Vocal Experimentation in the Juvenile Songbird Requires a Basal Ganglia Circuit

Songbirds learn their songs by trial-and-error experimentation, producing highly variable vocal output as juveniles. By comparing their own sounds to the song of a tutor, young songbirds gradually converge to a stable song that can be a remarkably good copy of the tutor song. Here we show that vocal variability in the learning songbird is induced by a basal-ganglia-related circuit, the output of which projects to the motor pathway via the lateral magnocellular nucleus of the nidopallium (LMAN). We found that pharmacological inactivation of LMAN dramatically reduced acoustic and sequence variability in the songs of juvenile zebra finches, doing so in a rapid and reversible manner. In addition, recordings from LMAN neurons projecting to the motor pathway revealed highly variable spiking activity across song renditions, showing that LMAN may act as a source of variability. Lastly, pharmacological blockade of synaptic inputs from LMAN to its target premotor area also reduced song variability. Our results establish that, in the juvenile songbird, the exploratory motor behavior required to learn a complex motor sequence is dependent on a dedicated neural circuit homologous to cortico-basal ganglia circuits in mammals

Control of Vocal and Respiratory Patterns in Birdsong: Dissection of Forebrain and Brainstem Mechanisms Using Temperature

Learned motor behaviors require descending forebrain control to be coordinated with midbrain and brainstem motor systems. In songbirds, such as the zebra finch, regular breathing is controlled by brainstem centers, but when the adult songbird begins to sing, its breathing becomes tightly coordinated with forebrain-controlled vocalizations. The periods of silence (gaps) between song syllables are typically filled with brief breaths, allowing the bird to sing uninterrupted for many seconds. While substantial progress has been made in identifying the brain areas and pathways involved in vocal and respiratory control, it is not understood how respiratory and vocal control is coordinated by forebrain motor circuits. Here we combine a recently developed technique for localized brain cooling, together with recordings of thoracic air sac pressure, to examine the role of cortical premotor nucleus HVC (proper name) in respiratory-vocal coordination. We found that HVC cooling, in addition to slowing all song timescales as previously reported, also increased the duration of expiratory pulses (EPs) and inspiratory pulses (IPs). Expiratory pulses, like song syllables, were stretched uniformly by HVC cooling, but most inspiratory pulses exhibited non-uniform stretch of pressure waveform such that the majority of stretch occurred late in the IP. Indeed, some IPs appeared to change duration by the earlier or later truncation of an underlying inspiratory event. These findings are consistent with the idea that during singing the temporal structure of EPs is under the direct control of forebrain circuits, whereas that of IPs can be strongly influenced by circuits downstream of HVC, likely in the brainstem. An analysis of the temporal jitter of respiratory and vocal structure suggests that IPs may be initiated by HVC at the end of each syllable and terminated by HVC immediately before the onset of the next syllable.United States. National Institutes of Health (Grant R01 DC009183)United States. National Institutes of Health (Grant R01 MH067105

Structural and molecular interrogation of intact biological systems

Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease

Dopamine neurons modulate neural encoding and expression of depression-related behaviour

Major depression is characterized by diverse debilitating symptoms that include hopelessness and anhedonia1. Dopamine neurons involved in reward and motivation are among many neural populations that have been hypothesized to be relevant, and certain antidepressant treatments, including medications and brain stimulation therapies, can influence the complex dopamine system. Until now it has not been possible to test this hypothesis directly, even in animal models, as existing therapeutic interventions are unable to specifically target dopamine neurons. Here we investigated directly the causal contributions of defined dopamine neurons to multidimensional depression-like phenotypes induced by chronic mild stress, by integrating behavioural, pharmacological, optogenetic and electrophysiological methods in freely moving rodents. We found that bidirectional control (inhibition or excitation) of specified midbrain dopamine neurons immediately and bidirectionally modulates (induces or relieves) multiple independent depression symptoms caused by chronic stress. By probing the circuit implementation of these effects, we observed that optogenetic recruitment of these dopamine neurons potently alters the neural encoding of depression-related behaviours in the downstream nucleus accumbens of freely moving rodents, suggesting that processes affecting depression symptoms may involve alterations in the neural encoding of action in limbic circuitry

Bilateral Injections of the NMDA Receptor Antagonist AP5 into RA Significantly Reduced Song Variability

<div><p>(A) Excitatory synaptic inputs to RA from LMAN and HVC are mediated by a different mix of glutamate receptor types (see text). Using AP5 we could block LMAN input while only partially inactivating HVC input.</p> <p>(B) Eight sequential renditions of one song syllable in a juvenile zebra finch (63 dph) before and after AP5 injection into RA. Note the rapid fluctuations in pitch, the appearance of noisy acoustic structure, and variations in syllable duration before injection. The average variability scores <i>(V)</i> before and after injections for the eight shown syllable renditions were 0.50 and 0.25, respectively.</p> <p>(C) Following injection of AP5 into RA, fluctuations in acoustic structure were substantially reduced. Variability scores of 11 syllables in four birds before and after injection of AP5 into RA.</p> <p>(D) Time course of acoustic variability following drug injection averaged over all identifiable syllables for the bird in (B).</p></div

