Bottom-up activation of the vocal motor forebrain by the respiratory brainstem

Abstract

Brainstem motor structures send output commands to the periphery and are dynamically modulated by telencephalic inputs. Little is known, however, about ascending brainstem control of forebrain motor structures. Here, we provide the first evidence for bottom-up activation of forebrain motor centers by the respiratory brainstem. We show that, in the avian vocal control system, activation of the brainstem inspiratory nucleus paraambigualus (PAm), a likely homolog of the mammalian rostral ventral respiratory group, can drive neural activity bilaterally in the forebrain vocal control nuclei HVC (used as a proper name) and the robust nucleus of the arcopallium (RA). Furthermore, this activation is abolished by lesions of nucleus uvaeformis (Uva), a thalamic nucleus necessary for song production. We identify a type of bursting neuron within PAm whose activity is correlated, in an Uva dependent manner, to bursting activity in RA, rather than to the respiratory rhythm, and is robustly active during the production of stimulus evoked vocalizations. Because this ascending input results in cross-hemisphere activation, our results suggest a crucial role for the respiratory brainstem in coordinating forebrain motor centers during vocal production.

Figure 1.

Diagram of the avian song system emphasizing its bilateral organization and the bilateral projections from the brainstem to the forebrain. The figure emphasizes two pathways crossing from one half of the brain to the other. The first is the connection from RA through the vocal respiratory network (DM and PAm) and Uva (blue). The second is the pathway from RA to DMP to the contralateral MMAN (green). Both pathways converge on the contralateral HVC. Sites of stimulation (RA, DM, and PAm), recording (Uva and RA), and lesions (Uva) have been added. Additional recording (HVC and MMAN) and lesion (MMAN) sites were used in this study but are not shown here for clarity. Also not shown are nuclei of the anterior forebrain pathway which are necessary for song learning and maintenance. NIf, Nucleus interfacialis of the nidopallium.

Figure 2.

Stimulation of the respiratory brainstem activates contralateral Uva, RA, and HVC. A, Effect of PAm stimulation on activity in contralateral Uva and RA. Middle, PSTH of RA activity after 100 stimulations in PAm of which four sample multiunit traces are shown above the histogram. Stimulation occurred at time 0 and the stimulus artifact has been removed from the neural traces and the PSTH. The bottom panel represents the PSTH of activity recorded simultaneously in Uva contralateral to the PAm stimulation site. In this example, stimulation consisted of two 400 μs wide, 60μA biphasic pulses within 2 ms. B, Effect of DM stimulation on activity in contralateral Uva and RA. PSTH of activity recorded in the contralateral RA and Uva after stimulation in the left DM. C, Latency for contralateral RA responses after stimulation in PAm and DM. Latency was measured from the sum of the poststimulus traces, each rectified and smoothed with a Gaussian filter, as the time from the beginning of stimulation to the time to reach half of the peak value of this summed trace. D, Latency to activate both the contralateral (white) and ipsilateral (gray) HVC after stimulation in PAm. Latency distribution for C and D is shown as a box-and-whisker plot.

Figure 3.

Lesions of Uva block brainstem to forebrain signal propagation. A, PSTH showing activity after stimulation in the left PAm recorded in the contralateral RA, before and after electrolytic lesions of the right Uva. B, Comparison of evoked responses before and after Uva lesion. Measurements of evoked responses in RA (contralateral to stimulation) are shown for birds receiving stimulation in PAm (n = 4). The y-axis values indicate the mean peak-to-baseline ratio for each group (see Materials and Methods). The dotted line represents the mean control peak-to-baseline value calculated before Uva lesions (n = 4), and the gray area represents two SDs from this mean. Error bars represent SEM. Asterisks indicate significance for the group by one-tailed paired t tests, wherein each bird was paired with either its own control peak-to-baseline value, or paired before and after Uva lesions. C, Comparison of evoked responses from stimulation in DM (n = 9) before and after Uva lesions.

Figure 4.

Cross-hemisphere activation is partially dependent on the Uva-mediated relay pathway. A, PSTHs of right RA activity after stimulation in the left RA, before (top) and after (bottom) lesion of the intermediate right Uva. In this example, the lesion attenuates the RA response and causes a shift in peak latency. B, Mean peak-to-baseline ratios for six birds with stimulation delivered in one RA and recordings made in the contralateral RA. Mean control value ± two SDs is shown as a dotted line surrounded by a gray box, and was calculated from randomly sampled RA traces before the Uva lesion. Asterisks represent significance by t tests. Error bars indicate SE. C, Latency to the evoked response from RA stimulation, recorded in the contralateral RA, measured as time to half peak.

Figure 5.

