The schematics of a bifurcation diagram and its use in experimental design.

<p>A computational model for slow motor gestures predicts the existence of three regions of the parameter space [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005544#pbio.2005544.ref005" target="_blank">5</a>]. For parameters in each region, qualitatively different solutions (different behaviors) are expected. One of the parameters is related to the animal’s growth. As the second parameter is varied, different solutions can be found at early stages of development (light grey arrow), and only one solution type is expected later (dark grey arrow). Placing marmoset infants in a heliox atmosphere, Zhang and Ghazanfar mimic the reversal of a parameter that correlates with development, recovering the lost behaviors (green arrow).</p

Effects of temperature on modeled neurons: Bursting emergence.

<p>(A) Correction parameter Q with temperature sensitivity that multiplies every constant rate in the model. Processes are three times faster at 30°C than at 20°C where parameters were obtained by experiments in slices [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005699#pcbi.1005699.ref067" target="_blank">67</a>]. At 40°C rates are three times faster than at 30°C. (B) Maximal conductances are also changed by parameter Q. Dotted line corresponds to a , used for HVC_<i>X</i> and HVC_<i>INT</i>, and full line to which modeled HVC_<i>RA</i>. (C) Behavior of neurons in response to an applied current of 12ms for HVC_<i>X</i> and HVC_<i>INT</i> and 16ms for HVC_<i>RA</i>. At 40°C there is a remarkable similarity with intracellular measurements in vivo, where HVC_<i>X</i> and HVC_<i>RA</i> have bursting behavior (Long et al. [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005699#pcbi.1005699.ref066" target="_blank">66</a>]). Decreasing temperature affects the interspike interval, which widens in all cases, leading to a reduced number of spikes. At 20°C HVC_<i>RA</i> fails to spike. (D) Exploration of longer (30ms) and higher current inputs show bursts that terminate solely by intrinsic current properties at normal temperature for HVC_<i>X</i> and HVC_<i>RA</i> having the same duration as real neurons. The latter retains bursting up to 30°C and fails to spike at 20°C. HVC_<i>X</i> loses bursting offset before, at around 36°C. The short pulses have the same duration as in (A) 50ms after long pulse for HVC_<i>X</i> and HVC_<i>INT</i> and 100ms after for HVC_<i>RA</i>, show a spike number reduction at this short latency compared with (A). Bars beneath traces show duration of pulse.</p

Spike widening in single neuronal model.

<p>(A) Intracellular voltage evolution of a single spike for the three modeled neurons across temperatures shows an increase in its width with the same current input as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005699#pcbi.1005699.g003" target="_blank">Fig 3C</a>. The time window used to plot is identical to the one used in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005699#pcbi.1005699.g001" target="_blank">Fig 1A and 1C</a> for the extracellular measurements, and the positive peak is aligned to 1ms and waveforms are shifted (less than 5mV) to match at peak amplitude. (B-C) Width increase for the three neuronal types was computed for the first spike of the traces in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005699#pcbi.1005699.g003" target="_blank">Fig 3C</a>. Width is calculated as the spike width above a threshold of 55 and 60% for HVC_<i>X</i> and HVC_<i>RA</i> and from the hyperpolarization to a uprising threshold of 30% for HVC_<i>INT</i>. These were selected to match the measured experimental width at 40°C (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005699#pcbi.1005699.s002" target="_blank">S2A Fig</a>). In (C) we show the same normalized to the width at 40°C. We see that the widening effect is of more than 100% for HVC_<i>INT</i> and about 50 and 35% in HVC_<i>X</i> and HVC_<i>RA</i> respectively for the 10°C range explored.</p

Effects of temperature on HVC neurons.

