Characterization of synaptically connected nuclei in a potential sensorimotor feedback pathway in the zebra finch song system

Abstract

Birdsong is a learned behavior that is controlled by a group of identified nuclei, known collectively as the song system. The cortical nucleus HVC (used as a proper name) is a focal point of many investigations as it is necessary for song production, song learning, and receives selective auditory information. HVC receives input from several sources including the cortical area MMAN (medial magnocellular nucleus of the nidopallium). The MMAN to HVC connection is particularly interesting as it provides potential sensorimotor feedback to HVC. To begin to understand the role of this connection, we investigated the physiological relation between MMAN and HVC activity with simultaneous multiunit extracellular recordings from these two nuclei in urethane anesthetized zebra finches. As previously reported, we found similar timing in spontaneous bursts of activity in MMAN and HVC. Like HVC, MMAN responds to auditory playback of the bird's own song (BOS), but had little response to reversed BOS or conspecific song. Stimulation of MMAN resulted in evoked activity in HVC, indicating functional excitation from MMAN to HVC. However, inactivation of MMAN resulted in no consistent change in auditory responses in HVC. Taken together, these results indicate that MMAN provides functional excitatory input to HVC but does not provide significant auditory input to HVC in anesthetized animals. We hypothesize that MMAN may play a role in motor reinforcement or coordination, or may provide modulatory input to the song system about the internal state of the animal as it receives input from the hypothalamus.

Figure 1. Schematic of the feedback loop to HVC through MMAN.

For clarity, anatomical connections are only shown for the right hemisphere. HVC projects to the robust nucleus of the archipallium (RA), which projects to the (DMP). DMP projects bilaterally to MMAN. RA also projects to premotor nuclei that control the syrinx and respiration (PAm and RAm). Abbrev: DMP, dorsomedial nucleus of the posterior thalamus; MMAN, medial magnocellular nucleus of the nidopallium; nXIIts, tracheosyringeal part of the hypoglossal nucleus; PAm, paraambigualis; RAm, retroambigualis.

Figure 2. Spontaneous bursts of action potentials are correlated in HVC and MMAN.

A. Simultaneously recorded spontaneous activity in the ipsilateral MMAN and HVC. The top two traces are an expansion of the recording in the bottom two traces (dotted line). B. Coronal section showing the lesion (arrow) of the recording site for experiment in A. Note the location of the lesion medial to the lateral magnocellular nucleus of the anterior nidopallium (LMAN; dotted line) and in between the mesopallial lamina (LaM) and lamina pallio-subpallialis (LPS). Scale bar, 500 µm. The dashed vertical line denotes the midline. Dorsal is upward.

Figure 3. Auditory-evoked action potential activity in MMAN and HVC.

Simultaneous multiunit activity from ipsilateral MMAN and HVC in response to playback of the bird's own song (BOS), the BOS in reverse (REV), and conspecific (CON) song. For both HVC and MMAN, top row, raw data for a single playback of each song; middle row, raster plot of activity to thirty iterations of each song; bottom row, peri-stimulus time histogram (PSTH) of the cumulative response to each song playback. Bin size = 25 ms. The response strength (RS) for each response is given, * indicates a RS that was significantly greater than 0 (one-tailed t-test; p<0.05). For MMAN response to REV, p = 0.06; CON, p = 0.08.

Figure 4. Comparison of auditory responses and song selectivity in simultaneously recorded multiunit activity in MMAN and HVC.

A. Comparison of the response strength in HVC and MMAN. HVC had a significantly larger RS to BOS than did MMAN (*; paired t-test, p<0.05). The response to REV and CON in HVC and MMAN were not significantly different (REV, p = 0.17; CON, p = 0.47). B. Z-score for auditory-evoked activity in HVC and MMAN. HVC had a significantly higher z-score to BOS than did MMAN (*; paired t-test, BOS, p<0.01). The response in HVC and MMAN to REV and CON were not significantly different (REV, p = 0.20; CON, p = 0.15). C. Comparison of z-scores from simultaneous recorded activity in HVC and mMAN. Black diagonal line is the unity line. D. Both HVC and MMAN were selective for BOS vs REV and BOS vs CON. Selectivity was defined as d′>0.5 (dashed horizontal line). E. HVC was more selective for BOS versus CON than simultaneously recorded MMAN, as many points lie above the unity line than expected at random (p<0.05). The points for BOS versus CON did not lie significantly above the unity line than expected at random (p>0.05). The grey bars demark non-significant d′ values for MMAN and HVC; i.e., −0.5>d′>0.5.

Figure 5. Stimulation of MMAN functionally excites the ipsilateral HVC.

A. Example of the short latency response from MMAN stimulation to HVC response. Top trace is an exemplar of the raw HVC response to MMAN stimulation (large artifact). The dotted grey line denotes the spike threshold set by the user. Middle trace, raster plot of responses to thirty stimulus pulses in MMAN. Grey bar indicates time in which stimulus artifacts were removed from the plot. Bottom trace, PSTH of HVC response to MMAN stimulation. B. Longer latency response in HVC to MMAN stimulation. Top two traces, raw exemples of two HVC responses to stimulation of MMAN. Middle trace, raster plot to of HVC response to 30 MMAN stimulations. Grey bar indicates time in which stimulus artifacts were removed from the plot. Bottom trace, PSTH of the cumulative response in HVC to MMAN stimulation. For both A and B, bin size = 1 ms. A and B are from two different birds.

Figure 6. Inactivation of MMAN resulted in little change in HVC auditory responses.

A. Example of auditory evoked activity in MMAN and HVC before (left) and with (right) GABA application to MMAN. Top trace, PSTH of HVC response to ten iterations of BOS playback. Bin size, 25 ms. Middle trace, single, raw example of multiunit activity in HVC. Bottom trace, sonogram of BOS. B. Location of GABA application in MMAN approximated by rhodamine labeling (arrows). LMAN is outlined to the right of the dye (dotted semicircle, *). Scale bar, 200 µm. The dashed vertical line denotes the midline. Dorsal is upward. C. Average response of HVC to BOS presentation before (pre), during (GABA) and after (post) BOS presentation. In two experiments (filled squares) GABA application to MMAN did not produce a significant change in HVC response. In one experiment (open squares) there was a significant decrease in the HVC response to BOS during GABA application compared to pre and post GABA application (ANOVA, p<0.5, Tukey post-hoc). In one bird (open stars) the response to BOS increased throughout the duration of the experiment (pre, GABA, and post were all significantly different than each other; p<0.5, ANOVA, Tukey post-hoc). D. Normalized responses of data shown in C.

Figure 7. GABA inactivation of the area ventral to MMAN resulted in a reduction in auditory responses in HVC.

A, Left, HVC response to ten iterations of BOS before, during, and after GABA application. Right, normalized response to BOS. B. Location of the GABA injection (*). Dye was located ventral to LPS and medial to Area X (outlined by dotted line). The midline is indicated by the line (arrow). Scale bar, 200 µm. M, medial; V, ventral.