Recurrent interactions between the input and output of a songbird cortico-basal ganglia pathway are implicated in vocal sequence variability

Abstract

Complex brain functions, such as the capacity to learn and modulate vocal sequences, depend on activity propagation in highly distributed neural networks. To explore the synaptic basis of activity propagation in such networks, we made dual in vivo intracellular recordings in anesthetized zebra finches from the input (nucleus HVC, used here as a proper name) and output [lateral magnocellular nucleus of the anterior nidopallium (LMAN)] neurons of a songbird cortico-basal ganglia (BG) pathway necessary to the learning and modulation of vocal motor sequences. These recordings reveal evidence of bidirectional interactions, rather than only feedforward propagation of activity from HVC to LMAN, as had been previously supposed. A combination of dual and triple recording configurations and pharmacological manipulations was used to map out circuitry by which activity propagates from LMAN to HVC. These experiments indicate that activity travels to HVC through at least two independent ipsilateral pathways, one of which involves fast signaling through a midbrain dopaminergic cell group, reminiscent of recurrent mesocortical loops described in mammals. We then used in vivo pharmacological manipulations to establish that augmented LMAN activity is sufficient to restore high levels of sequence variability in adult birds, suggesting that recurrent interactions through highly distributed forebrain-midbrain pathways can modulate learned vocal sequences.

Figure 1.

Correlated spontaneous activity at the input and output structures of the BG pathway suggests a bidirectional interaction. A, Schematic of the song system showing the song motor pathway (red) and BG pathway (blue) and intracellular recording sites in HVC and LMAN. B, C, Spontaneous membrane potential recordings of HVC_RA–LMAN (B) and HVC_X (hyperpolarized)–LMAN (C) neuron pairs (action potentials are clipped) show correlated synaptic activity. D, Some possible shapes of coherency functions. E, F, The average coherency function of spontaneous activity recorded from HVC_RA–LMAN pairs (E, n = 15 of 17 pairs significant, p < 0.05) and HVC_X–LMAN pairs (F, n = 21 of 27 pairs significant, p < 0.05). Shaded region, ±1 SD of population data. Crosses indicate the peak coherency function for individual pairs with ±3 SD of jackknife variance. G, An example of an HVC_shelf–LMAN paired recording. H, The average coherency function of spontaneous activity recorded from HVC_shelf–LMAN pairs (E, n = 0 of 4 pairs significant, p > 0.05). All p values are Bonferroni-corrected.

Figure 2.

Electrical stimulation of LMAN induced synaptic responses in HVC. A, Schematic diagram of the recording and stimulation configuration. B, C, Population averages of the synaptic responses evoked in HVC_RA (B) and HVC_X (C) neurons by brief electrical stimulation in LMAN (10–20 μA, 400 μs, bipolar electrodes). Shaded region, ±1 SEM; crosses indicate the peak responses of individual cells with ±3 SD of jackknife variance. HVC_RA: n = 12 of 12 cells showed significant responses; HVC_X: n = 29 of 31 cells showed significant responses; p < 0.01.

Figure 3.

Local pharmacological manipulation of activity provides evidence of bidirectional interactions between HVC and LMAN. A, In vivo intracellular recording of an HVC_X (red) and LMAN (blue) neuron pair and extracellular recording in LMAN (black) after BMI (10 mm) injection into LMAN. B, Average HVC_X neuron voltage aligned to BMI-induced action potential burst onset in LMAN shows an HVC_X-following peak (t = 21.3 ± 2.9 ms, n = 7 pairs). Shaded region, ±1 SD; crosses indicate the peak responses for individual cells with jackknife variance; square with error bar indicates median with quartile. C, In vivo intracellular recording of an HVC_X (red) and LMAN (blue) neuron pair and extracellular recording in HVC (black) after BMI injection into HVC. D, Average LMAN neuron voltage changes aligned to onset of BMI-induced burst in HVC shows an LMAN-following peak (t = 16.7 ± 5.7 ms, n = 8 pairs). E, The averaged coherency function of HVC_X-LMAN pairs when BMI was applied to either HVC (red, n = 8 pairs) or LMAN (blue, n = 7 pairs).

