A higher sensory brain region is involved in reversing reinforcement-induced vocal changes in a songbird

Abstract

Many animals exhibit flexible behaviors that they can adjust to increase reward or avoid harm (learning by positive or aversive reinforcement). But what neural mechanisms allow them to restore their original behavior (motor program) after reinforcement is withdrawn? One possibility is that motor restoration relies on brain areas that have a role in memorization but no role in either motor production or in sensory processing relevant for expressing the behavior and its refinement. We investigated the role of a higher auditory brain area in the songbird for modifying and restoring the stereotyped adult song. We exposed zebra finches to aversively reinforcing white noise stimuli contingent on the pitch of one of their stereotyped song syllables. In response, birds significantly changed the pitch of that syllable to avoid the aversive reinforcer. After we withdrew reinforcement, birds recovered their original song within a few days. However, we found that large bilateral lesions in the caudal medial nidopallium (NCM, a high auditory area) impaired recovery of the original pitch even several weeks after withdrawal of the reinforcing stimuli. Because NCM lesions spared both successful noise-avoidance behavior and birds' auditory discrimination ability, our results show that NCM is not needed for directed motor changes or for auditory discriminative processing, but is implied in memorizing or recalling the memory of the recent song target.

Figure 1.

Pitch reinforcement and recovery. A, We drove the pitch of zebra finch song syllables up (down) by broadcasting WN stimuli when pitch was below (above) a manually set threshold. Shown is the WN amplitude as a function of pitch on days with aversive reinforcement of low-pitch (blue) and of high-pitch (black) syllables. We indicate the pitches of three syllables by green vertical lines (I, II, and III); these syllables are also delineated by green boxes in B. For reference, overlaid is the pitch distribution (red) produced during the last day of pitch reinforcement (this day is highlighted by a red star in C and indicated by red lines in D–F). B, Sample sound spectrogram of a zebra finch song bout (high sound intensities shown in yellow and low intensities in red-black). The first (I) highlighted target syllable is an escape, the second (II), triggered full amplitude WN; and the third (III), triggered low-amplitude WN. C, Pitch trajectory in a control bird that restores baseline pitch (black dotted line) within 5 d after WN is switched off (red star). Shown are daily pitch averages (black dots), the daily SDs (vertical bars), and the aversive reinforcement zone (gray-shaded area). The last baseline day is marked by a black star and day 5 after WN off by a blue star. D, E, Pitch traces of the target syllable averaged over all renditions produced on (1) the last baseline day (black); (2) the last WN day (red, cut at the onset of WN, dashed red line); and (3) the fifth day after WN off (blue). All traces are aligned to the last of the three pitch measurements (time origin). The black vertical lines delimit the interval in which pitch measurements are taken, and the black dashed lines delimit the 95th percentile of that interval relative to syllable onset. Shaded areas indicate ±1 SD. E, Inset (gray rectangle) in D showing the increase in pitch (red) after reinforcement and the recovery thereafter (blue). Note that the bird also increased pitch outside the reinforced interval (outside the 95th percentile), revealing nonlocal effects of pitch reinforcement. F, Histograms demonstrating that daily detection time jitter (mean-subtracted) was small and consistent across days (colored as in D, E).

Figure 2.

Histology demonstrating NCM lesions. A, Nissl-stained sagittal brain sections from four birds killed 1, 7, 14, and 32 d (from left to right) after ibotenic acid injections into NCM (these sections were taken from birds that did not participate in the experiment). The lesioned areas (delimited by gray ellipses and colored arrowheads) are defined by absence of large cell bodies. Dashed lines indicate the approximate paths of the injection pipettes. Bottom, Zoomed in view of the area inside the rectangles on top. On day 32 after surgery (rightmost) a glial scar is clearly visible in the middle of the rectangle (green arrowheads). B, Left, Overlay of the ellipses in A (with colors matching the arrowhead colors in A). As a reference, we also drew a black circle of radius corresponding to a 500 nl sphere (injected acid volume). Right, Ellipse area (colored dots) as a function of postsurgery survival time, shown together with an exponential fit (gray curve). C, Example sagittal brain section of a bird perfused 28 d after ibotenic acid injection, showing the fits (dashed lines) to the ventricle (V) and the LaM (black arrowheads). The estimated injection site is marked by a blue dot. White arrowheads indicate the area where tissue damage is still visible. D, Estimated injection sites (blue dots, left hemisphere; red dots, right hemisphere) for all NCM-lesioned birds (six were pitch reinforced, two were not). For three hemispheres we were unable to establish the injection site. The NCM region is highlighted in yellow.

Figure 3.

NCM lesions impair pitch restoration. Left, Four sham-lesioned birds exhibit pitch recovery trajectories (blue curves) that quickly converge toward baseline pitch (zero residual pitch), whereas trajectories in six NCM-lesioned birds do not converge (red curves). Residual pitch trajectories are normalized such that 1 (dashed black line) corresponds to pitch on the last WN day and 0 corresponds to baseline pitch. The 15 late recovery days are marked by a gray-shaded area, and the WN off event by a red dashed line. Right, In sham-lesioned birds the late-recovery residuals both are closer to zero (blue dots correspond to late-recovery averages in individual birds) and fluctuate less than corresponding residuals in NCM-lesioned birds (red dots; vertical bars indicate ±1 SD of late recovery residuals).

Figure 4.

NCM-lesioned birds show no deficit in auditory and motor performance. A, Schematic of the song discrimination setup. During trial sessions, when the experimental bird (in the foreground) sits on the perch, the computer triggers random playback of one of two songs, one of which is followed by an air puff coming from a tube just next to the perch. B, An NCM-lesioned bird successfully discriminates the two songs (shown are data from the fifth day of training; Bird A1 in Table 1). Below the time line (top), two song spectrograms show the nonpuffed song (upper) and the puffed song (lower, with air puff noise visible between 3 and 4 s into the trial). Bottom plot, The bird-on-perch probability as a function of time in the trial (daily performance of 600 trials) shows that on trials without air puffs the bird remains throughout on the perch in more than 80% of the trials (blue curve), whereas on air puff trials the bird stays on the perch for 3 s in only about 60% of cases (black curve). The significant difference between black and blue curves (p < 0.01, gray background) before air puff onset indicates that the bird is able to discriminate between the two songs. C, Consistent auditory discrimination performance over consecutive days in one example bird (same bird as in B). We report the performance as probability difference P(perch|nopuff) − P(perch|puff) of the bird being on the perch at the 3 s mark on trials without and with air puff (black line), together with the number of trials done on each day (orange). The low number of playbacks on the first training day is often due to the bird getting used to the air puff and not quickly returning on the perch for more trials. From the second training day the bird's performance is positive and significant (black stars: p < 0.01). D, NCM-lesioned birds are able to modify pitch to avoid WN. Shown are d′ pitch changes from baseline (black curves) for two birds that received bilateral NCM lesions 19 and 20 d before WN onset (shifted up) and for two birds that received lesions 3 and 10 d earlier (shifted down). All birds quickly changed pitch beyond the 1% significance range, gray-shaded rectangle). E, Scatter plot (one dot per bird) of Wiener Entropy averaged over all renditions of the target syllable on the last baseline day (horizontal axis) and on postlesion day 20 (vertical axis). Blue, sham-lesioned birds; red, NCM-lesioned birds. F, No motor deficit following NCM lesions is revealed by visual inspection of song motif spectrograms (0–8 kHz) on baseline days, on postlesion day 1, and on postlesion day 20 (N = 6 birds from left to right). Green bars highlight target syllables.