone-induced increased activity in PL was evident from the very first second after tone onset, whereas significant freezing did not occur until two seconds after tone onset. To assess the earliest tone response latency in PL, we examined activity in 100-ms bins. PL tone responses were not evident until after 100 ms after tone onset (see ). These findings argue against the hypothesis that PL tone responses are induced by freezing.
To further test our hypothesis that PL activity parallels freezing, we took advantage of within-subject variability to performed activity-behavior correlations for single PL neurons. For each neuron-rat pair, activity and freezing were averaged across 5 trials during early extinction. This analysis showed that changes in PL activity across the tone paralleled changes in freezing. As shown in , more neurons showed positive correlations with freezing than negative correlations (66% vs. 34%; binomial probability test, p = 0.007). Furthermore, 24% of the activity-freezing pairs showed significant positive correlations (r’s > 0.55, p’s < 0.01), whereas only 4% showed significant negative correlations (r’s < −0.55) (proportion of significant positive vs. negative correlations, χ2 = 7.85, p = 0.005). Altogether, the present findings support the hypothesis that PL activity mediates conditioned fear responses.
Single-neuron examples of prelimbic activity versus freezing
Fear conditioning increases bursting and synchrony in prelimbic neurons
In addition to increased rate during the tone, PL neurons showed increased bursting following conditioning. As shown in , levels of bursting during the tone increased with conditioning, and tended to reduce with extinction training (; F(2,36) = 3.69, p = 0.035; habit vs. early ext, p = 0.029; early ext vs. late ext, p = 0.19). Training-induced changes in bursting paralleled changes in firing rate during the tone (; F(2,36) = 11.7, p < 0.001; habit vs. early ext, p < 0.001; early ext vs. late ext, p = 0.0013), although these increases were not correlated with each other across neurons (see ; r = −0.09, df = 17, p = 0.71). This suggests that increased bursting was not an epiphenomenon of increased rate. In contrast to the tone, spontaneous activity during pretone periods did not differ across phases (pretone bursting: F(2,36) = 0.73, p = 0.49; pretone rate: F(2,36) = 1.32, p = 0.28).
Conditioning increased bursting and synchrony in prelimbic neurons
Cross-correlations between simultaneously recorded neurons showed increased synchrony during tone periods as a function of conditioning. Correlograms for 36 of 147 cell pairs (24%) showed significant peaks within 100 ms of reference spikes (see Material and Methods for analysis on cross-correlations). The average correlogram for these 36 cell pairs (corrected with shift-predictors) is shown in . Compared to the habituation phase, early extinction showed significantly more activity around reference spikes (t(35) = 2.69, p = 0.011), consistent with increased spike synchrony. Together with increased bursting, this suggests that conditioning increases the impact of PL on its target structures.
Failure to recall extinction is associated with an increase in the percentage of tone responsive neurons in the prelimbic cortex
Does PL activity influence recall of extinction? To address this question, we examined tone responsiveness of PL neurons twenty-four hours after extinction training. Similar to our previous study (Burgos-Robles et al., 2007
), freezing levels at recall were bimodally distributed across rats, with a cut-off at 50% freezing (see ). Of 15 rats tested, 8 rats showed less than 50% freezing, indicating good recall of extinction (Low-Fear subgroup), and 7 rats showed more than 50% freezing, indicating poor recall of extinction (High-Fear subgroup). Repeated measures ANOVA confirmed a main effect of subgroup at the recall phase (F(1,13)
= 37.9, p
< 0.001), and revealed no significant subgroup difference during conditioning (F(1,13)
= 0.47, p
= 0.50) and extinction training (F(1,13)
= 3.70, p
Failure to recall extinction was associated with increased prelimbic tone responses
We therefore compared excitatory PL tone responses in these two subgroups of rats. As shown in , high-fear rats showed significantly larger PL tone responses at recall than low-fear rats (t(16) = 2.16, p = 0.046). Furthermore, high-fear rats exhibited a somewhat higher percentage of tone responsive neurons during conditioning (32% vs. 22%), extinction training (37% vs. 19%) and recall (47% vs. 28%, see ). However, due to low n’s these proportions did not reach significance. Thus, increased conditioning in PL neurons may contribute to poor recall of extinction the following day.