Figure 4.

HVC responses to LMAN bursting activity are blocked by Area X inactivation, but not by inactivation of RA and Ad. A, Left, Schematic diagram of the stimulation, recording and inactivation pipettes. Right, An example of LMAN BMI (top) and Area X Lidocaine (bottom) injections visualized by dextran fluorescent dyes mixed in the drug solution [LMAN; dextran Alexa Fluor 488 (Invitrogen), Area X, dextran Texas Red (Invitrogen)]. B, Top left, Traces of MUA recorded in HVC, Area X, and LMAN before and after Lidocaine injection (4%, 40–80 nl) in Area X. Bottom left, Averaged HVC MUA response to LMAN burst onset before (solid) and after (red) Area X inactivation. Right, Normalized HVC peak MUA responses before Area X inactivation, during Area X inactivation (Area X(−)), and upon recovery (∼2 h). C, Left, Schematic diagram of the stimulation and recording configuration used in RA/Ad inactivation experiments. Right, An example of Lidocaine injection sites in RA and Ad/Av visualized by dextran Texas Red mixed in the Lidocaine solution. D, Top left, Traces of MUA recorded in HVC, RA, and LMAN before and after Lidocaine injection (4%, 40–80 nl) in RA. Middle and bottom left, Averaged HVC MUA response to LMAN burst onset before (solid) and after (red) RA inactivation and before and after Ad/Av inactivation (bottom). Right, Normalized HVC peak MUA responses before RA inactivation, during RA inactivation (RA(−)), and during both RA and Ad/Av inactivation (RA,Ad(−)). E, Left, Histological verification of a stimulation site in Ad. Right, Example traces of HVC extracellular recording with electrical stimulation in RA or Ad (both 10 μA). F, Average HVC MUA response to RA stimulation (n = 2 stimulation sites from two birds) and Ad/Av stimulation (n = 4 stimulation sites from two birds). **p < 0.01.

Figure 5.

Electrical or chemical stimulation of LMAN drives responses in NIf and MMAN, but not in Uva. A, Schematic diagram of the stimulation and recording configuration. B, Traces of MUA extracellularly recorded in NIf, MMAN, and Uva before and after electrical stimulation in LMAN (10–20 μA; marked by arrow). C, Extracellular or intracellular recordings of activity in NIf, MMAN, Uva, and HVC following chemically-induced bursting activity in LMAN (BMI, 10 mm; 4.6–32 nl; 100–197 μm radius sphere). D, E, Population averages of the MUA histograms of NIf, MMAN, HVC, and Uva activity aligned to electrical stimulation (D) or the onset of BMI-induced bursting (E) in LMAN. Significant responses to either form of LMAN stimulation were detected in NIf, MMAN, and HVC, but not in Uva. F, Latency plots of multiunit extracellular activity evoked in NIf, MMAN, and HVC, showing median, first, and third quartiles.

Figure 6.

Unilateral electrical or chemical (BMI) stimulation of LMAN evokes responses in the ipsilateral but not contralateral HVC. A, Schematic diagram of the stimulation and recording configuration, including the recurrent brainstem pathway that is one possible pathway that may link LMAN to HVC. B, An example of the lateralized response evoked in HVC by electrical stimulation in LMAN. Bilateral HVC recordings made in the same bird reveal that unilateral LMAN stimulation evokes responses only in the ipsilateral HVC. C, An example of the lateralized response evoked in HVC by BMI-induced bursting activity in LMAN. D, Population average of simultaneously recorded ipsilateral and contralateral HVC MUA aligned to LMAN stimulation (red lightning bolt): all ipsilateral HVC recordings showed significant responses (n = 4 hemispheres, p < 3. × 10⁻¹⁸ with Bonferroni), whereas no contralateral HVC recordings showed significant responses (n = 4 hemispheres, p > 0.01). E, Population average of HVC MUA aligned to the onset (dashed vertical line) of bursting activity induced in LMAN by focal BMI injection. All ipsilateral HVC recordings showed significant responses (n = 2 birds, p < 1.8 × 10⁻²¹ with Bonferroni) following the LMAN burst onset, whereas no contralateral HVC recordings showed significant responses (n = 2 birds, p > 0.01). RVL, Rostral ventrolateral medulla; DMP, dorsomedial nucleus of posterior thalamus.

