1 2 3 A role for descending auditory cortical projections in songbird 4 vocal learning 5 6 7 8 Yael Mandelblat-Cerf*, Liora Las*, Natalia Denissenko*, and Michale S. Fee† 9 10 McGovern Institute for Brain Research 11 Department of Brain and Cognitive Sciences 12 Massachusetts Institute of Technology 13 Cambridge, MA 14 15 16 * These authors contributed equally 17 18 †To whom correspondence should be addressed: 19 [email protected] 20 MIT 46-5133 21 77 Massachusetts Ave 22 Cambridge, MA 02139 23 (617) 324-0173 (phone) 24 (617) 324-0256 (fax) 25 26 27 28 Abstract (149 words): 29 Many learned motor behaviors are acquired by comparing ongoing behavior with 30 an internal representation of correct performance, rather than using an explicit external 31 reward. For example, juvenile songbirds learn to sing by comparing their song with the 32 memory of a tutor song. At present, the brain regions subserving song evaluation are not 33 known. Here we report several findings suggesting that song evaluation involves an 34 avian ‘cortical’ area previously shown to project to the dopaminergic midbrain and other 35 downstream targets. We find that this ventral portion of the intermediate arcopallium 36 (AIV) receives inputs from auditory cortical areas, and that lesions of AIV result in 37 significant deficits in vocal learning. Additionally, AIV neurons exhibit fast responses to 38 disruptive auditory feedback presented during singing, but not during nonsinging periods. 39 Our findings suggest that auditory cortical areas may guide learning by transmitting song 40 evaluation signals to the dopaminergic midbrain and/or other subcortical targets. 41 Introduction 42 Most human behaviors, such as speech, music, or athletic performance, are learned 43 through a gradual process of trial and error. In all of these behaviors, the motor actions 44 are shaped during learning by an internal model of good performance (Wolpert et al., 45 1995). While a great deal has been learned about the neural mechanisms by which 46 external rewards, such as food or juice drops, are represented in the brain and might 47 shape future behavior (Schultz et al., 1997, Hikosaka, 2007), little is known about how 48 the brain evaluates its own performance, where such self-evaluation is computed and how 49 these signals might shape future behavior. 50 Here we use the songbird as a model system to understand how complex 51 behaviors can be learned by self-evaluation of motor performance, rather than relying on 52 explicit external rewards such as food or social reinforcement. Songbirds learn their 53 vocalizations by storing a memory, or ‘template’, of their tutor’s song (Doupe and Kuhl, 54 1999). By listening to themselves sing and comparing their own song to this template 55 (Konishi, 1965), they gradually converge to what can be a nearly exact copy of the tutor’s 56 song. Vocal learning requires a basal-ganglia forebrain circuit called the anterior 57 forebrain pathway (AFP), the output of which projects to cortical premotor vocal areas 58 (Scharff and Nottebohm, 1991, Nottebohm et al., 1976, Bottjer et al., 1984). The AFP is 59 crucial for driving learned changes in song and shaping plasticity in the song motor 60 pathway (Brainard and Doupe, 2000, Andalman and Fee, 2009, Warren et al., 2011). A 61 key component of this vocal learning circuit is Area X, a basal ganglia homologue 62 containing both striatal and pallidal components (Person et al., 2008). 63 Of critical importance in understanding the mechanisms of vocal sensorimotor 64 learning in the songbird is to determine where song is evaluated and how the resulting 65 evaluation signals are transmitted to the AFP to shape learning. Some hypotheses have 66 emphasized the possible role of HVC (used as a proper name), a premotor cortical circuit 67 that receives auditory inputs and also projects to Area X (Prather et al., 2008, Troyer and 68 Doupe, 2000, Roberts et al., 2012, Mooney, 2006). While the involvement of HVC in 69 evaluating ongoing song remains an open question, several lines of evidence suggest that 70 HVC does not transmit error-related signals to the AFP (Kozhevnikov and Fee, 2007, 71 Hessler and Doupe, 1999), or receive auditory inputs (Hamaguchi et al., 2014) during 72 singing. 73 Another possibility is suggested by the large projection to Area X from 74 dopaminergic nuclei in the midbrain—the ventral tegmental area (VTA) and substantia 75 nigra pars compacta (SNc) (Person et al., 2008) (Figure 1A). Inspired by the recently 76 hypothesized role of dopaminergic signaling in reinforcement learning (Schultz et al., 77 1997, Tsai et al., 2009, Bayer and Glimcher, 2005, Houk et al., 1994), we and others have 78 proposed that this dopaminergic input to Area X may play a role in song learning by 79 signaling vocal errors (Gale et al., 2008, Fee and Goldberg, 2011, Hara et al., 2007, Gale 80 and Perkel, 2010). Notably, a recent anatomical study in songbirds has described a 81 projection to the dopaminergic midbrain from the arcopallium (Gale et al., 2008), an 82 avian cortical region containing subtelencephalically-projecting neurons analogous to 83 those in deep layer-5 of mammalian cortex (Dugas-Ford et al., 2012, Karten, 2013). 