Nucleus Uvaeformis (Uva) is a small polysensory nucleus in the caudal thalamus that is reported to be necessary for song production. Uva is a recipient of somatosensory information from the trigeminal system, visual input from the optic tectum, auditory information from the lateral lemniscus, cholinergic input from the medial habenula, and information about respiratory timing from the ventral respiratory column (Coleman et al., 2007; Faunes and Wild, 2017; Wild and Gaede, 2016; Wild et al., 2010; Wild, 1994; Akutagawa and Konishi, 2005). Its inputs to HVC are thought to be pivotal in song motor control (Moll et al., 2023; Danish et al., 2017; Coleman and Vu, 2005). However, how Uva synaptically influences HVC activity is still poorly understood. We found that Uva neurons are robustly transduced by our AAV expressing eGtACR1 (Figure 2A). Thalamic regions around Uva do not project to HVC, allowing us to selectively map Uva’s input to the three classes of HVC-PNs.

In acute brain slices we observed glutamatergic oEPSCs in all three classes of retrogradely identified HVC-PNs (27/66 HVC_X, 43/51 HVC_RA, 14/28 HVC_Av neurons; Figure 2B, Figure 2—figure supplement 1). Optical stimulation of Uva axons resulted in significantly lower probability of observing oEPSCs in HVC_X and HVC_Av than in HVC_RA neurons (Figure 2B, Figure 2—figure supplement 1). When we held the membrane voltage to +10 mV, we also observed relatively delayed GABAergic currents (53.93% probability, Figure 2—figure supplement 1), consistent with disynaptic inhibition. Uva axon stimulation elicited oIPSCs mainly in cells where we observed oEPSCs (oIPSC contingent with oEPSC: HVC_X, 100.0%, HVC_RA 96.6%, HVC_Av, 95.8%; Figure 2B).

Despite the markedly higher probability of eliciting currents in HVC_RA neurons, oEPSC and oIPSC amplitudes were comparable across all the HVC-PN classes (Figure 2C, D, Figure 2—figure supplement 1). The excitation/inhibition ratio revealed that Uva terminal stimulation generally led to higher amplitude oIPSCs than oEPSCs (Figure 2E, Figure 2—figure supplement 1).

We next examined if HVC-PN classes receive monosynaptic input from Uva. Recent studies have suggested different views for how Uva may influence the propagation of activity in HVC. Uva has been proposed to provide high-frequency (30–60 Hz) synchronous synaptic inputs that facilitate activity propagation across HVC_RA premotor neurons (Hamaguchi et al., 2016). In another proposal, Uva selectively drives activity in only a fraction,~16%, of HVC_RA neurons which are active during syllable transitions (Moll et al., 2023). Consistent with the higher likelihood of polysynaptic excitation in HVC_RA neurons, we found that oEPSC returned after application of TTX +4 AP in all HVC_RA neurons tested (HVC_RA: 6/6 neurons) while HVC_X and HVC_Av were infrequently found to receive monosynaptic input from Uva (HVC_X: 1/5 cells, HVC_Av: 2/5 cells, Figure 2F). Our synaptic circuit mapping provides the first conclusive evidence that Uva makes monosynaptic connections with HVC_RA neurons. Given that we observed oEPSCs in 84.3% of recorded HVC_RA and found monosynaptic connections from Uva onto 100% of the HVC_RA neurons tested, our data further suggests that Uva is unlikely to make monosynaptic connections with only a small percentage of HVC_RA neurons.

Previous studies have been equivocal to the excitatory, inhibitory, or neuromodulatory nature of the Uva to HVC circuitry (Moll et al., 2023; Coleman et al., 2007). We did not observe monosynaptic oIPSCs in any cell class, suggesting that Uva provides only a glutamatergic input to HVC (HVC_X: 0/3, HVC_RA: 0/5, HVC_Av: 0/4, data not shown). We combined retrograde labeling from HVC with in-situ hybridization labeling on brain slices. This revealed that Uva neurons projecting to HVC (Uva_HVC) selectively expressed the glutamatergic marker SLC17A6, but not the GABAergic marker GAD1 (Colquitt et al., 2021; Figure 2G). Interestingly, our labeling did not identify any GABAergic neurons within Uva, indicating that Uva_HVC may function as an excitatory relay of diverse polysensory input pathways. Although we cannot exclude the possibility that Uva neurons may also co-release neuropeptides, our results define the excitatory synaptic connectivity between Uva and HVC PNs.