Figure 7.

Inactivating NIf and/or MMAN fails to block propagation of BMI-induced bursting activity from LMAN to HVC. A, A schematic of the stimulation, inactivation, and recording configuration. B, The peak amplitude of depolarizing postsynaptic potentials following each LMAN burst are plotted before, during, and after NIf inactivation [NIf GABA (250 mm)]. C, LMAN burst-triggered averaged dPSP responses in HVC before (blue), during [NIf GABA (red)], and after (recovery; cyan) NIf inactivation (traces are plotted with SEM, using the same data from A). D, Population average of the normalized dPSP distribution before, during, and after NIf inactivation. For this plot, both LMAN electrical stimulation cases (n = 5 birds, triangles) and LMAN BMI cases (n = 3 birds, circles) are pooled together. NIf inactivation augmented dPSP amplitude of HVC projection neurons [n = 4 out of 5 birds in LMAN stimulation, n = 3 out of 3 birds in LMAN BMI, significant increases during NIf inactivation (p < 0.0001)]. E, Schematic diagram of the experiment. F, Average HVC MUA aligned to the onset of BMI-induced bursts in LMAN in four conditions; control, when NIf was inactivated with muscimol (5 mm) (NIf(−)), when MMAN was inactivated with muscimol (MMAN(−)), and when NIf and MMAN were inactivated with muscimol (NIf(−) MMAN (−)). Inactivating NIf and MMAN did not stop the propagation of activity from LMAN to HVC. G, Peak responses of HVC MUA aligned to LMAN burst onset did not show significant differences under the four conditions (ANOVA, p = 0.66). **p < 0.01.

Figure 8.

The dorsal midbrain dopaminergic area A11 displays properties consistent with a role in conveying activity from LMAN to HVC. A, Confocal images of TH-positive neurons in A11 (red; top left). HVC-projecting A11 (A11_HVC) neurons (green; top right) were labeled by injecting Dextran Alexa Fluor 488 tracer into HVC. Most of the A11_HVC neurons near the midline (0–700 μm) are TH-positive (merged; bottom right); scale bar is 50 μm and applies to all confocal images. Bottom left, A drawing of a transverse section showing A11_HVC (red) and a more laterally-positioned population of TH-negative HVC-projecting neurons distributed over the central gray (GCt; green). B, MUA traces of HVC, A11, and LMAN recorded while bursting activity was induced in LMAN by BMI and NIf was inactivated with muscimol. LMAN-burst triggered average (bottom) of the firing rate histogram shows that activity in A11 follows the burst onset in LMAN earlier than the activity in HVC. C, Population average of LMAN burst-triggered PSTH of MUA recorded in A11 and HVC [n = 5 triple recordings in 5 birds; A11: 5 out of 5 significant (p < 0.0006); HVC: 4 out of 5 significant (p < 1. × 10⁻⁰²¹)]. D, Top, Short latency excitatory synaptic responses evoked in an HVC_X neuron by electrical stimulation of A11 (10 μA; dashed red line). Synaptic response onsets for multiple trials recorded from this cell are plotted with circles. Bottom, Distribution of the synaptic latency (n = 17 cells, 5.7 ± 0.05 ms).

Figure 9.

