3.1. Location of stimulating electrodes
summarizes the placements of all stimulating electrodes. Of the 16 animals that were trained in the BLA/1.0 protocol, 11 had electrode placements within the BLA. The five animals with placements outside of the BLA failed to exhibit EEG desynchronization and served as a nonBLA stimulation control group, referred to hereafter as the nonBLA/1.0 group. All five of the animals trained in the BLA/1.6 protocol had placements within the BLA.
Fig. 3 Electrode placements for each animal for each group. BLA/1.0, n = 11; BLA/1.6, n = 5; nonBLA/1.0, n = 5. Basal lateral amygdala (BLA); posterior basal lateral amygdala (BLAP); ventral basal lateral amygdala (BLAV); lateral amygdala (LA); basomedial amygdala (more ...)
3.2. Effects of BLA stimulation on frequency tuning
Stimulation of the BLA did induce frequency tuning shifts. provides an example of the type of shift induced in the BLA/1.0 group over the 75 min of post-training recording. The pre-training BFmax was 11.3 kHz, the frequency chosen for the CS was 4.0 kHz which was slightly beyond the frequency receptive field. Immediately after training, the BFmax exhibited a slight shift away from the CS, resulting in an SI score of –0.06. However, the tuning curve became broader and now extended down to and beyond the CS frequency. Also, the shift away from the CS was a result of the loss of responsiveness at the BFmax. With the passage of time, further development was evident. By 45 min after training, the BFmax had shifted to the CS frequency, resulting in an SI score of 1.0. Also, responses to the higher frequencies were diminished. At 75 min after training, the BFmax remained at the CS frequency, the cells no longer responded to the pre-training BFmax, and the frequency receptive field became more sharply tuned around the CS frequency. shows the differences between post-training and pre-training tuning. Immediately after training, the greatest loss of response was at the pre-training BFmax, while conversely, the greatest increase in response was to the CS frequency. The tuning shift continued to develop in the absence of further training. This pattern became stronger by 45 min post-training and was accentuated at 75 min post-training.
Fig. 4 (A) Example of tuning curves recorded from an animal in the BLA/1.0 group. Top row is the pre-training tuning curve with a BFmax that was at 11.3 kHz; the training frequency (CS) selected was 4.0 kHz. Immediately after training, there was a large decrease (more ...)
In contrast, the BLA/1.6 group did not develop CS-specific tuning shifts. presents an example. Although the CS (6.7 kHz) was at the edge of the frequency RF, similar to the example of the BLA/1.0 animal in , there was no comparable tuning shift. The SI values were –0.1, 0.0 and 0.1 immediately, 45 min and 75 min after training, respectively (). Similarly, the differences in tuning between the post-training period and the pre-training period exhibited no pattern of specific decrease at the pre-training BFmax or specific increase at the CS frequency ().
Fig. 5 (A) Examples of tuning curves recorded from an animal in the BLA/1.6 group. Tuning Training shifts were negligible in this group. The top row is the pre-training tuning curve with a BFmax at 1.7 kHz; the chosen was 6.7 kHz. Immediately after training, (more ...)
presents the distribution of SI scores of both the BLA/1.0 and the BLA/1.6 groups at each time period. The BLA/1.0 mean SI score for the pre-training tuning was 0.07 (±0.05) indicating tuning stability before training. However, immediately after training, the mean SI of this group rose to 0.27 (±0.09), showing a substantial CS-directed tuning shift; however this shift was not statistically significant from baseline (Wilcoxen signed-ranks, z = 1.62, p = .11). The mean SI score increased at 45 min post-training, to 0.44 (±0.10), revealing a characteristic of neural consolidation, i.e., continued increase in strength in the absence of further training which was significant from baseline (z = 2.94, p = .003). Further, the 45 min post-training time point was also significantly greater than the immediate post-training time point (z = 2.27, p = .023). This marked shift was retained at the 75 min retention interval. Although it was somewhat reduced, i.e., SI = 0.36 (±0.08), the shift was significantly greater than baseline (z = 2.76, p = .006) (), and there was no difference between the 45 and 75 min retention intervals (z = 0.11, p = .91).
Fig. 6 The distribution of SI scores across the pre-training and post-training periods. (A) BLA/1.0 tuning. Note the increase in positive SI scores indicating tuning shifts toward the CS frequency. (B) BLA/1.6 tuning. Note the lack of CS-directed tuning shifts (more ...)