We next examined the synaptic transmission between the nucleus NIf and the three classes of HVC-PNs. NIf is a higher order polysensory DVR nucleus located adjacent to the primary auditory DVR (Field L2 - region that receives thalamic input from the avian homologue of the medial geniculate nucleus). NIf is the recipient of afferents from Uva and multiple areas of the auditory DVR and has an essential role in relaying auditory signals to HVC (Coleman and Mooney, 2004). The synaptic connection between NIf and HVC is obligatory in a young male zebra finch’s ability to form a memory of their father’s song (Zhao et al., 2019; Roberts et al., 2012). This memory is used as birds evaluate their song performances, permitting vocal imitation of song (Ikeda et al., 2020). Optogenetic manipulations of NIf inputs to HVC are also sufficient to implant song memories that guide song syllable imitation (Zhao et al., 2019). Although NIf is selectively active during singing, it is not necessary for producing song in adulthood or the song imitation process once the memory of a father’s song is acquired in juvenile birds (Zhao et al., 2019; Roberts et al., 2012; Mackevicius et al., 2020; Otchy et al., 2015).

To map synaptic connections from NIf to HVC, we expressed eGtACR1 in NIf. While the surrounding area, Field L, has been described to project to the ventral border of HVC (Vates et al., 1996; Fortune and Margoliash, 1995; Kelley and Nottebohm, 1979), we recorded from visually identified, retrogradely labeled HVC projection neurons. Area Avalanche is the only other nearby brain region that also sends projections to HVC, and we carefully checked for a lack of expression in this region in all brain hemispheres used in this study (Figure 3A). This provides confidence that the currents we measured are evoked by stimulating neurotransmission from NIf terminals. Light stimulation (1ms) reliably elicited glutamatergic oEPSCs in all three classes of HVC projection neurons (78.6% probability, Figure 3B, Figure 3—figure supplement 1). We examined oIPSCs in a subset of neurons and found strong GABAergic currents evoked by optical stimulation in all three classes of projection neurons (65.9% probability, Figure 3B, Figure 3—figure supplement 1). oIPSCs were predominantly found in cells in which we also observed oEPSCs (oIPSC contingent with oEPSC: HVC_X 90.0%; HVC_RA 76.5%, HVC_Av 94.1%; Figure 3B).

We found oEPSC and oIPSC amplitudes to be comparable between HVC_X and HVC_Av PNs (Figure 3C and D, Figure 3—figure supplement 1). However, HVC_RA neurons had lower amplitude currents than the other HVC-PN classes (Figure 3C and D, Figure 3—figure supplement 1). When we computed the amplitude of oEPSCs and oIPSCs for each cell to calculate the excitation/inhibition, we observed that optogenetic stimulation of NIf axonal terminals evoked relatively higher amplitude oIPSCs than oEPSCs in all HVC-PN classes (Figure 3E, Figure 3—figure supplement 1).

We next examined monosynaptic connectivity from NIf to each HVC-PN. We found that application of TTX followed by 4AP reliably returned oEPSCs in all three HVC projection subtypes (monosynaptic oEPSCs: HVC_X: 5/5, HVC_RA: 4/5, HVC_Av: 5/5, Figure 3F). This widespread monosynaptic connectivity is in stark contrast to thalamic excitatory inputs from Uva that preferentially target HVC_RA neurons. Similar to Uva projections, we did not observe monosynaptic oIPSCs (HVC_X: 0/5, HVC_RA: 0/2, HVC_Av: 0/3, data not shown), suggestive of a glutamatergic projection from NIf to HVC. We combined retrograde labeling from HVC with in situ hybridization labeling on brain slices. NIf neurons projecting to HVC (NIf_HVC) selectively expressed the glutamatergic marker SLC17A6, but not the GABAergic marker GAD1 (Colquitt et al., 2021; Figure 3G). This finding is consistent with previous studies indicating that NIf projections result in disynaptic inhibition onto HVC projection neurons (Coleman and Mooney, 2004).

