Cortical Betz cells analogue in songbirds utilizes Kv3.1 to generate ultranarrow spikes

Complex motor skills in vertebrates require specialized upper motor neurons with precise action potential (AP) firing. To examine how diverse populations of upper motor neurons subserve distinct functions and the specific repertoire of ion channels involved, we conducted a thorough study of the excitability of upper motor neurons controlling somatic motor function in the zebra finch. We found that robustus arcopallialis projection neurons (RAPNs), key command neurons for song production, exhibit ultranarrow spikes and higher firing rates compared to neurons controlling non-vocal somatic motor functions (AId neurons). This striking difference was primarily due to the expression of a high threshold, fast-activating voltage-gated K+ channel, Kv3.1 (KCNC1). RAPN properties thus mirror those of the sparse, specialized Betz cells in the motor cortex of humans and other primates, which also fire ultranarrow spikes enabled by Kv3.1 expression. These large layer 5 pyramidal neurons are involved in fine digit control and are notably absent in rodents. Our study thus provides evidence that songbird RAPNs and primate Betz cells have convergently evolved the use of Kv3.1 to ensure precise, rapid AP firing required for fast and complex motor skills.

dendrites are cut off by the slicing procedure, the results suggest that AId neurons may receive 153 more numerous, perhaps diverse, synaptic inputs that are differentially filtered compared to 154 RAPNs. Finally, we calculated the surface to volume (S/V) ratio. For RAPNs we find 1.5-2.3 µm -1 155 and for AId neurons 2.5-2.9 µm -1 (Supplementary Table 1). These values are similar to those 156 found using EM 3-D reconstructions in mouse cortical neurons (1.63 µm -1 ) but lower than found 157 in astrocytes (4.4 µm -1 (Cali et al., 2019)), which may have metabolic energy consequences for 158 RA nuclei, which stain heavily for metabolic markers (Adret & Margoliash, 2002).  Table 1). However, AId neurons had 184 an AP half-width that was twice as broad as the RAPN APs ( Fig. 2C-D and 2F-G; Table 1). The 185 shorter half-width of RAPNs could be due to either a faster AP depolarization and/or repolarization 186 rate. To determine which phase of the AP was responsible for this difference, we derived phase 187 plane plots from averaged spontaneous APs and compared the maximum rates of depolarization 188 and repolarization. At room temperature, the maximum rate of repolarization was 76% larger in 189 RAPNs compared to AId neurons ( Fig. 2E; Table 1), whereas the maximum rate of depolarization 190 was only 29% larger in RAPNs (Table 1). At 40 o C the difference in the maximum depolarization 191 rate disappeared, while the maximum repolarization rate was 37% greater in RAPNs ( Fig. 2H; 192 Table 1). These results suggest that the relatively slower AP repolarization is predominantly 193 responsible for the broader AP half-width of AId neurons compared to the ultranarrow RAPN APs.

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We next examined the evoked firing properties by delivering increasing, step-wise 1 sec current 196 injections to RAPNs and AId neurons ( Fig. 3A & C, examples at +500 pA). As expected, a higher 197 firing rate was observed in both brain regions at physiological temperature compared to room 198 temperature. In the absence of injected current, both RAPNs and AId neurons fired spontaneous 199 APs (spontaneous APs in RAPNs also observed in extracellular slice recordings; (Wood, Lovell,   channel inhibitors tetraethylammonium (TEA) and 4-aminopyridine (4-AP) (Rettig et al., 1992). If 234 the differences in AP half-width and maximum repolarization rate between RAPNs and AId 235 neurons are due to the expression of Kv3.1, we would expect a more significant AP broadening 236 upon exposure to either of these compounds in RAPNs than in AId neurons.

