Figure 1.
Three simple syllabic calls of P. p. rubigenosus containing different patterns of FMs.
Amplitude envelops (above) and spectrograms (below) for (A) bent upward FM, (B) single humped FM and (C) descending rippled FM.
Figure 2.
Schematic showing the CS presentation paradigm and HR recordings and analysis.
(A) Illustration of the fear-conditioning paradigm. The CS+ was paired with shock to one leg. Either vocalization or leg movement terminated the US (see text for details). (B) The safety signal (CS−) was not paired with shock. (C) Band passed EKG trace showing HR recordings. (D) The filtered and amplified signal (top) used to generate beat markers (middle) that correspond (vertical dashed line) to the time of threshold crossing (horizontal dashed line). Bottom panel shows time of beat markers converted to instantaneous HR before and during presentation of CS (shaded segment) in a single trial. Note the consistently variable, but gradual increase in HR. (E) Averaged response curve obtained after CS presentations. HR increased dramatically after CS onset, and steadily declined over the next 40 s.
Figure 3.
Fear-induced learning employing vocalization as the operant conditioning paradigm.
(A–B) Raster plots for event markers and PSTHs of vocalization markers to show selective suppression of spontaneous echolocation by CS+ presentations. (A) Echolocation pulses are suppressed during playback of the CS+. The bat vocalized with short latency after delivery of a shock pulse at the end of the CS+. Unfilled rectangle and vertical grey bar indicates timing and duration of the stimulus. (B) The CS− did not influence the rate of echolocation.
Figure 4.
Fear-induced learning employing leg movement as the operant conditioning paradigm.
(A) Raster plots and PSTHs of leg movement transiently increased during the CS+ (UFM) and decreased following CS+ offset, whereas (B) the CS− (DFM) did not influence the frequency of leg movement. FM direction is indicated by arrow. Box plots of leg flexion events analyzed in 5 animals (C) showing a significant difference in leg flexion between the CS+ and CS−. Solid lines in boxes are medians, boxes surround 50% data and whiskers are 5th and 95th percentiles.
Figure 5.
Two examples of learning curves using HR recordings.
Lines are smoothed (0.5 s and 1.0 s sliding window in A and B, respectively). Line plot of HR is expressed as percentage change above average pre-stimulus values representing the “learning curve” for first three training sessions in two bats. Each bat acquired a robust and relatively stable response on the third day that was rapidly re-established on day 7 in the first bat (A).
Figure 6.
Cumulative line plots for representative examples of peri-stimulus HRs in response to conditioning with three different FMs.
CS+ was a steep DFM (40 to 30 kHz sweep in 10 ms; rate 1.0 kHz/ms) FMs used as CS− differed (A) in direction (UFM), (B) in duration (100 ms) and rate (0.1 kHz/ms), and (C) in bandwidth (60 kHz to 10 kHz) and rate (0.5 kHz/ms). Line plots indicate instantaneous HR to the presentation of FM stimuli. Each black trace represents the HR in response to CS+, and each grey trace shows HR on trials with one of the three CS− stimuli. CS onset occurs at time zero (vertical dashed line). (D) Bar graph of data to show ∂HR for CS+ (black bar) versus CS− (grey bar) for each of the same three stimulus conditions. Error bars represent standard error of the mean.
Figure 7.
Dynamic correlation between FM rate and HR responses.
(A) The traces represent the average instantaneous HR of one bat to 10 presentations of a CS− and CS+ without shock. The vertical grey bars mark the presentation period of the stimulus. The solid black bars at the upper-right corner in each panel denote the rate of the FM. (B) Regression plot (averaged from three bats) for FM rates ranging from a pure tone (0 kHz/ms) to 1.0 kHz/ms (step size of 0.1 kHz/ms). HR obtained to CS+ with shock during training (filled black circle) was not included when fitting the regression line. The data were best fit by a straight line suggesting that HR increased linearly with increasing rates approaching CS+. No significant difference was found in the HR evoked by CS+ with shock (filled black circle) and the CS+ without shock (target FM, filled grey circle) independent samples t-test, P = 0.992).
Figure 8.
Two examples of US induced enhancement of the LFP to CS+.
Line plots show averaged LFPs evoked by FM sweeps before (grey trace) and after (black trace) conditioning. (A) LFP responses after 100 repetitions of the target stimulus CS+ (a single DFM at a rate of 0.6 kHz/ms). (B) LFP response to CS−, and (C) LFP response to a sequence of 5 FM sweeps (CS+) pre- and post-conditioning presented at rate of 40 Hz; onset is indicated by vertical dashed line. Responses were obtained from depths of 3.71 mm (A and B) and 3.50 mm (C) from the brain surface and ∼3.1 mm from the midline. Vertical dashed lines and black/grey diagonal bars indicate stimulus onset and offset. Labels indicate enhanced peaks P1 to P3. D. Charting (left) and photomicrograph (right) of a histological section in the transverse plane to show the location of the electrolytic lesion (arrow) at a LFP recording site. AC: auditory cortex, BLA: baslolateral amygdala, ECT: ectorhinal cortex, FPC: frontoparietal cortex; MGB: medial geniculate body; MHB: medial habenular nucleus, NIC: nucleus of the internal capsule, PIR: piriform cortex, PRH: perirhinal cortex, RS: rhinal sulcus. SCN: suprachiasmatic nucleus, SF: Sylvian fossa, SOR: retrochiasmatic part of the supraoptic nucleus, v; fourth ventricle.
Figure 9.
Schematic showing major brain regions involved in the generation of a fear-conditioned response.
Associations between the CS and US are created in the BLA from auditory and somatosensory inputs. Leg movement is mediated vja projections of the BLA to the dorsal striatum [84], whereas HR and vocalizations are mediated by parallel pathways from the BLA to motor centers within the brainstem via the centromedial amygdala [85], [86]. BLA has been proposed as a locus for associative plasticity [87]. Solid lines indicate direct neuronal projections; dashed lines indicate indirect (multilevel) projections. Connectivity diagram does not show reciprocal connections between amygdala and auditory brain regions. Sensory processing areas and inputs are indicated in light blue, motor centers and outputs are indicated in green and sensorimotor interface (amygdala) in bluish green.