<p>(A) Two different types of single units were recognized in terms of their spike shape. 12 Fast Spikers (FS, orange) and 18 Regular Spikers (RS, blue) (B) Classifications were made based on peak to peak wave width (<i>p</i>2<i>p</i> width). FS (orange circles) have a more variable and higher spike rate than RS (blue triangles are the five with the highest rates, and the blue rectangles the five with the lowest rates, the eight blue circles are intermediate rates cells). (C) Widening of the spike shape of normalized waveforms across temperatures for three example FS (top) and two RS (bottom). We see that RS_<i>hf</i> units show a bigger width change than RS_<i>lf</i>. Color scale shows temperature. (D) Normalized <i>full</i> width increase of the spike shape across temperatures. FS almost doubles the spike width, while RS_<i>hf</i> and RS_<i>lf</i> only increase ∼40% and ∼20% respectively at the lowest temperature. Points are mean ± s.e.m. (p* < 0.05, p** < 0.02 values for two tailed t-test made at each temperature) (E) Normalized spike rate decrease across temperature, where we also include Multiunits (MU). We do not observe significant differences between them (two tailed t-test p> 0.1). Points are mean ± s.e.m. (F) Patterns of inter-spike-interval (ISI) activity across temperature for the single units shown in (C), left to right corresponding to top to bottom. Three different types of histogram appear for all measured FS, where ISIs change non trivially (not only a distributional shift). The first one belongs to a FS with firing rate of 5.9Hz, measured over 8 minutes. ISI depletes at intervals higher than 20ms, but retain its lower than 10ms peak at lower temperatures. Second column is a neuron with a firing rate of 3.3 Hz measured for 4.8 min. This neuron shows no bursting behavior and the ISI shifts to the right and depletes. Third column is a neuron with firing rate of 2.4Hz measured for 6.4 min, where no changes are evident across temperatures. It lacks the second timescale from around 20ms. Last two columns show the behavior of the two RS, firing at 2.4Hz and 0.7Hz measured for 4 min. We see the evolution of a clear shift to the right of the distribution over the first temperatures, until they get depleted at lower temperatures. (G) Cumulative distribution of the ISI of the three types of fast spiking neurons. The only distributions that change significantly with respect to the one at normal temperature are FS 1 and FS 2, which have the second timescale (p* < 0.0005 Kolmogorov Smirnov test, ns: not significant, alpha value is strict to account for fewer counts at lower temperatures). We can see from FS 1 that this timescale starts to disappear at 36°C and at around 10ms of ISI. Bins are 1ms.</p

Temperature effects from a single synaptic input produce delays.

<p>(A) Voltage traces for the three neuronal types in red, and synaptic input in blue across different temperatures are aligned to 0ms at current injection start. Single bursts can be seen for the excitatory types and a similar number of spikes for the inhibitory HVC_<i>INT</i>. All neurons present interspike interval increases and HVC_<i>RA</i> also shows a spike onset delay with decreasing temperatures. Vertical line is a reference aligned to the peak of the first spike in each trace. (B) Current inputs in blue from a single synaptic model fed with the voltage traces of HVC_<i>X</i> and HVC_<i>RA</i>. Evolution of HVC_<i>INT</i> voltage in red shows a single spike elicited after the third synaptic current peak. Latencies increase when lowering temperature. Time origin and vertical line are the same as in A. (C) Delay for spike onset for the five traces in A and B relative to the timing of the first spike at normal temperature. HVC_<i>RA</i> and HVC_<i>INT</i> only have slight delays for the constant applied current, while HVC_<i>RA</i> displays an almost 5ms delay to burst onset at 30°C. For the synaptic model HVC_<i>INT</i> has delays that go up to 6ms and 12ms for the two excitatory input types. (D) Maximum absolute synaptic current elicited decreases in a similar fashion for the two excitatory inputs and decreases more than twofold in the range of temperatures explored. The change in slope of the HVC_<i>RA</i> to HVC_<i>INT</i> curve at around 34°C is due to the slight distortion of last spike in HVC_<i>RA</i>. (E) Spiking pattern of HVC_<i>INT</i> at 40°C for decreasing current at steps of -200pA shows the characteristic sensitivity of the inhibitory interneurons to input currents. (F) Interspike interval (ISI) of HVC_<i>INT</i> for different currents and temperatures. We see that it is more sensitive to currents than temperatures. Inset panels show that at 1000 and 2000pA ISI changes less than 0.3ms for different temperatures, and shows changes above 1ms only below 500pA. Currents were generated with 25ms pulses decreasing at -100pA steps in a single simulation with 200ms intervals between pulses. ISI was computed only where there was at least two spikes, which happened below -300pA.</p

Changes in model ISI distributions from poisson excitable inputs.

