A micro-CT-based method for quantitative brain lesion characterization and electrode localization

Lesion verification and quantification is traditionally done via histological examination of sectioned brains, a time-consuming process that relies heavily on manual estimation. Such methods are particularly problematic in posterior cortical regions (e.g. visual cortex), where sectioning leads to significant damage and distortion of tissue. Even more challenging is the post hoc localization of micro-electrodes, which relies on the same techniques, suffers from similar drawbacks and requires even higher precision. Here, we propose a new, simple method for quantitative lesion characterization and electrode localization that is less labor-intensive and yields more detailed results than conventional methods. We leverage staining techniques standard in electron microscopy with the use of commodity micro-CT imaging. We stain whole rat and zebra finch brains in osmium tetroxide, embed these in resin and scan entire brains in a micro-CT machine. The scans result in 3D reconstructions of the brains with section thickness dependent on sample size (12–15 and 5–6 microns for rat and zebra finch respectively) that can be segmented manually or automatically. Because the method captures the entire intact brain volume, comparisons within and across studies are more tractable, and the extent of lesions and electrodes may be studied with higher accuracy than with current methods

Mesoscopic patterns of neural activity support songbird cortical sequences

Time-locked sequences of neural activity can be found throughout the vertebrate forebrain in various species and behavioral contexts. From time cells in the hippocampus of rodents to cortical activity controlling movement, temporal sequence generation is integral to many forms of learned behavior. However, the mechanisms underlying sequence generation are not well known. Here, we describe a spatial and temporal organization of the songbird premotor cortical microcircuit that supports sparse sequences of neural activity. Multi-channel electrophysiology and calcium imaging reveal that neural activity in premotor cortex is correlated with a length scale of 100 microm. Within this length scale, basal-ganglia-projecting excitatory neurons, on average, fire at a specific phase of a local 30 Hz network rhythm. These results show that premotor cortical activity is inhomogeneous in time and space, and that a mesoscopic dynamical pattern underlies the generation of the neural sequences controlling song

Unstable neurons underlie a stable learned behavior

Motor skills can be maintained for decades, but the biological basis of this memory persistence remains largely unknown. The zebra finch, for example, sings a highly stereotyped song that is stable for years, but it is not known whether the precise neural patterns underlying song are stable or shift from day to day. Here we demonstrate that the population of projection neurons coding for song in the premotor nucleus, HVC, change from day to day. The most dramatic shifts occur over intervals of sleep. In contrast to the transient participation of excitatory neurons, ensemble measurements dominated by inhibition persist unchanged even after damage to downstream motor nerves. These observations offer a principle of motor stability: spatiotemporal patterns of inhibition can maintain a stable scaffold for motor dynamics while the population of principal neurons that directly drive behavior shift from one day to the next

Unstable neurons underlie a stable learned behavior

Motor skills can be maintained for decades, but the biological basis of this memory persistence remains largely unknown. The zebra finch, for example, sings a highly stereotyped song that is stable for years, but it is not known whether the precise neural patterns underlying song are stable or shift from day to day. Here we demonstrate that the population of projection neurons coding for song in the premotor nucleus, HVC, change from day to day. The most dramatic shifts occur over intervals of sleep. In contrast to the transient participation of excitatory neurons, ensemble measurements dominated by inhibition persist unchanged even after damage to downstream motor nerves. These observations offer a principle of motor stability: spatiotemporal patterns of inhibition can maintain a stable scaffold for motor dynamics while the population of principal neurons that directly drive behavior shift from one day to the next

A micro-CT-based method for characterising lesions and locating electrodes in small animal brains

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12-15 and 5-6 µm per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies

Mesoscopic patterns of neural activity support songbird cortical sequences.

Time-locked sequences of neural activity can be found throughout the vertebrate forebrain in various species and behavioral contexts. From "time cells" in the hippocampus of rodents to cortical activity controlling movement, temporal sequence generation is integral to many forms of learned behavior. However, the mechanisms underlying sequence generation are not well known. Here, we describe a spatial and temporal organization of the songbird premotor cortical microcircuit that supports sparse sequences of neural activity. Multi-channel electrophysiology and calcium imaging reveal that neural activity in premotor cortex is correlated with a length scale of 100 µm. Within this length scale, basal-ganglia-projecting excitatory neurons, on average, fire at a specific phase of a local 30 Hz network rhythm. These results show that premotor cortical activity is inhomogeneous in time and space, and that a mesoscopic dynamical pattern underlies the generation of the neural sequences controlling song

Data used in PLoS Biology article " Mesoscopic Patterns of Neural Activity Support Songbird Cortical Sequences"

<p>MATLAB data files containing spike, LFP, and calcium imaging data collected from HVC in singing zebra finches.  All files are in the MATLAB .mat format, see README.txt for details.</p

Three hypothetical models for the spatiotemporal organization of the song premotor code in HVC.

<p>The spatial organization of neural activity in HVC in singing birds is unknown. The geometry of neural activity could be described by three schematics that form a continuum: <b>a,</b> a random, disorganized geometry [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002158#pbio.1002158.ref008" target="_blank">8</a>]; <b>b,</b> functional clustering (i.e., nearby cells code for similar elements) with a characteristic length scale [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002158#pbio.1002158.ref027" target="_blank">27</a>]; and <b>c,</b> traveling waves [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002158#pbio.1002158.ref028" target="_blank">28</a>].</p

A stereotyped spatiotemporal 30 Hz pattern underlies premotor cortical activity during song.

<p>Trial-averaged 25–35 Hz LFP patterns on two consecutive days for six electrodes spaced by 175 μm along the mediolateral axis of HVC. Each tick mark indicates the timing of the local LFP peak, and the color indicates the phase relative to the average phase of all electrodes at that time point. (See breakout illustration top right.) See also <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002158#pbio.1002158.s004" target="_blank">S4 Fig</a>.</b></p

Inhibitory neuron firing patterns and three models for projection neuron/inhibitory neuron relationships.

<p><b>a,</b> Raster plots of three nearby (<200 μm) inhibitory neurons recorded during singing from the same bird. Synchronous pauses are marked with a black triangle. Shown to the right are three conceptual models of timing relationships between inhibitory and projection neuron activity. <b>b,</b> Interneuron firing patterns are uncorrelated in local ensembles, leading to a net local inhibition that is weakly modulated in time. <b>c,</b> Gaps in inhibitory neuron firing activity are locally correlated, leading to synchronous pauses in inhibition, but these pauses are unrelated to projection neuron activity. <b>d,</b> Gaps in inhibitory neuron firing activity are locally correlated, and contribute to defining projection neuron firing times. PN, projection neuron; INT, interneuron.</p