Inactivation of both A11 and NIf blocks propagation of activity from LMAN to HVC. A, After inactivating NIf with muscimol (5 mm), BMI-induced LMAN bursting activity still drives HVC responses (left). Inactivating A11 with GABA (250 mm, ∼ 500 nl during orange bar) induced HVC shutdown ∼15 s after the onset of GABA injection (middle). After HVC shutdown, no response was detected in HVC and A11, despite continued bursting activity in LMAN. B, Averaged HVC MUA aligned to LMAN burst onsets (n = 3). C, Normalized HVC MUA responses aligned to LMAN burst onset were significantly reduced after HVC shutdown (n = 3, paired t test, p < 0.012). Injecting GABA near the center of the A11 population strongly attenuated HVC responses to LMAN bursting (circles: ∼75% reduction). A muscimol injection centered ∼300 μm ventral to the center of the distribution of A11 cells reduced the HVC response by 50% (square). An injection of muscimol placed ∼1200 μm lateral from midline did not evoke any state change in HVC (asterisk). D, Top, Schematic diagram of the drug/tracer (250 mm GABA or 5 mm muscimol mixed with Dextran Alexa Fluor 594) injection sites, based on post hoc visualization of tracer and retrograde labeling from HVC with Dextran Alexa Fluor 488. Distribution of HVC-projecting A11 and more lateral GCt neurons are shown in green. The medial region contains TH-positive HVC-projecting cells (A11, red), while TH-negative HVC-projecting cells are more laterally distributed (light green). Bottom, The GABA injection site (red) and retrograde-labeled cells from tracer-injection in HVC (green) recovered 5 d after the inactivation experiment. *p < 0.05.

Figure 10.

Augmenting LMAN activity with BMI infusion increases sequence variability and syllable entropy in adult zebra finches. A, Adult song recorded when saline was infused in LMAN, and typical transition diagram of song recorded under these conditions. B, Examples of variable syllable sequences recorded when BMI (10 mm) was bilaterally infused in LMAN. C, Syllable transition matrix recorded on saline (left) and BMI (right) treatment days. D, Sequence consistency scores of all normal hearing adults (n = 4 of 4, circles) and deafened adults (n = 2 of 2, triangles) significantly decreased with BMI infusion in LMAN (p < 0.01, Mann–Whitney U test). E, Song continuity of adults decreased with BMI treatment in LMAN (saline vs BMI days, p < 0.01, Mann–Whitney U test, n = 4 of 4 normal birds and n = 2 of 2 deafened birds). F, Examples of syllable level variability on control and BMI injection days. G, Distribution of the log entropy (Wiener Entropy) of four syllables from bird or811m. H, Syllable entropy significantly increased on BMI treatment days (n = 17 syllables from three birds; linear regression; slope = 0.81, 95% confidence limit 0.71–0.91). **p < 0.01.

Figure 11.

A, Fluorescent photomicrograph of muscimol-BODIPY (1 mm) infused through dialysis probe visualized in a fixed tissue section. B, Fluorescent level shown in log₁₀ scale. The exponential decay distribution of the fluorescent level indicates a 90% drop in concentration per 287 μm from the probe. C, Examples of an MMAN lesion visualized by Nissl-staining (left) and distribution of HVC projecting MMAN neurons (MMAN_HVC; right) retrogradely labeled from the tracer injection in HVC (Dextran Alexa Fluor 488, 42–96 nl). D, Top, Distribution of MMAN_HVC neurons as a function of the distance from the midline (n = 177 cells from four birds). Bottom, Cumulative probability distribution of MMAN_HVC neurons and corresponding lesion size for each hemisphere (6 hemispheres from 3 birds). E, Sequence consistency score of MMAN-lesioned birds significantly decreased with BMI treatment in LMAN (saline vs BMI days, p < 0.01, Mann–Whitney U test, n = 3 of 3 birds). Song continuity decreased with BMI treatment in LMAN in 2 of 3 birds (saline vs BMI day, p < 0.01, Mann–Whitney U test, n = 2 of 3 birds).

Figure 12.

Similar structure of mammalian and songbird cortico-BG circuitry emphasizing mesocortical feedback. Midbrain dopaminergic cell groups in both mammals and birds provide recurrent input to the pallium: VTA neurons project to the prefrontal cortex and primary motor cortex in mammals, and A11 neurons project to HVC in songbirds. sp, Subpallidum; vp, ventral pallidum.