Morphology of axonal projections from the high vocal center to vocal motor cortex in songbirds

Complex motor behaviors such as running, playing the piano, and producing speech require the brain to execute stereotyped neural sequences. Understanding how the brain comes to control a series of complicated movements is essential for developing treatments for learning disorders and motor dysfunction. In adult male zebra finches (Taenopygia guttata), the cortical nucleus HVC (high vocal center; located in the nidopallium; Reiner et al., 2004) generates highly stereotyped and precisely timed activity patterns that control learned vocal output (Nottebohm et al., 1976; Simpson and Vicario, 1990; Vu et al., 1998; Hahnloser et al., 2002; Wild, 2004; Ashmore et al., 2005). The projection from HVC to RA (robust nucleus of the arcopallium; in motor cortex; Fig. 1) provides an interesting model for understanding how sparse firing codes in HVC influence the relatively “dense” firing patterns in RA to control a fixed motor sequence (Yu and Margoliash, 1996; Dave and Margoliash, 2000). HVC receives auditory input indirectly via primary auditory cortex and directly via two higher-level auditory regions of cortex, nucleus interface of the nidopallium (NIf) and caudal mesopallium (CM) (Vates et al., 1996; Cardin and Schmidt, 2004a,b; Coleman and Mooney, 2004; Bauer et al., 2008; Wang et al., 2010; Akutagawa and Konishi, 2010). Individual HVCRA neurons produce brief bursts of action potentials at highly precise times during the production of song as well as when the bird hears its own song played back (Hahnloser et al., 2002, 2006; Prather et al., 2008). Figure 1 Schematic of song-control pathways in adult zebra finches. Song-control circuits include the HVC-RA vocal motor pathway in adult birds (boxes shaded gray) and a cortico-basal ganglia-thalamo circuit that is necessary for vocal plasticity (LMAN→Area ... HVC contains two separate populations of projection neurons. One population projects to a basal ganglia pathway necessary for plasticity in vocal behavior (Bottjer 2004; Brainard, 2004). As indicated above, the second population sends axons to the motor-cortical nucleus RA (Nottebohm et al., 1976, 1982; Bottjer et al., 1989; Johnson and Bottjer, 1993, 1995; Foster and Bottjer, 1998), which in turn drives activity in vocal motor and respiratory hindbrain regions to produce stereotyped learned vocal output (Wild, 1993, 2004; Sober et al., 2008) (Fig. 1). The axonal projections of HVC are nontopographic: tracer injections into single subregions of HVC produce a uniform distribution of anterograde label throughout RA rather than in a specific restricted location (Fortune and Margoliash, 1995; Vates and Nottebohm, 1995; Foster and Bottjer, 1998). Interestingly, other projections to and from RA are highly topographic (Vicario, 1991; Johnson et al., 1995; Vates and Nottebohm, 1995). Dorsal RA is innervated by axons from the medial portion of LMAN (lateral magnocellular nucleus of the anterior nidopallium; Fig. 1) and projects to mid- and hindbrain regions that control respiration (Wild, 1993, 2004). Intermediate RA is innervated by intermediate LMAN and projects to rostral hypoglossal motor neurons, which project to ventral muscles of the vocal organ (syrinx); ventral RA is inner-vated by lateral LMAN and projects to caudal hypoglossal motor neurons, which innervate the dorsal muscles of the syrinx (Vicario, 1991). Ventral syringeal muscles control the fundamental frequency of vocalizations and dorsal syringeal muscles control the timing and amplitude of sound production (Goller and Suthers, 1996; Suthers and Zollinger, 2004). During song production, HVCRA neurons produce a brief burst of spikes at a single point during each rendition of a song motif (Hahnloser et al., 2002; Fee et al., 2004), whereas RA neurons produce ≈10–12 bursts (Yu and Margoliash, 1996; Leonardo and Fee, 2005; Sober et al., 2008). Different combinations of HVCRA neurons burst at different timepoints in the song. This pattern has given rise to a synaptic-chain model for vocal production in which ensembles of HVCRA neurons burst sequentially and trigger the next group of neurons in the sequence to fire (Hahnloser et al., 2002; Fee et al., 2004; Long et al., 2010). This model suggests that different ensembles of HVCRA neurons fire at specific points in the song to control a short segment or subsyllable of song, and that each neuron contributes to only a small proportion of song production. We analyzed the morphological characteristics of single HVCRA axons to determine how connections from HVC to RA are organized. In accord with the idea that single HVC neurons contribute to a brief interlude of the song pattern, HVCRA axons exhibited an extremely simple morphology with limited branching patterns. We also reconstructed axons projecting from HVC to the area surrounding anterior and ventral RA, RA cup (Kelley and Nottebohm, 1979; Vates et al., 1996). RA cup receives inputs both directly and indirectly from auditory cortex and projects to thalamic, midbrain, and pontine nuclei of the ascending auditory pathway (Mello et al., 1998). We found that individual axons projecting to RA cup also extended branches into RA, indicating that sensory and motor integration may occur directly at the level of RA.