Together, these results indicate that intratelencephalic DVR circuits are excitatory and that NIf provides excitatory glutamatergic monosynaptic projections onto all three known classes of HVC-PNs. Given that monosynaptic connectivity across regions of the DVR has not previously been examined, this suggests the possibility that, in contrast to the thalamo-DVR pathways, projections between regions of the DVR might not have cell type selectivity.

To better define the specificity of regional DVR projections, we next mapped synaptic connectivity of two other HVC afferents, mMAN and Av. The DVR is comprised of three large pallial fields separated by distinct lamina: the nidopallium, mesopallium, and the arcopallium. HVC, NIf, and mMAN reside in the nidopallium, and Av is in the mesopallium. HVC and NIf are in caudal regions of the DVR’s nidopallium, while mMAN is located at anterior portions of the DVR, some 4–5 mm from HVC. Av, on the other hand, is only ~1.5 mm from HVC but is in a distinct pallial field.

Cell-type-specific synaptic connectivity between nidopallial regions and between mesopallial and nidopallial regions is poorly understood; however, the connectivity of mMAN and Av is better described than other portions of these pallial fields. mMAN receives synaptic input from the thalamic nucleus DMP and is part of a vocal motor DVR-thalamo-DVR loop (mMAN >HVC > RA>DMP > mMAN), a circuit architecture similar to premotor and motor control-related cortico-thalamo-cortical loops in mammals. Although the synaptic connectivity between mMAN and HVC has not been well studied, lesions of mMAN in juvenile zebra finches result in song imitation deficits and lesions in adult Bengalese finches increase song syntax variability, suggesting a role in selection and planning of vocal motor sequencing (Foster and Bottjer, 2001; Koparkar et al., 2024). Av is one of the more recently described song-related regions, and unlike Uva, NIf, and mMAN, it is reciprocally connected to HVC. The role of Av is still poorly explored, but it is understood to be embedded in a higher order auditory portion of the DVR and thought to be an important node in the circuit comparing efferent copies of motor commands from HVC to auditory feedback (Bauer et al., 2008; Akutagawa and Konishi, 2010).

While evaluating connectivity between HVC, mMAN, and Av, we discovered that mMAN also provides a strong projection to Av (Figure 4). Approximately half of the identified projection neurons in mMAN project to Av, and our tracing experiments reveal that there are only partially overlapping classes of projection neurons in mMAN: mMAN_HVC PNs, mMAN_Av PNs, and mMAN_HVC/AV PNs. This newly identified pathway appears positioned to provide the auditory system with information about song motor commands via a DVR-thalamo-DVR loop (RA >DMP > mMAN>Av). It also opens the potential for synaptic loops relaying through HVC between these regions.

Brain regions nearby mMAN do not send projections to HVC, and we were able to achieve robust expression of eGtACR1 in mMAN (Figure 5A), allowing us to selectively examine the synaptic connectivity between mMAN and HVC-PNs. Optical stimulation of mMAN axon terminals in brain slices revealed glutamatergic oEPSCs in all retrogradely identified projection neuron classes, albeit with significantly lower success rate compared to what we observed when stimulating NIf terminals (48.15% probability; mMAN vs. NIf oEPSC probability: Fisher’s exact test p<0.001). In addition, we found that oEPSCs were more likely evoked in recordings from HVC_X and HVC_Av than from HVC_RA neurons (Figure 5B, Figure 5—figure supplement 1).

Optogenetic stimulation of mMAN axon terminals also revealed GABAergic currents in all three HVC-PN classes (48.24% probability). Similar to NIf and Uva inputs, oIPSCs elicited by mMAN axons were most often found in cells where oEPSCs were also observed (oIPSC contingent with oEPSC: HVC_X: 93.5%, HVC_RA: 96.4%, HVC_Av: 90.9%; Figure 5B).

