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Title: Genetically-identified cell types in avian pallium mirror core principles of excitatory 1
and inhibitory neurons in mammalian cortex 2
3
Authors: Jeremy A. Spool1, Matheus Macedo-Lima1,2, Garrett Scarpa1, Yuichi Morohashi3, 4
Yoko Yazaki-Sugiyama3, and Luke Remage-Healey1
5
6
Affiliations: 7
1Neuroscience and Behavior, Center for Neuroendocrine Studies, University of Massachusetts, 8
Amherst, MA 01003, USA. 9
2CAPES Foundation, Ministry of Education of Brazil, 70040-020, Brazil DF. 10
3Okinawa Institute of Science and Technology (OIST) Graduate University, Okinawa, Japan. 11
12
Corresponding author: Luke Remage-Healey; healey@cns.umass.edu 13
14
15
Abstract: In vertebrates, advanced cognitive abilities are associated with a highly developed 16
telencephalic pallium. In mammals, the six-layered neocortex of the pallium is composed of 17
excitatory neurons and inhibitory interneurons, organized across layers into microcircuits. These 18
organizational principles are proposed to support efficient, high-level information processing. 19
Comparative perspectives across vertebrates provide a lens to understand what common 20
features of pallium are important for complex cognition. For non-mammalian vertebrates that 21
exhibit complex cognitive abilities, such as birds, the physiology of identified pallial cell types 22
and their circuit organization are largely unresolved. Using viral tools to target excitatory vs. 23
inhibitory neurons in the zebra finch auditory association pallium, we systematically tested 24
predictions derived from mammalian neocortex. We identify two segregated neuronal 25
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populations that exhibit profound physiological and computational similarities with mammalian 26
excitatory and inhibitory neocortical cells. Specifically, despite dissimilarities in gross 27
architecture, avian association pallium exhibits neocortex-typical coding principles, and 28
inhibitory-dependent cortical synchrony, gamma oscillations, and local suppression. Our 29
findings suggest parallel evolution of physiological and network roles for pallial cell types in 30
amniotes with substantially divergent pallial organization. 31
32
33
34
Main Text: The vertebrate pallium is considered essential for advanced cognitive abilities. 35
Unlike other sectors of the vertebrate brain, the cytoarchitecture and elaboration of pallial 36
domains vary immensely across vertebrate classes, and even within closely related taxa1. In 37
mammals, for example, dorsal pallium develops into a six-layered structure called neocortex, 38
the elaboration of which is associated with advanced cognitive abilities2,3. In cartilaginous fishes, 39
some taxa have a layered pallium, while in other species the organization of pallium is not at all 40
obvious, and difficult to separate from subpallial structures1. Large swaths of bird pallium are 41
unlayered4,5 (Fig. 1A; though see 6–9 for evidence of lamination in auditory pallium and 42
hyperpallium), yet several species of birds, including parrots and corvids, exhibit cognitive 43
abilities on par or exceeding capabilities of our closest primate relatives10. Deep characterization 44
of the molecular properties and developmental origins of pallial cells has advanced our 45
understanding of pallial evolution (e.g.11–18). However, a key piece missing is an examination of 46
the physiology of pallial cell types in non-mammalian species. In this study, we begin to address 47
this gap by interrogating the physiological and network phenotypes of two cell types in avian 48
auditory association pallium. 49
While there remains a lack of consensus over how the majority of the avian pallium is 50
related to mammalian pallium, there are unquestionable similarities in pallial circuit organization 51
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and function between the two taxa. The adult function and connectivity of the avian pallium 52
appears highly similar to mammalian neocortex, including thalamic input to primary pallial 53
structures, high interconnectivity of pallial regions, and output to subpallial brain structures19–23. 54
Brain regions in the avian pallium perform functions of similar cognitive complexity to functions 55
ascribed to neocortex, including but not limited to encoding memory of complex, ecologically-56
relevant sensory stimuli, vocal learning, encoding numerical information, cross modal 57
associative learning, and decision-making based on abstract rules24–33. Brain molecular 58
expression analyses have identified striking similarities between birds and mammals at the level 59
of pallial cell types12,17,34. The physiology of pallial cell types in birds is unresolved. Currently, the 60
identification of cell types strongly resembling mammalian cortical cell-types in avian pallium is 61
putative. For example, extracellular recordings in avian pallium consistently identify broad-62
spiking neurons with sparse firing rates that have selective receptive fields for ecologically-63
relevant stimuli, and narrow-spiking neurons with higher firing rates that are less selective 64
among stimuli7,28,35–42. Such similar circuit organization and function could reflect homologous 65
structures and cell types in birds and mammals, convergent evolution of cell types originating 66