The BLA/1.6 mean SI score for pre-training tuning was 0.03 (±0.04), also indicating frequency stability before training. However, BLAstm did not produce substantial tuning shifts in this group. Immediately after training the mean SI was 0.09 (±0.23) (z = 1.29, p = .20); 45 min later it was 0.07 (±0.12) (z = 1.50, p = .13). At 75 min after training, the SI was 0.02 (±0.16) indicating a slight shift away from the CS, which was not significant from baseline (z = 1.08, p = .28) ().
The groups did not differ in pre-training SI values, (z = 0.83, p = .41, ) or in SI values immediately after training (z = 1.93, p = .054). However, the BLA/1.0 group had significantly greater tuning shifts thereafter: 45 min post-training, (z = 2.28, p = .023); 75 min post-training, (z = 2.71, p = .007, ). In summary, the BLA/1.0 group showed a shift towards the CS that continued to develop overtime but the BLA/1.6 group did not.
SI scores (±SE) for all three groups, across time. The BLA/1.0 group had a significantly larger SI score than the BLA/1.6 and the nonBLA/1.0 groups at 45 and 75 min after training. *p < .05; **p < .01.
3.3. BLA specificity
To determine if stimulation of the BLA was necessary for tuning shifts, we examined the nonBLA/1.0 group, which was trained in the same protocol but had placements outside of the BLA (). summarizes the SI scores across time of both groups, plus the BLA/1.6 group. The pre-training SI score of the nonBLA/1.0 group was 0.02 (±0.04) indicating tuning stability before training and was not significantly different from the BLA/1.0 group (z = 0.71, p = .48). Immediately after training, the nonBLA/1.0 group showed essentially no shift (SI = 0.02 ± 0.14). Although the BLA/1.0 group exhibited a substantial tuning shift (SI = 0.27 ± 0.09), as noted previously, this proved not to be statistically different from the nonBLA/1.0 group (z = 1.60, p = .11). However, the BLA/1.0 group had significantly greater shifts at 45 min (z = 2.85, p = .004) and 75 min (z = 2.52, p = .012). Moreover, the nonBLA/1.0 group never exhibited any change from baseline at any post time period (Wilcoxen signed-ranks; post immediate, z = 0.91, p = .36; 45-min post, z = 0.80, p = .42; 75-min post, z = 0.31, p = .75). Therefore, tuning shifts were found only for stimulation of the BLA itself.
3.4. Other potential factors
Although the differences in training protocol between the BLA/1.0 and BLA/1.6 groups (e.g., CS–BLAstm interval of 1.0 vs. 1.6 s) appear to be responsible for the differences in representational plasticity, other factors might have been involved (). If some frequencies are more “plastic” than others are, then differences in the pre-training BFmax frequencies might have affected the results. However, the groups did not differ significantly (z = 0.866, p = .387). Another potential factor is the octave distance between the pre-training BFmax and the CS frequency. For example, if the CS frequency were closer to the BFmax in the BLA/1.0 group, then it might have been easier to induce a tuning shift than in the BLA/1.6 group. However, there was no significant difference in the octave distance (z = 0.13, p = .90). If the level of BLAstm current were higher in the BLA/1.0 group that could explain the group differences in tuning shifts. However, the intensity of stimulation was not different between groups (z = 0.61, p = .55).
Comparison of groups BLA/1.0 and BLA/1.6 on predisposing factors and magnitude of evoked discharges.
The level of responsivity might have been a factor. To assess this possibility the evoked firing rates were analyzed within and between both groups. The BLA/1.0 mean firing rate did not change significantly throughout the recording session (Friedman χ2 = 2.48, p = .48). Similarly, there were no significant differences in the firing rate of BLA/1.6 across time (χ2 = 1.63, p = .65). Finally, firing rates between groups were evaluated for each time period and were not significantly different ().
BLA/1.0 and nonBLA/1.0 groups were also compared on several factors other than differential electrode placements. There were no significant differences between groups for BFmax frequency or CS–BF octave distance (). Stimulation current was significantly higher in the nonBLA/1.0 (z = 3.91, p = .0005) because they did not exhibit EEG desynchronization during electrode placement (see Section 2), although attempts were made to elicit this effect at higher currents. However, a higher current level cannot account for failure to obtain tuning shifts. A comparison of evoked firing rates revealed no significant differences immediately or 45 min after training, but a higher rate for the nonBLA/1.0 group at 75 min after training (). A higher rate of response shows that the absence of tuning shifts is not due to a lack of responsivity. In toto, the evidence indicates that stimulation of the BLA along with the BLA/1.0 training paradigm is necessary to produce large and enduring CS-specific tuning shifts.
Comparison of groups BLA/1.0 and nonBLA/1.0 on predisposing factors and magnitude of evoked discharges.