While oEPSCs amplitudes across HVC-PN classes were statistically indistinguishable (Figure 5C), we found that oIPSCs in HVC_Av had significantly higher amplitude than those evoked in HVC_RA neurons (Figure 5D, Figure 5—figure supplement 1). Despite this difference, when computing the E/I ratio, we did not see any significant difference across HVC-PNs, and like the other inputs examined, GABAergic currents had higher amplitude than glutamatergic currents (Figure 5E, Figure 5—figure supplement 1).

We next asked whether mMAN monosynaptically contacted any class of HVC-PNs. We found evidence that mMAN monosynaptically contacts all three classes of HVC PNs. The most reliable monosynaptic transmission is between mMAN and HVC_Av neurons and HVC_X neurons (HVC_RA: 2/5, HVC_X: 5/7, HVC_Av: 6/6, Figure 5F). We did not observe monosynaptic oIPSCs (HVC_X: 0/5, HVC_RA, 0/5, HVC_Av: 0/6, data not shown), and confirmed that mMAN neurons projecting to HVC are glutamatergic using in-situ hybridization combined with retrograde tracing from injection of tracer in HVC (Figure 5—figure supplement 1). Together, this data indicates that mMAN predominantly provides robust monosynaptic excitatory input to HVC_Av and HVC_X neurons. HVC_Av neurons only comprise ~3% of the neurons in HVC (Roberts et al., 2017). Therefore, finding that 100% of the HVC_Av neurons tested receive monosynaptic input from mMAN, suggesting that this is likely to be a highly selective synaptic target of mMAN inputs to HVC. Given that we have now also identified a projection from mMAN directly to Av, this supports a model in which mMAN is providing Av song-related signals directly and indirectly via projections onto HVC_Av neurons.

Lastly, we mapped the synaptic inputs from Av onto HVC-PNs. Avalanche lacks clear anatomical boundaries, and our viral expression did spread into surrounding brain regions, but only brain hemispheres in which we could verify a lack of expression in NIf were included in this study (Figure 6A). Whole-cell patch-clamp recordings from identified HVC-PN classes in brain slices revealed glutamatergic oEPSCs in all three cell classes, yet most of the cells were not excited by Av terminals (39.18% probability; Av vs. NIf oEPSC probability: Fisher’s exact test p<0.001). However, the probability of evoking oEPSCs was similar among HVC-PN classes (Figure 6B, Figure 6—figure supplement 1). We tested the presence of oIPSCs in a subset of cells and found GABAergic currents in all three projection subtypes (43.24% probability, Figure 6—figure supplement 1). As in other pathways already described, we observed oIPSCs mainly in HVC-PNs also showing oEPSCs (oIPSC contingent with oEPSC: HVC_X 100.0% HVC_RA 90.0%, HVC_Av 84.6%; Figure 6B).

While oEPSCs amplitudes were broadly similar among HVC-PN classes (Figure 6C), oIPSCs had significantly smaller amplitude in HVC_RA compared to those evoked in HVC_X neurons (Figure 6D, Figure 6—figure supplement 1). Consistently, this was also reflected in a significantly higher E/I ratio in HVC_RA compared to HVC_X. Similar to the other afferents, Av axon terminal stimulation elicited stronger GABAergic currents than glutamatergic currents across HVC-PN classes (Figure 6E, Figure 6—figure supplement 1).

When examining which HVC-PN neuron classes received monosynaptic input from Av, we consistently observed monosynaptic transmission onto HVC_X neurons (HVC_X 4/5 cells monosynaptically contacted). Monosynaptic transmission onto HVC_RA and HVC_Av was less common (HVC_RA 2/5, HVC_Av 2/6; Figure 6F). Previous reports indicated that Av neurons projecting to HVC are excitatory (Roberts et al., 2017). Consistent with this, we didn’t observe any monosynaptic oIPSC across HVC-PNs (monosynaptic oIPSCs: HVC_X 0/4, HVC_RA 0/3, HVC_Av 0/3, data not shown) and further confirmed that Av_HVC neurons are glutamatergic by in situ hybridization labeling of retrogradely identified neurons (Figure 6—figure supplement 1). Our data shows that Av provides monosynaptic excitatory input to HVC_X neurons with high frequency and makes monosynaptic connections with HVC_RA and HVC_Av neurons less frequently.

