Article Figures & Data
Figures
- Figure 1.
Large-scale mapping vocalization-evoked brainstem activity using Neuropixels probes and c-fos expression. A, Schematic of vocalization mapping by electrical stimulation in the PAG and Neuropixels recordings from brainstem neurons of an anesthetized rat. PAG stimulation-elicited vocalizations and respiration were simultaneously recorded throughout the experimental session. Various acoustic stimuli were presented and repeated 20 times by an ultrasonic dynamic speaker. µA, microampere; USV, ultrasonic vocalization. B, Representative spectrogram of ultrasonic vocalizations. Left, Modulated high-frequency vocalization; sounds with a fundamental frequency >35 kHz were classified as high-frequency vocalizations. Right, A low-frequency vocalization, for example, fundamental frequency <35 kHz. C, Vocalization-related respiration and neuronal activity in the NRA in the caudal medulla. Top, Sonogram of a low-frequency call, evoked by microstimulation in PAG. Bottom, A well-isolated unit in the NRA shows vocalization-related excitation during vocalization. D, Schematic showing stimulating electrodes placed in the PAG in the c-fos experiments. E, Schematic of the c-fos experimental procedure. F, Representative immunofluorescence section of the rat NRA stained with the neuronal nuclear marker NeuN (green) and the neuronal activation marker c-fos (red), and the merged confocal picture in the vocalized animal group.
- Figure 2.
Overview of vocalization-related activity detected by large-scale recording and c-fos expression. A1, Schematic representation of a penetration in a coronal section of the rat anterior brainstem. A2, A drawing (horizontal view) of the rat brainstem modified from a rat brain atlas (Paxinos and Watson, 2006). Three different areas of the posterior brainstem are colored differentially. A3, Schematic representation of a penetration in a coronal section of the rat posterior brainstem. B, Bar graphs illustrating the proportion of seven different types of responses of units in each brainstem region. A total of 3345 well-isolated single units were included in the data shown here. C, Quantitative analyses of the ratio between total number of double-labeled neurons (c-fos/NeuN-positive cells) and total number of neurons (NeuN-positive cells) in each experimental group. The density of activated neurons was evaluated as none if no double-labeled neurons were found in a brain region, low (+) if 0–20% double-labeled neurons were detected in a brain region, modest (++) if 20–40% double-labeled neurons were detected in a brain region, and high (+++) if 40–60% double-labeled neurons were detected in a brain region. 7n, Facial nerve.
- Figure 3.
A vocalization-activated and a vocalization-inhibited nucleus retroambiguus neuron. A, Left, Neuropixels fluorescent track and superimposed probe (black-white dashed line). Red circle represents assigned channel for the units depicted in B–D. Right, Reference brainstem coronal view as in Figure 2A3. B, Vocalization-excited NRA neuron. Top, Ultrasonic vocalization. Bottom, Raw recording trace; blue sorted waveforms from the unit. C, Neural responses of the excited neuron triggered to vocalization onset. Trials are sorted by call length. D, Neural responses of the excited neuron triggered to vocalization offset. Trials are sorted by call length. E, Left, Neuropixels fluorescent track and superimposed probe (black-white dashed line). Red circle represents assigned channel for the units depicted in F–H. Right, Reference brainstem coronal view as in Figure 2A3. F, Vocalization-inhibited NRA neuron. Conventions as in B. G, Neural responses of the inhibited neuron triggered to vocalization onset. Trials are sorted by call length. H, Neural responses of the inhibited neuron triggered to vocalization offset. Trials are sorted by call length.
- Figure 4.
Response patterns of IRt and MdDP neurons. A, Left, Neuropixels fluorescent track and superimposed probe (black-white dashed line). Green circle represents assigned channel for the units depicted in B–D. Right, Reference brainstem coronal view as in Figure 2A3. B, Mainly activated IRt neurons were observed. Top, Ultrasonic vocalization. Bottom, Raw recording trace; blue sorted waveforms from the unit. C, Neural responses of the IRt neuron triggered to vocalization onset. Trials are sorted by call length. D, Neural responses of the IRt neuron triggered to vocalization offset. Trials are sorted by call length. E, Left, Neuropixels fluorescent track and superimposed probe (black-white dashed line). Purple circle represents assigned channel for the units depicted in F–H. Right, Reference brainstem coronal view. F, Representative activated MdDP neuron. Conventions as in B. G, Neural responses of the MdDP neuron triggered to vocalization onset. Trials are sorted by call length. H, Neural responses of the MdDP neuron triggered to vocalization offset. Trials are sorted by call length.
- Figure 5.
Population responses from NRA, IRt, and MdDP. A, IRt population onset responses. Top, Single neuron responses with z-scored firing rate values. Bottom, Mean response using raw firing rate values. Red, activated neurons; blue, inhibited neurons. B, IRt population offset responses. Top, Single neuron responses with z-scored firing rate values. Bottom, Mean response using raw firing rate values. Red, activated neurons; blue, inhibited neurons. C, NRA population onset responses. Same as A. D, NRA population offset responses. Same as B. E, MdDP population onset responses. Same as A. F, MdDP population offset responses. Same as B.
- Figure 6.
Nucleus retroambiguus discharges related to call intensity. A, Top, Waveform representation of a loud call from a recording dataset. Bottom, Discharges of a well-isolated unit in the NRA showing vocalization-related excitation; sorted spike waveforms are depicted in blue. B, Top, Waveform representation of a low-amplitude call from the same recording dataset shown in A. Bottom, Discharges of the same unit in the NRA as shown in A. C, Raster plot of NRA neurons, where calls are sorted by intensity (red window reflects the time window used to compute the correlation on B). Upper blue line represents the example in A, and lower blue line represents the example in B. Red line represents the beginning of the call. D, Scatter plot of firing rate and call intensity. Red line represents linear regression between the two variables. r2 = 0.2745. E, Population r2. Box plots of the cumulated r2 by region. Crosses represent outliers. *p < 0.05, ***p < 0.001. F, Percentage of neurons per region with significant r2 > 0.1; NRA, 18% (10 of 55 units; MdDP, 1.6% (1 of 61 units). G, Scatter plot of firing rate before and during the call. r2 = 0.9435, p = 0.001. Fr, firing rate
- Figure 7.
Summary figure. A, Overview of the possible role of different caudal medulla areas involved in vocalization, based on present data. B, Population average onset responses of NRA, IRt, and MdDP.
