Noradrenergic Control of Gene Expression and Long-Term Neuronal Adaptation Evoked by Learned Vocalizations in Songbirds

Norepinephrine (NE) is thought to play important roles in the consolidation and retrieval of long-term memories, but its role in the processing and memorization of complex acoustic signals used for vocal communication has yet to be determined. We have used a combination of gene expression analysis, electrophysiological recordings and pharmacological manipulations in zebra finches to examine the role of noradrenergic transmission in the brain’s response to birdsong, a learned vocal behavior that shares important features with human speech. We show that noradrenergic transmission is required for both the expression of activity-dependent genes and the long-term maintenance of stimulus-specific electrophysiological adaptation that are induced in central auditory neurons by stimulation with birdsong. Specifically, we show that the caudomedial nidopallium (NCM), an area directly involved in the auditory processing and memorization of birdsong, receives strong noradrenergic innervation. Song-responsive neurons in this area express α-adrenergic receptors and are in close proximity to noradrenergic terminals. We further show that local α-adrenergic antagonism interferes with song-induced gene expression, without affecting spontaneous or evoked electrophysiological activity, thus dissociating the molecular and electrophysiological responses to song. Moreover, α-adrenergic antagonism disrupts the maintenance but not the acquisition of the adapted physiological state. We suggest that the noradrenergic system regulates long-term changes in song-responsive neurons by modulating the gene expression response that is associated with the electrophysiological activation triggered by song. We also suggest that this mechanism may be an important contributor to long-term auditory memories of learned vocalizations

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

Local action of noradrenaline modulates song-induced gene expression in NCM.

<p>a) Camera lucida drawing of a parasagittal section containing NCM, field L2a and CMM (about 500 µm from the midline), indicating the site of injections (pipette and dashed circle). b) Schematic representation of the experimental design depicting the timing of stereotaxic injection into NCM of awake restrained birds in relation to silence and song stimulation periods. c) Autoradiograms of adjacent parasagittal sections from a representative bird that received an injection of phentolamine or vehicle (opposite hemispheres) into NCM, hybridized with <i>zenk</i>, <i>c-fos</i> or <i>hat2</i> riboprobes. d) Fold-induction values for <i>zenk</i>, <i>c-fos</i> and <i>hat2</i> in the NCM of vehicle- (white bars) or phentolamine-injected (gray bars) hemispheres in birds stimulated for 10 min with conspecific song normalized to the corresponding injected hemispheres in unstimulated controls. For abbreviations, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036276#pone-0036276-g001" target="_blank">Figs 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036276#pone-0036276-g002" target="_blank">2</a>. Scale bar in a: 2 mm.</p

Noradrenergic modulation of song-induced gene expression and long-lasting changes in NCM auditory neurons.

<p>Undisturbed α-adrenergic transmission is required for song-induced gene expression and the long-term maintenance of song-adaptation yet does affect short-term habituation, indicating separate mechanisms. Blocking the downstream transcription (actinomycin) and translation events (cycloheximide) also results in disruption of long-lasting adaptation.</p

Systemic injections of α-adrenergic receptors antagonists block song-induced gene expression.

<p>a) Schematic representation of the experimental design depicting the timing of systemic injections (10 min prior to and 10 min after stimulation onset), and the overnight sound isolation (grey bar) and stimulation periods (30 minutes, patterned bar); b) Camera lucida drawing of a parasagittal brain section containing NCM at the level analyzed. c and d) Autoradiographic images of brain sections from unstimulated controls and song-stimulated birds systemically injected with saline or phentolamine (c) and saline or propanolol (d), hybridized with <i>zenk</i> riboprobes. e–f) Song fold-induction estimates of <i>zenk</i> mRNA based on quantitative autoradiography in birds injected with phentolamine (e) or propanolol (f) compared to vehicle (saline); fold-induction values were calculated by dividing values in song-stimulated birds by unstimulated controls for each drug treatment. Abbreviations: Cb, cerebellum; Hp, hippocampus; H, hyperpallium; NCM, caudomedial nidopallium; S, septum. Scale bar: 1 mm.</p

Expression of α-adrenergic receptors in NCM.

<p>a) Camera lucida drawing of a parasagittal brain section containing NCM and CMM (about 100 µm from the midline), indicating the position of the photomicrographs in b and c. b–c) Dark-field view of emulsion autoradiograms of brain sections hybridized with radioactively-labeled <i>ADRA1d</i> and <i>1a</i> riboprobes, respectively; shown is an area corresponding to the rectangle in a. d) High-magnification view of double <i>in situ</i> hybridization for <i>ADRA1d</i> (emulsion grains) and <i>zenk</i> (green fluorescence); arrows point to double-labeled cells and arrowhead indicates a single-labeled <i>ADRA1d</i>-positive cell.</p

Alpha-adrenergic blockade does not affect short-term responses in NCM.

<p>a) Representative responses to a song stimulus shown as raw multi-unit activity (green), and as RMS (black, 20 ms bins) for 6 simultaneously recorded sites (3 in the phentolamine hemisphere, 3 in the control hemisphere). b) Schematic representation of the experimental design for short-term adaptation: initial playback of songs from set A followed by phentolamine injection and then playback of songs from both sets A and B in shuffled order; dashed bar indicates the period of recordings. c) The change in NCM spontaneous activity is plotted as the ratio of activity recorded before phentolamine injection to activity during injection for both the control and phentolamine-injected hemispheres. A ratio of 1 indicates no change. d) The change in response amplitude to songs in set A from the end of training to the beginning of testing after drug injection is plotted as a ratio for both control and phentolamine-injected hemispheres. A ratio of 1 indicates no change. e) Response amplitude to songs in set B when novel during drug injection is shown for both control and phentolamine-injected hemispheres. f) Change in response to songs in set B when novel and when familiar after training during phentolamine injection is plotted as a ratio for both control and phentolamine-injected hemispheres. Similar adaptation (ratio <1, dotted line) is seen in both hemispheres.</p

Alpha-adrenergic blockade disrupts long-term adaptation in NCM.

<p>a) Schematic representation of the experimental design depicting exposure to training songs followed the next day (20–21 hr later) by electrophysiological recordings during playback of both familiar training songs and novel songs in control and phentolamine-injected hemispheres. b) Familiarity indexes (the ratio between adaptation rates for novel songs and songs heard by the animal 20–21 h earlier, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036276#s2" target="_blank">Methods</a>) are shown for both the control and phentolamine-injected hemispheres.</p