Taken together, this research provides a rich dataset mapping the polysynaptic and monosynaptic connectivity of the input and output pathways of HVC – the best studied portions of the avian song circuitry – as well as a newly discovered pathway between DVR circuits that are important for vocal learning (Figure 7).

Experiments described in this study were conducted using adult male zebra finches (130–500 days post hatch). All procedures were performed in accordance with protocol 2016–101562 G approved by the Animal Care and Use Committee at UT Southwestern Medical Center.

The following adeno-associated viral vectors were used in these experiments: rAAV2/9/CBh-FLP and rAAV-CBh-eGtACR1-mScarlet (IDDRC Neuroconnectivity Core at Baylor College of Medicine or UT Southwestern AAV Viral Vector Core). All viral vectors were aliquoted and stored at –80 °C until use.

CBh-eGtACR1-mScarlet was generated by inserting KpnI and AgeI restriction sites flanking EF1a in the pAAV-EF1a-F-FLEX-mNaChBac-T2A-tdTomato (a gift from Dr. Massimo Scanziani; Addgene plasmid # 60658) through PCR cloning. The CBh promoter was isolated from pX330-U6-Chimeric_BB-CBh-hSpCas9 (a gift from Dr. Feng Zhang; Addgene plasmid # 42230) by digestion of the KpnI and AgeI restriction sites and inserted into the KpnI/AgeI restriction sites of the pAAV-EF1a-F-FLEX-mNaChBac-T2A-tdTomato backbone. Next, we isolated mScarlet from pmScarlet_C1 (a gift from Dr. Dorus Gadella; Addgene plasmid # 85042) through PCR amplification, inserting a BamHI restriction site into the 5’ end of the ORF. We digested the amplified fragments with BamHI and BsrGI, inserting the digested fragments into BamHI/BsrGI restriction sites of the pAAV-CBh-F-FLEX-mNaChBac-T2A-tdTomato backbone. eGtACR1 was then amplified from pAAV-CAG-DIO-NLS-mRuby3-IRES-eGtACR1-ST (a gift from Dr. Hillel Adesnik; Addgene plasmid # 109048) with a BmtI restriction site on the 5’ end and an AflII restriction site on the 3’ end, avoiding amplification of the soma-targeting sequence in the original plasmid. We then digested the amplified fragment with BmtI and AflII, inserting the fragment into the BmtI/AflII sites of the pAAV-CBh-F-FLEX-mNaChBac-T2A-mScarlet.

For CBh- Flpo, we amplified the FLP ORF from pCAG- Flpo (a gift from Dr. Massimo Scanziani; Addgene plasmid # 60662) with an AgeI restriction site on the 5’ end. We then digested the amplified fragment with AgeI and EcoRI, inserting the fragment into the AgeI/EcoRI sites of the CBh-eGtACR1-mScarlet.

Constructs were verified with Sanger sequencing.

All surgical procedures were performed under aseptic conditions. Birds were anaesthetized using isoflurane inhalation (0.8–1.5%) and placed in a stereotaxic surgical apparatus. The centers of HVC, NIf, mMAN, and RA were identified with electrophysiological recordings, and Area X, Av, and Uva were identified using stereotaxic coordinates (approximate stereotaxic coordinates relative to interaural zero and the brain surface: head angle, rostral-caudal, medial-lateral, dorsal-ventral (in mm). HVC: 45°, AP 0, ML ±2.4, DV –0.2–0.6; NIf: 45°, AP 1.75, ML ±1.75, DV –2.4–1.8; mMAN: 20°, AP 5.1, ML ±0.6, DV –2.1–1.6; RA: 70°, AP –1.5, ML ±2.5, DV –2.4–1.8; X: 45°, AP 4.6, ML ±1.6, DV –3.3–2.7; Av: 45°, AP 1.65, ML ±2.0, DV –0.9; UVA: 20°, AP 2.8, ML ±1.6, DV –4.8–4.2).