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To test this prediction, we recorded spontaneous APs from RAPNs and AId neurons in frontal 239 slices before and after exposure to 500 µM TEA (Fig. 4A-B) and 100 µM 4-AP ( Fig. 4E-F   Evoked AP firing was also more affected by TEA and 4-AP in RAPNs than in AId neurons. Upon 256 exposure to TEA, RAPNs showed significant decreases in the evoked firing rate ( Fig. 5A; 35 ± 257 4.1% decrease in spikes per sec, 29.3 ± 10.4% decrease in instantaneous firing and 33.7 ± 3.8% 258 decrease in steady-state firing frequency upon the +500 pA current injection), whereas trends, 259 but no significant effects, were seen in AId (Fig. 5B). Upon exposure to 4-AP, RAPNs also showed 260 significant decreases in the evoked firing rate ( Fig. 5C; 36.8 ± 10.9% decrease in spikes per sec, 261 63.7 ± 4.8% decrease in instantaneous firing frequency and 36.5 ± 12.9% decrease in steady-262 state firing frequency upon the +500 pA current injection), whereas trends, but no significant 263 effects, were seen in AId (Fig. 5D).

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Our results with TEA and 4-AP alone, however, could not rule out the possible contributions of 266 other K + channels that are also hypersensitive to either TEA (Kv1, Kv7, and large-conductance

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To confirm that the regional differences in our current clamp recordings were indeed due to 280 differences in the outward voltage gated K + current (IK+), we performed voltage clamp recordings, 281 under conditions that minimize space clamping issues (details in Methods, (Zemel et al., 2021)).

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After pharmacologically isolating IK+, we delivered sequential 200 ms test pulses to -30 mV and 0 283 mV, from a 5 sec holding potential at -80 mV, to preferentially activate low threshold IK+ or both

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Informed by our current clamp pharmacology experiments, we next tested the prediction that IK+ 298 would be more attenuated by sub-millimolar concentrations of TEA in RAPNs than in AId neurons.

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Upon washing in 500 µM TEA onto slices we saw decreases in the peak current from neurons in 300 both brain regions (Fig. 6D), with RAPNs showing a larger fold-change in both the peak of the A-  and delayed rectifier components (Fig. 6G). By dividing the current 8 ms after the initial A-type 307 peak by the peak A-type current we found that the degree of inactivation was indistinguishable 308 between RAPNs and AId neurons ( Supplementary Fig. 6). Notably, the time to peak of the A-type 309 component was preserved (Mean ± SE: 4.5 ± 0.4 ms vs. Mean ± SE: 5.8 ± 0.4 ms for RAPNs and 310 AId neurons, respectively), with RAPNs maintaining a trend toward smaller values (Fig. 6H).

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Importantly, whether measuring the A-type current peak (

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The evidence presented thus far is consistent with the ultra-fast APs unique to RAPNs being

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The predicted zebra finch KCNC3 protein showed only moderate conservation with human 347 (68.32% residue identity), some domains including the BTB/POZ and transmembrane domain 348 being fairly conserved, but spans of residues on the N-terminal and C-terminal regions being 349 highly divergent. Notably, the N-terminal inactivation sequence (Rudy & McBain, 2001) of KCNC3 350 appears to be absent in the zebra finch ( Supplementary Fig. 9).

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We next performed in situ hybridization for all identified KCNC/Kv3.x family members in adjacent 353 frontal brain sections from adult male zebra finches. We replicated our previous finding that Kv3.1

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Interestingly, whereas AUT5 had no effects on the instantaneous firing frequency in RAPNs, the 406 steady-state firing frequency (as measured for the last two spikes recorded during the 1 sec +500 407 pA current injection) increased substantially (Mean ± SE: 24.3 ± 8.2% increase). Taken together, 408 these results are again consistent with the finding of higher Kv3.1 expression in RAPNs than in 409 AId, and further support the role for Kv3.1 in the specialized fast firing properties of RAPNs.