Analysis of the Effect of Bilateral LMAN Inactivation on Song Variability

<div><p>(A) Consecutive renditions of a repeating song motif of 0.5 s duration in a juvenile bird (59 dph) arranged vertically. Note the large variations in acoustic structure within individual syllables before LMAN inactivation (left). Following TTX injection into LMAN, the acoustic variability is dramatically reduced (middle), only to return to the original level by the following day (right). Numbers below each column indicate the variability index (See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030153#s4" target="_blank">Materials and Methods</a> section) calculated for the four renditions of the syllables shown.</p> <p>(B) Scatter plot of variability scores before and during LMAN inactivation with TTX (red) and muscimol (blue). Also shown are results for bilateral TTX injection into MMAN (black; see text), and saline injection into LMAN (green).</p> <p>(C) Time course of variability reduction following TTX (red) and muscimol (blue) injections show a time dependence that reflects the known in vivo pharmacology of the respective agents. Data were averaged over four identified syllables and taken from the same bird over consecutive days (dph = 70 and 71; muscimol inactivation followed by TTX inactivation).</p> <p>(D) Distribution of variability scores for all syllables analyzed in the TTX and muscimol experiments (25 unique syllables, six birds) before (black) and during (red) LMAN inactivation in juvenile birds. Shown for comparison are the variability scores for adult zebra finch syllables (18 syllables, 4 birds; undirected song, green; directed song, light blue). Dots represent raw data, while the lines are smoothed running averages.</p> <p>(E) TTX inactivation of LMAN significantly increased syllable sequence stereotypy. Sequence stereotypy scores (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030153#s4" target="_blank">Materials and Methods</a>) for six birds before (black) and after (red) TTX injections into LMAN. For comparison, the average stereotypy score for adult birds singing directed song was 0.95 (<i>n</i> = 4 birds).</p></div

Inactivation of LMAN Significantly Reduces Vocal Experimentation, Making the Otherwise Variable Song of the Juvenile Zebra Finch Highly Stereotyped

<div><p>(A) Two major pathways in the vocal control system of the songbird. The motor pathway (gray) includes motor cortex analogs HVC and RA, while the AFP (white), a basal ganglia thalamo-cortical circuit, consists of Area X, the dorsolateral anterior thalamic nucleus (DLM), and LMAN, which, in turn, projects to RA. To inactivate the output of the AFP, injections of TTX and muscimol (red bolus) were made into LMAN.</p> <p>(B) Examples of a juvenile zebra finch song (57 dph) showing large variability in the sequence and acoustic structure of song syllables.</p> <p>(C) Inactivating LMAN with TTX produces an immediate reduction of sequence and acoustic variability, revealing a highly stereotyped song produced by the motor pathway.</p> <p>The song snippets shown in (B) and (C) are from consecutive song bouts, immediately before and 1 h after drug injection. Songs are displayed as spectral derivatives calculated as described [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030153#pbio-0030153-b36" target="_blank">36</a>]. The frequency range displayed is 0–8.6 kHz. For audio of song bouts before and during LMAN inactivation in this bird, refer to Audios <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030153#sa001" target="_blank">S1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030153#sa002" target="_blank">S2</a>, and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030153#sa003" target="_blank">S3</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030153#sa004" target="_blank">S4</a>, respectively.</p></div

Song-Aligned Firing Patterns of RA-Projecting LMAN Neurons in Singing Juvenile Zebra Finches Are Highly Variable

<div><p>(A) Three successive renditions of a 67-d-old bird's song motif. Displayed under each spectrogram is the simultaneously recorded voltage waveform of an antidromically identified RA-projecting LMAN neuron (verified by collision testing). Average syllable variability for the three motifs is 0.31. Motif alignment was done at the onset (yellow lines) of syllable C.</p> <p>(B) Raster plot showing the spike patterns for 50 consecutive motif renditions for the same cell as in (A). The motifs from (A) are indicated in green.</p> <p>(C) Relative frequency of inter-spike intervals during singing (black) and non-singing (blue) for all the 17 identified projection neurons (units are intervals per second; bin size is 0.04 log units).</p> <p>(D) Distribution of spike-train correlations across all pairs of motifs for the cell in (B) (solid red line). Correlations calculated with random time shifts added to the spike trains have a similar distribution (dashed red line; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030153#s4" target="_blank">Materials and Methods</a>). Also shown is the correlation distribution for the population of identified projection neurons (solid black line; mean correlation indicated by solid arrowhead), and for the population with random time shifts added (dashed black line). In comparison, spike trains of neurons in premotor nucleus RA of the adult bird are highly stereotyped (from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030153#pbio-0030153-b23" target="_blank">23</a>]; mean correlation indicated by open arrowhead).</p></div

Multiple convergent hypothalamus–brainstem circuits drive defensive behavior

The hypothalamus is composed of many neuropeptidergic cell populations and directs multiple survival behaviors, including defensive responses to threats. However, the relationship between the peptidergic identity of neurons and their roles in behavior remains unclear. Here, we address this issue by studying the function of multiple neuronal populations in the zebrafish hypothalamus during defensive responses to a variety of homeostatic threats. Cellular registration of large-scale neural activity imaging to multiplexed in situ gene expression revealed that neuronal populations encoding behavioral features encompass multiple overlapping sets of neuropeptidergic cell classes. Manipulations of different cell populations showed that multiple sets of peptidergic neurons play similar behavioral roles in this fast-timescale behavior through glutamate co-release and convergent output to spinal-projecting premotor neurons in the brainstem. Our findings demonstrate that homeostatic threats recruit neurons across multiple hypothalamic cell populations, which cooperatively drive robust defensive behaviors