84 Here we set out to examine the role of the cortical area projecting to VTA/SNc in 85 song learning. First, we characterized the spatial extent of the arcopallial neurons 86 projecting to VTA or SNc, and characterized the inputs to these neurons from auditory 87 cortical fields. We then carried out lesions targeted to this area to examine the effect on 88 tutor imitation and on vocal plasticity that occurs after deafening. Finally, we recorded 89 from VTA/SNc-projecting neurons in the arcopallium of singing birds and observed fast 90 transient responses to disruptive auditory feedback during singing. Altogether, our 91 findings suggest that descending auditory cortical projections may play a role in song 92 evaluation via projections to the dopaminergic midbrain and/or other subcortical targets. 93 Results 94 An auditory cortical pathway to the dopaminergic midbrain 95 To elucidate the spatial pattern of cortical neurons projecting to the dopaminergic 96 midbrain in songbirds, we made small injections of a retrograde tracer (cholera toxin 97 subunit β, CTB) into VTA and SNc (n=12 zebra finches, Figure 1B, see Methods). 98 Consistent with an earlier report (Gale et al., 2008), we observed a distinct mass of 99 retrogradely-labeled cell bodies within the ventral-most extent of the intermediate 100 arcopallium. Labeled neurons were distributed in a complex pattern that was highly 101 consistent across birds (Figure 1C and Figure 1-figure supplement 1). The main cluster of 102 labeled neurons was arranged in a stem-like column ventral to RA (robust nucleus of the 103 arcopallium), while a distinct ‘stripe’ of labeled neurons was observed 300-400µm 104 anterior to the main cluster. A smaller number of labeled neurons formed a thin shell 105 around the dorsal surface of RA, as previously described (Gale et al., 2008). We will 106 refer specifically and collectively to the arcopallial areas containing neurons retrogradely- 107 labeled from the dopaminergic midbrain, described above, as the ventral intermediate 108 arcopallium (AIV). 109 Injections of anterograde tracer (HSV viral vector expressing green fluorescent 110 protein, GFP) within the anterior and posterior clusters of AIV neurons each revealed 111 axonal arborization intermingled with TH-positive neurons in both VTA and SNc (Figure 112 1D,E, n=6 birds). 113 It is important to elucidate the anatomical relation between the area we identify as 114 AIV and the dorsal arcopallium (Ad), which has previously been implicated in vocal 115 learning (Bottjer and Altenau, 2010), and also in locomotory control (Feenders et al., 116 2008, Jarvis et al., 2013) (called lateral intermediate arcopallium, LAI, in the latter 117 studies). Ad was labeled by injections of anterograde tracer (Alexa 488 conjugated 118 dextran 10MW) into the nidopallium lateral to LMAN (LMAN-shell), and AIV was 119 retrogradely-labeled in the same birds by injection of CTB into VTA and SNc (n=8 120 hemispheres). In coronally-sectioned tissue, Ad was seen as a band of labeled fibers 121 extending lateral to RA (Figure 1F), as previously described. Neurons retrogradely- 122 labeled from VTA/SNc were visible as a wedge of cell bodies ventrolateral to RA and 123 ventromedial to Ad. Few labeled cell bodies were observed within the region of labeled 124 fibers in Ad, although some retrogradely-labeled neurons were seen in the thin ‘neck’ of 125 labeled fibers connecting RA and Ad. Injections of retrograde tracer (CTB) into the 126 anterior or posterior parts of AIV (n=5) revealed no retrogradely-labeled neurons in the 127 nidopallium adjacent to LMAN (LMAN-shell). Thus, AIV and Ad appeared to be largely 128 distinct non-overlapping regions. 129 To further elucidate the possible cortical afferents to AIV, we made injections of 130 retrograde tracer (CTB) into different locations anterior and ventral to RA (n=10 birds). 131 Following injections into the anterior parts of AIV (n=5 birds), retrogradely-labeled 132 neurons were reliably observed in auditory cortical areas HVC-shelf, caudal mesopallium 133 (CM) and L1 (Figure 2A-F and Figure 2-figure supplement 1A-C) (Vates et al., 1996, 134 Mello et al., 1998, Kelley and Nottebohm, 1979). We note that, while these previous 135 studies have identified inputs to ventral arcopallium (RA-cup) from L1 and HVC-shelf, 136 the projection described here from CM is novel. Following injections into the more 137 posterior parts of AIV (n=5 birds), retrogradely-labeled neurons were observed in the 138 caudal nidopallium (NC) posterior to HVC-shelf (Figure 2G,H).