Conjunction of vocal production and perception regulates expression of the immediate early gene ZENK in a novel cortical region of songbirds

Abstract

The cortical nucleus LMAN (lateral magnocellular nucleus of the anterior nidopallium) provides the output of a basal ganglia pathway that is necessary for acquisition of learned vocal behavior during development in songbirds. LMAN is composed of two subregions, a core and a surrounding shell, that give rise to independent pathways that traverse the forebrain in parallel. The LMAN(shell) pathway forms a recurrent loop that includes a cortical region, the dorsal region of the caudolateral nidopallium (dNCL), hitherto unknown to be involved with learned vocal behavior. Here we show that vocal production strongly induces the IEG product ZENK in dNCL of zebra finches. Hearing tutor song while singing is more effective at inducing expression in dNCL of juvenile birds during the auditory-motor integration stage of vocal learning than is hearing conspecific song. In contrast, hearing conspecific song is relatively more effective at inducing expression in adult birds, regardless of whether they are producing song. Furthermore, ZENK+ neurons in dNCL include projection neurons that are part of the LMAN(shell) recurrent loop and a high proportion of dNCL projection neurons express ZENK in singing juvenile birds that hear tutor song. Thus juvenile birds that are actively refining their vocal pattern to imitate a tutor song show high levels of ZENK induction in dNCL neurons when they are singing while hearing the song of their tutor and low levels when they hear a novel conspecific. This pattern indicates that dNCL is a novel brain region involved with vocal learning and that its function is developmentally regulated.

Fig. 1.

Major axonal connections made by core and shell regions of LMAN. A: the cortical nucleus LMAN provides the output of a basal ganglia pathway from Area X (and surrounding medial striatum) to DLM (in dorsal thalamus) to LMAN. Area X contains both striatal and pallidal neurons. The core region of LMAN (light gray) projects to RA within the area of avian brain that is analogous to motor cortex and RA projects in turn to descending motor circuits that activate vocal and respiratory muscles. In contrast, the shell region of LMAN (dark gray) projects to Ad, an area of motor cortex adjacent to RA. Ad does not make direct connections to hindbrain vocal-motor circuits, but makes a variety of axonal projections, including a projection to DLM that forms a recurrent loop from LMAN_shell → dNCL → Ad → DLM_VM → LMAN_shell. The shell region of LMAN projects to both Ad and to a region of polymodal association cortex, dNCL, which projects in turn to Ad, such that Ad receives both direct and indirect projections from LMAN_shell. Separate groups of neurons in DLM (dorsolateral [DL] vs. ventromedial [VM]) project to either core or shell, respectively, and core and shell regions of LMAN project to RA vs. Ad, such that these connections form parallel, apparently independent projections that traverse the forebrain. Core and shell regions of LMAN also project to Area X and surrounding medial striatum, respectively. Not shown for the sake of clarity is a projection from Ad to DMP (part of a dorsal thalamic zone that includes both DLM and DMP); DMP projects to MMAN, which projects in turn to HVC (see Supplemental Fig. S1). B: changes in the volume encompassed by core (light gray) and shell (dark gray) regions of LMAN. The volume of LMAN_core decreases slightly (but significantly) despite maintaining a fixed number of projection neurons. The volume of LMAN_shell exhibits pronounced growth during early stages of vocal learning (20–35 days of age), followed by substantial regression between 35 days and adulthood (90 days). Data are replotted from Johnson et al. (1995). LMAN, lateral magnocellular nucleus of the anterior nidopallium; MMAN, medial magnocellular nucleus of the anterior nidopallium; c, core region; s, shell region; Area X, Area X of the medial striatum; HVC, high vocal center; RA, robust nucleus of the arcopallium; Ad, dorsal arcopallium; DLM, medial dorsolateral nucleus of the thalamus (DL contains neurons in the dorsolateral portion of DLM that project to LMAN_core, whereas VM contains neurons in the ventromedial portion of DLM that project to LMAN_shell); DMP, dorsomedial nucleus of the posterior thalamus; dNCL, dorsal region of the caudolateral nidopallium (see Reiner et al. 2004).

Fig. 2.

Schematic illustration of dNCL. Cross section through the caudal telencephalon (after Bottjer et al. 2000) showing RA and Ad within the arcopallium and dNCL in the dorsolateral nidopallium The projection from dNCL to Ad is topographic, as shown here by injection sites in midmedial (black) and midlateral (gray) Ad that produced corresponding clusters of retrogradely labeled cells in dNCL (note that the extreme lateral and medial regions of dNCL do not include labeled cells in this figure). The borders of dNCL are not apparent in Nissl-stained sections nor in our immunostained tissue. In experiment 1, we therefore extrapolated the location of dNCL based on the location of Ad and our knowledge of the path of axons from dNCL to Ad. We counted ZENK+ cells within a sampling window (gray box), the size and placement of which were chosen to be conservative in the sense that it would exclude extreme dorsal, ventral, medial, and lateral regions of dNCL. In this way we avoided counting immunoreactive cells outside the borders of dNCL. In experiment 2, we counted cells within dNCL by locating our sampling window over regions that included the highest density of retrogradely labeled neurons, depending on the quality and location of the injection site. Neurons outside dNCL are never labeled by injections into Ad (Bottjer et al. 2000).

Fig. 3.

ZENK induction in dNCL in response to singing and hearing. Number of ZENK+ cells per mm³ within dNCL (means ± SE) in juvenile birds (∼55 days of age) in the 4 groups of experiment 1.

Fig. 4.

ZENK induction in dNCL as a function of age and playback in singing birds. A: number of ZENK+ cells per mm² within dNCL (means ± SE) in singing juvenile (JUV) vs. adult (ADL) birds during playback of tutor (TUT) vs. conspecific (CON) song. B: the percentage of ZENK+ cells among retrogradely labeled neurons within dNCL. Retrogradely labeled cells (Ad-projecting neurons) were examined separately such that the incidence of double-labeled cells was expressed as the ratio of double-labeled cells to retrogradely labeled cells (see methods).

Fig. 5.

ZENK induction in dNCL neurons that project to Ad. Representative photomicrographs of ZENK+ cells, retrogradely labeled cells (showing projection neurons in dNCL that send axons to Ad), and the merge showing double-labeled cells within dNCL from an adult singing bird in the CON playback group (Dg633, top panels) and an adult control bird that neither sang nor heard playback (Lg716, bottom panels). Arrows show examples of double-labeled neurons.

Fig. 6.

ZENK induction in dNCL as a function of age and playback in singing and nonsinging birds. Number of ZENK+ cells per mm² within dNCL (means ± SE) in juvenile and adult birds that heard different types of playback (tutor song vs. conspecific song: TUT vs. CON). The majority of birds in each group sang, but some did not (see text), allowing a comparison of the effects of playback in singing vs. nonsinging birds. Data from Fig. 4A are replotted here for comparison.