Characterization of respiratory phase relationship in RA and PAm respiratory neurons. A, Simultaneous recordings of air sac pressure (top), single-unit activity in left PAm (middle), and unit activity in contralateral RA (bottom). Expiration (exp) is indicated as a positive deflection in the air sac pressure trace, whereas inspiration (insp) is indicated as negative deflections. The dotted line indicates ambient pressure. PAm units fire during or just preceding the negative deflection phase of respiration indicating that these units are phase locked to the inspiratory phase of respiration. B, A polar plot of spikes counted (radius) in an 80 s trace at different phases of respiration (angle). The count of PAm spikes is shown in black, and the RA spike count is shown in gray. In this figure, 0° is the approximate peak of the inspiratory phase. C, Mean vector strength for eight RA (left) and eight PAm inspiratory (right) neurons, compared with the mean of the vector strengths calculated by shuffling the spike times of each neuron's trace. The asterisk represents significance by two-tailed paired t tests. D, A canonical air sac pressure trace is shown for one respiratory cycle. The mean phase-relative timing of eight PAm inspiratory neurons is overlaid on the trace as a vertical dotted line. The surrounding gray box represents two SDs from this mean.

Figure 6.

Identification of nonrespiratory bursting neurons in PAm that are spatially near inspiratory neurons. Multiple unit activity was recorded in PAm (center trace) simultaneously with activity in the contralateral RA (bottom trace) and with air sac pressure (top trace). The arrow indicates the time of simultaneous bursting in RA and the nonrespiratory PAm unit. Also visible in the background of the PAm electrode are units that fire in phase with inspiration. The thick gray line is a rectified and smoothed version of the PAm trace after the burst spikes have been removed, highlighting the respiratory rhythm of this background activity. In this example, the respiratory cycle was longer when a burst occurred than when the PAm unit remained silent. Changes in respiratory cycle length were occasionally observed during bursts in PAm and/or RA; however, this phenomenon was not consistently seen. exp; Expiration; insp, inspiration.

Figure 7.

Spontaneous activity of nonrespiratory neurons in PAm is correlated with bursting in the contralateral RA. A, Simultaneous recordings of two single units in the left PAm while maintaining the same unit in the contralateral RA. For the left pair, the recording electrode was placed closer to a site showing the characteristic nonrespiratory bursting pattern. Bursts in these neurons occur at approximately the same time as bursts (and subsequent pauses) in RA. In the right pair, a respiratory neuron recorded several minutes after the unit on the left shows a complete lack of correlated activity with the same RA neuron. B, Two PETHs are shown, representing activity recorded in the right RA of one bird. In the top PETH, RA activity was aligned to spikes from the nonrespiratory PAm neuron. In the bottom PETH, RA activity was randomly selected from the same recording. Significant peaks in the top histogram (dark gray) indicate that RA activity is high around the time of bursts in the PAm nonrespiratory bursting neurons, but shows no correlation with the shuffled control. The horizontal dotted lines indicate the mean height of histogram peaks generated from randomly selected 50 ms periods of RA activity. The gray areas indicate two SDs from that mean. The vertical dotted lines delineate the window of time for all PETHs used to calculate the values shown in D. C, Autocorrelation plots of PAm (top) and RA (bottom) activity used in B, showing values up to 0.1 (of 1.0). The absence of peaks at time lags greater or smaller than 0 indicate that the activity seen in the PETHs does not result from intrinsic correlation in RA or PAm. D, Comparison of peak/baseline ratios for RA activity 50 ms after alignment (0–50 ms) to nonrespiratory bursting neurons (left), and respiratory neurons (middle), and when the nonrespiratory aligned RA activity is randomly shuffled (right). Error bars indicate SD. Asterisks indicate significance by paired t test.

Figure 8.

Nonrespiratory PAm neurons are active during vocalization evoked under anesthesia. A, An example single-unit recording made from PAm. The raster at the top shows spike times (black) from one PAm neuron overlaid on periods of vocal production (gray) for 40 instances of DM stimulation-evoked vocalizations. The sonogram beneath the raster represents a sample vocalization. The neural trace below represents sample activity from the PAm neuron during that vocalization. The light gray box surrounding the sample indicates the vocalization period for this example. The PSTH below the trace was compiled from the 40 trials. To examine only vocalization-related activity and exclude activity resulting from direct trans-synaptic activation, the first 50 ms of activity after stimulation (dark gray box) was ignored when performing the analysis shown in C and D. B, A multiunit example from another bird. A longer period of stimulation was used (600 ms) than in A. Unlike in A, activity at this site was elevated at an earlier time point relative to vocalization, indicating a heterogeneous temporal relationship between PAm nonrespiratory neurons and EVOC production. C, Mean ± SD of neural activity in PAm recorded during multiple instances of DM stimulation evoked vocalizations (black) and paired baseline periods (immediately before each stimulation, white) for five birds. D, Population activity (grand mean ± SD) recorded during evoked vocalizations for PAm (left) and HVC (right). PAm activity was gathered from five sites in four birds, and HVC activity was gathered from four sites in three birds. Asterisks indicate significance by paired t test.