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Our results demonstrate that RAPNs and AId neurons exhibit some common properties, including

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Calculations of membrane capacitance (Cm) allowed us to estimate the average surface area of 455 RAPNs and AId neurons (Table 1), whereas 3-D reconstructions using ShuTu allowed us to also 456 estimate shrinkage-corrected surface areas (Supplementary Table 1

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Based on their larger overall spine surface area, it appears that AId neurons are investing more 459 "resources" on spines than RAPNs, despite having proportionately smaller individual spines. We 460 suggest that the sparse population of large spines in adult male RAPNs may limit filtering of 461 synaptic inputs compared to AId neurons, a factor that could at least partly contribute to the highly

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We also note striking differences between the properties of finch RAPNs and AId neurons

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Notably, however, these antagonists have known effects on other members of this ion channel 502 family, even at sub-millimolar concentrations (Kaczmarek & Zhang, 2017). In situ hybridization 503 showed higher expression of the Kv3.1 subunit in RAPNs than in AId neurons (Fig. 7), suggesting

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We also noted a modest depolarization of the AP peak, after-hyperpolarization and threshold with 513 AUT5 exposure. Considering this combination of changes was not observed in AId, this may be 514 an additional result of increases in Nav channel availability due to positive modulation of Kv3.1.

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The fact that the AUT5 effects on Kv3.1-expressing zebra finch neurons were similar to those 516 seen in mammals (Boddum et al., 2017) is not surprising, as the predicted peptide sequence of 517 finch Kv3.1 is ~96.5% identical to that of the human Kv3.1b splice variant (Supplementary Fig. 8).

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Songbirds and mammals diverged 300 million years ago, so this remarkable conservation 519 suggests that Kv3.1 channels may be optimized for enabling ultranarrow AP waveforms and high

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The combination of Navβ4, and its associated INaR, and Kv3 channels likely promote ultranarrow

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The AP waveforms in RAPNs and AId neurons are similar in several parameters, including 584 threshold, maximum depolarization rate, amplitude, peak and after-hyperpolarization (Table 1).

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These cells additionally have a multitude of common molecular correlates of excitability (e.g. high

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In conclusion, RAPNs in zebra finches exhibit many fundamental molecular and functional 601 similarities to primate Betz cells, that may be involved is fine digit movements (Tomasevic et al.,

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Patch clamp electrophysiology: RA and AId could be readily visualized in via infra-red differential 669 interference contrast microscopy (IR-DIC) (Fig. 2B). Whole-cell patch-clamp recordings were

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Recordings in which the resting membrane potential deviated by > 10 mV were discarded. We 699 note that recordings were not corrected for a calculated liquid junction potential of +9 mV.

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Estimated current clamp measurements of membrane capacitance (Cm) were calculated from the

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For voltage clamp recordings we attempted to limit the speed and space clamp error by 1) Cutting 708 thinner slices (~150 µm) to eliminate more processes, 2) decreased the intracellular K + 709 concentration to decrease the driving force and 3) compensated the series resistance to 1MΩ.  Fig. 2; (Zemel et al., 2021)). In order to isolate K + currents, slices 716 were exposed to bath applied CdCl2 (100 μM), TTX (1 μM), Picrotoxin (100 μM), CNQX (10 μM), 717 and APV (100 μM) for ~5 min prior to running voltage clamp protocols. After protocols were 718 applied, TEA (500 μM) was bath applied and the same voltage-clamp protocols were repeated 719 after K + currents were eliminated. K + currents were isolated by subtracting the TEA-insensitive 720 current traces from the initial traces. Capacitive currents generated during voltage-clamp   Fig. 1C). We excluded from the density estimation of 764 Supplementary Fig. 1C segments branching off the soma with fewer than 10 spines and any 765 segment shorter than 20 microns. We estimated the total dendritic area and length of each cell 766

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We estimated the surface area of the soma of each neuron by triangulating its 3-D structure. We

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We estimated the volumes of the dendrites using 3-D binary masks which we constructed from

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We estimated the volumes of the somas using an adaptation of our method for the surface areas.

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AUT5 was provided as a gift from Autifony Therapeutics (Stevenage, United Kingdom).

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Statistical data analysis and curve fitting: Data were analyzed off-line using IgorPro software 818 (Wave-metrics). Statistical analyses were performed using Prism 4.0 (GraphPad). Specific 819 statistical tests and outcomes for each analysis performed are indicated in the respective Figure   820 Legends and Tables

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Abbreviations-AP #: AP spikes produced per second, AP thresh.: AP threshold, AP amp.: AP 1063 amplitude as measured from the peak of the after-hyperpolarization to the AP peak, Max. depol.