scAAV9-Cre

Birdsong, like human language, consists of a sequence of temporally precise movements acquired through vocal learning. The learning of such sequential vocalizations depends on the neural function of the motor cortex and basal ganglia. Here, researchers used the songbird zebra finch as an animal model to explore the function of connections between the cortex-like (HVC) and basal ganglia (area X), which are connected via HVC(X) projection neurons and have temporally precise firing during singing. By specifically ablating HVC(X) neurons, juvenile zebra finches failed to imitate the acoustics of the guide syllables and developed songs that were temporally unstable with poor sequence consistency. In contrast, HVC(X)-ablated adults did not alter their learned song structure, but generated acoustic fluctuations and responded to auditory feedback disruption by the introduction of song deterioration, as did normal adults. These results suggest that cortico-basal ganglia inputs are important for learning the acoustic and temporal aspects of song structure but not for generating vocal fluctuations that help maintain already learned vocal patterns.

The researchers first injected scAAV9-Cre and scAAV9-FLExmRuby2 into area X and HVC, respectively, to test the induction rate and expression time of the target gene (i.e., mRuby2) in HVC_(X) neurons. One week after injection, selective expression of mRuby2 protein in HVC_(X) neurons was observed (Figure 1C). Neurotensin (NTS) mRNA was used as a marker of HVC_(X) neurons. The study confirmed that NTS mRNA was specifically expressed in HVC_(X) neurons labeled by retrograde cholera toxin B (CTB) injected into area X, but not in HVC(RA) neurons (Figure 1D). Using NTS labeling, a reliable estimation of residual HVC_(X) neuron number after ablation could be performed without additional retrograde labeling from area X to HVC. The researchers then injected scAAV9-Cre into area X of the test hemisphere and injected a mixture of scAAV9-FLEx-dtA and -caCasp into HVC. In the other hemisphere of the same animal, scAAV9-Cre and scAAV9-FLEx-mRuby2 were injected into area X and HVC, respectively, to serve as the control hemisphere. The number of HVC_(X) neurons was compared between the control and HVC_(X)-ablated hemispheres (Figure 1D). As a result, the residual number of HVC_(X) neurons was reduced to 23.9 to 57.2% in the ablated hemisphere when compared to the control HVC of the same individual. Although the cell ablation procedure failed to completely remove HVC_(X) neurons, the approach using the mixture of dtA and caCasp showed a similar or higher rate of effective reduction of target cells compared to previous efforts to perform cell ablation in songbirds.

Figure 1. Specific ablation of HVC_(X) neurons projecting to the basal ganglia area X. (Sánchez-Valpuesta M, et al., 2019)