<p>(A-C) Different input combinations (top) and evolution of the intracellular potential for HVC_<i>INT</i> (red) and synaptic current elicited (blue) for 40°C (middle) and 32°C (bottom). Many presynaptic inputs do not elicit spikes and spiking can happen in a “burst” like manner. (A) Input of 20 HVC_<i>RA</i> neurons and 60 HVC_<i>X</i> neurons. Firing rate at 40°C is 5.6 Hz, matching FS 1 unit in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005699#pcbi.1005699.g001" target="_blank">Fig 1F</a>. To achieve this behavior 30% of the input was made of bursts of 3 spikes, while the rest consisted of single spike events. (B) Input of 80 HVC_<i>X</i> neurons produces a firing rate at 40°C of 2.9 Hz with no “bursting” events, matching closely FS 2 unit in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005699#pcbi.1005699.g001" target="_blank">Fig 1F</a>. To achieve this behavior, 100% of the input was made of single spikes. (C) Input of 60 HVC_<i>RA</i> neurons and 20 HVC_<i>X</i> neurons. Firing rate at 40° is 2.4 Hz, matching FS 3 from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005699#pcbi.1005699.g001" target="_blank">Fig 1F</a>. 10% of the input was made of 3 spike bursts, 20% of 2 spike bursts while the rest consisted of a single spike. (D) ISI distributions across temperatures for connectivity in A shows a big peak at times below 10ms. (E)ISI distributions across temperatures for neuron in B shows the absence of the peak below 10ms. (F) ISI distributions across temperatures for connectivity in C shows a big peak at times below 10ms. (G-I) Cumulative distribution of the ISI of the three connectivities. The distributions with the peak below 10ms change significantly with respect to the one at normal temperature (p* < 0.0005 Kolmogorov Smirnov test, ns: not significant, alpha value is strict to account for fewer counts at lower temperatures). This is due to the slight shift to the right of this peak at lower temperatures. (J) Evolution across temperatures of the spike rate of the simulated neurons explored for different balances of HVC_<i>RA</i> neurons and HVC_<i>X</i> neurons, with a fixed total of 80. Percentage of single spike input varied in steps of 10% from 70% and double and triple spike bursts also varied in 10% steps. (K) Evolution of spike rate, but with the input rates of the excitatory neurons inversed. (L) Evolution of spike rate, with synaptic inputs stronger in 0.5nS. Data points are mean values and bars are s.e.m. ISI distributions are normalized for each temperature which is color coded in °C. Simulations were made of a duration of 4 minutes.</p

From transducer signals to binary vowel space.

<p>Upper right panel: sketch of Hall Effect transducers and magnets in the oral cavity. Transducers are marked with squares and magnets with circles. <i>Lips</i> (red): the transducer was attached to the center of the lower lip and the magnet was glued to the dental plastic replica, in between the central incisors. <i>Jaw</i> (green): magnet and transducer were glued to the dental replicas, in the space between the canine and the first premolar of the upper and lower teeth respectively. <i>Tongue</i> (blue): a cylindrical magnet was attached at a distance of 1.5 cm from the tip of the tongue. The corresponding Hall Effect transducer was glued to the dental plastic replica, at the hard palate, 1 cm right over the superior teeth (sagittal plane). Transducer wire was glued to the plastic replica and routed away to allow free mouth movements. Left, downwards: a spectrogram of the set of 5 vowels as pronounced by one of the subjects during a recording session (and frequency values for the first 2 formants) and the corresponding transducer signals for the lips, jaw and tongue. A binary code for each vowel can be defined by labeling the signal of each articulator as active (1) or inactive (0) as it reaches or not a predefined threshold (areas in color correspond to active motor coordinates). Lower right panel: resulting vowel-cube in the binary space. The edges of the cube represent an abstract space of size 8, were we explicitly locate the 5 vowels used in this work.</p

Connecting the vowel formants to anatomical coefficients.

<p>We use the mapping described by Story et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080373#pone.0080373-Story2" target="_blank">[6]</a> to find the coefficients (<i>q</i>₁, <i>q</i>₂) corresponding to the first average formants <i>F</i>₁ and <i>F</i>₂ (kHz) of the Spanish vowels pronounced by our participants. This allowed the construction of a simple affine map connecting each Spanish vowel from the discrete motor space to their corresponding vocal tract configuration.</p

Intact song and subject-driven, synthetic song.

<p>Intact pressure gesture and sonogram, with different colors and opacity of shading indicating the different syllables, and an arrow indicating the segment of the first syllable used for detection (upper panels). When the muted bird drives the syrinx, we see in the sonogram that synthetic sound is produced after the first syllable is detected and until recognition of the interruption of the motif (lower panels).</p

Illustration of the parameter fitting and calibration procedure.

<p>The thoracic air sac pressure is recorded together with the song; the pressure and corresponding sound of a bout are shown in (A). With these records, the temporal series that originate the syllables corresponding to the motif are constructed (B). After muting the bird and registering the pressure gesture as it attempts to produce a motif, the detection algorithm is tuned. In (C) we show the degraded pressure gesture, together with the correlation with the chosen segment of the intact pressure gesture. The segments pointed out by arrows indicate the detection of the intention to sing a motif. Song is synthesized during these periods by integration of the model with the parameters found previously. In (D), we show the pressure gesture of the muted bird and the output of the trigger/integrate algorithm.</p