Viral injections were performed using previously described procedures (Roberts et al., 2017; Roberts et al., 2012). Briefly, a cocktail of adeno-associated viral vectors (1:2 of rAAV-CBh-FLP and rAAV-DIO-CBh-eGtACR1, respectively) was injected into NIf, Uva, mMAN, or Av (1 nl/s, for a total of 1.5 µl/hemisphere). A minimum of 4 weeks after viral injections, fluorophore-conjugated retrograde tracers (Dextran 10,000 MW, AlexaFluor 488 and 568, Invitrogen; FastBlue, Polysciences) were injected bilaterally into Area X, Av, and RA. Tracer injections (160 nl, 5x32 nl, 32 nl/s every 30 s) were performed using previously described procedures (Roberts et al., 2017; Roberts et al., 2012; Xiao et al., 2018).

To test the functional expression of eGtACR1 in HVC afferent pathways, we performed extracellular recording of HVC activity in anesthetized birds previously injected with rAAV-DIO-CBh-eGtACR1 and rAAV-CBh-FLP (2:1). We performed the recordings under light isoflurane anesthesia (0.8%) with Carbostar carbon electrodes (impedance: 1670 microhms/cm; Kation Scientific). During the recordings, we lowered a 400 µm multimodal optical fiber to the brain surface overlaying HVC and delivered light stimulation (470 nm, ≈ 20 mW, 1 s). Signals were acquired at 10 KHz and filtered (high-pass 300 Hz, low-pass 20 KHz). We used Spike2 to analyze the spike rate (binned every 10ms) and build a peri-stimulus time histogram to evaluate the effect of light stimulation across trials (5–15 trials/hemisphere). We sampled a minimum of one site and a maximum of three HVC sites/hemisphere. Birds with weak or no optically evoked responses were excluded from further experiments.

After electrophysiological recordings, the slices were incubated in 4% PFA in PBS. Sections were then washed in PBS, mounted on glass slides with Fluoromount-G (eBioscience, CA, USA), and visualized under an LSM 880 laser-scanning confocal microscope (Carl Zeiss, Germany).

We used the hairpin chain reaction system from Molecular Instruments as previously described (Ben-Tov et al., 2023). Briefly, 5 days after having injected HVC with the retrograde tracer Fast Blue (150 nl/HVC, Polysciences), the brain was perfused with 4% paraformaldehyde (PFA), post-fixed at 4 °C for 12 hr, and dehydrated in 30% sucrose/4% PFA at 4 °C for 24 hr. The dehydrated brains were sagittally cryosectioned at 40 μm and collected into ice-cold 4% PFA. Sections were then incubated twice in PBS for 3 min, in 5% SDS/PBS for 45 min, three times in 2 X SSCT for 15 min, in Hybridization Buffer for 5 min, and finally in 10 nM each probe/Hybridization Buffer at 37 °C for 24 hr. The sections were then washed four times in Probe Wash Buffer for 15 min at 37 °C, three times in 2 X SSCT for 15 min at room temperature, then incubated in Amplification Buffer for 30 min at room temperature. Each Alexa fluor-conjugated hairpin was denatured in separated PCR tubes at 95 °C for 90 s and cooled down to room temperature at 2 °C/s. Sections were incubated in solutions containing 36 nM of each hairpin in Amplification Buffer at room temperature for 24 hr. The sections were then washed four times with 2 X SSCT for 15 min and mounted with mounting medium.

Electrophysiological data were analyzed with Clampfit (Molecular Devices). All data were tested for normality using the Shapiro-Wilk Test. Parametric and non-parametric statistical tests were used as appropriate. One-way ANOVA or the Kruskal-Wallis test was performed when comparisons were made across more than two conditions. Two-Way ANOVA or Mixed-effect analysis was used to compare multiple conditions across different groups. Fisher’s exact tests were used to compare the probability of finding optically evoked responses. Statistical significance refers to *p<0.05, ** p<0.01, *** p<0.001.

The Sankey diagram in Figure 7C was drawn with SankeyMATIC (https://www.sankeymatic.com/) setting ribbons proportional to the percentage of interrogated synaptic connections between each afferent area and HVCPN class displaying excitatory currents or lack thereof. From the hubs reporting existing synaptic connections (center), ribbons are scaled based on the percentage of monosynaptic and polysynaptic connectivity for each combination.