from nearby pallial compartments, or parallel evolution of circuit features from basal cell types 67
that existed in the last common ancestor of amniotes. 68
A definitive mapping of the physiology of pallial cells onto their molecular identity in birds 69
is critical for understanding the extent to which synaptic and computational properties track with 70
molecular phenotype compared to mammalian neocortical circuits. To date, physiological 71
dissection of non-mammalian pallium has been limited by a lack of tools to precisely identify and 72
control specific cell types. Here, we deployed viral tools in birds to dissect circuits in a 73
secondary auditory associative region of pallium, the caudomedial nidopallium (NCM; Fig. 1A). 74
NCM is highly interconnected, is involved in auditory memory and individual recognition, and the 75
auditory physiology of neurons in this region is well-described28,35–37,39,40. We identified two 76
classes of neurons with shared physiological features to neocortical excitatory neurons vs. 77
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inhibitory interneurons in mammals. Specifically, despite dissimilarities in anatomical 78
organization, this study reveals shared intrinsic physiological properties, auditory coding roles, 79
and network organization in which inhibition drives local suppression and synchronizes large-80
scale gamma oscillations. The extent of these similarities in birds and mammals may provide 81
insight into what pallial circuit features support complex cognition in amniotes. 82
83
Results 84
We established methods for viral optogenetics in the NCM of zebra finches (Taeniopygia 85
guttata) to test predictions derived from mammalian neocortical excitatory neurons vs. inhibitory 86
interneurons. In mammalian cortex, markers like calmodulin-dependent kinase (CaMKII ) 87
identify excitatory neurons, while the GABA-producing enzyme glutamate decarboxylase 1 88
(GAD1) and the homeobox-containing mDlx gene identify inhibitory neurons43–45. We first 89
injected zebra finch NCM with adeno-associated viruses driving opsin proteins under the control 90
of the CaMKII vs. GAD1 promoters, and observed clear segregation of transfected cells (Fig. 91
1B). The ratio of CaMKII vs. GAD1 abundance was 83:16, echoing the distribution of excitatory 92
and inhibitory cell types in mammalian neocortex (GABAergic neurons typically comprise ~10-93
20% of cortical neurons, e.g.46). Qualitatively, we observed that CaMKII cells were 94
morphologically variable with respect to dendritic spine density and thickness of dendritic 95
branches, while GAD1 cells were more often aspiny, with thinner processes (fig. S1). CaMKII 96
cells and GAD1 cells did not differ in soma size (Welch’s t29.4 = 0.66, P = 0.51; Fig. 1C). 97
Conventional antibody staining confirmed selective transduction of cell-type targets (Fig. 1D,E). 98
Thus, promoter-driven molecular cell identity segregates cell types in avian association pallium 99
as it does in mammalian neocortex. 100
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101
Fig. 1. Viruses targeting CaMKII and GAD1 promoters segregate cell types in avian 102 auditory association pallium. (A) Sagittal schematic of avian pallium (top left) showing 103 primary auditory cortex (Field L), and auditory association pallium (caudomedial mesopallium, 104 CMM; caudomedial nidopallium, NCM). In the schematic, left is rostral, right is caudal, top is 105 dorsal, and bottom is ventral. 20x confocal image (top right) shows non-laminar clustered 106 cytoarchitecture of NCM. Bottom: magnified from white box in top right (blue=DAPI; 107 white=NeuN). (B) Non-overlapping cell types identified by viral expression of fluorophores (left), 108 and 60x images of transduced CaMKII (green) and GAD1 (magenta) cells. (C) Cell soma area 109 at largest cross-sectional diameter. (D) Typical widefield view of viral injection site in avian 110 NCM. Shown is mDlx-GFP expression (top), parvalbumin immunolabeling (PV; middle), and 111 overlaid images (bottom) showing specificity (>66% viral cells co-labeled) and efficiency (>88% 112 PV cells co-labeled) of transduction. (E) 60x images of viral and antibody co-expression. Top: 113 CaMKII viral expression (green) colocalizing with CaMKII immunolabeling (orange). Bottom: 114 GAD1 viral expression (magenta) co-localizing with GABA (gold) and PV (white). White 115 arrowheads = co-localization. All scale bars = 50 microns. 116 117
We next examined the physiology of identified cell types in zebra finch NCM. 118
Mammalian excitatory neocortical neurons typically have phasic, accommodating responses to 119
depolarizing current injections and broad spike widths, whereas neocortical interneurons have 120
tonic responses and narrow spike widths47,48. In NCM whole-cell slice recordings (Fig. 2A), 121
depolarizing current steps elicited phasic firing profiles from CaMKII cells vs. tonic firing 122
profiles from GAD1 cells (Fig. 2B,C). Similar to phasic excitatory neurons in mammalian barrel 123
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and other cortices49–51, CaMKII cells responded with maximum 1-2 spikes (ISIs 48.8 +/- 26.6 124
ms; range 20-114 ms), in contrast to the tonically-responsive, non-accomodating profile of 125
GAD1 cells (ISIs 48.2 +/- 11.4 ms; range 35-70 ms) that exhibit very low adaptation ratios52: 126
1.105 +/- 0.02. CaMKII cells also had broader action potential widths compared to GAD1 cells 127
(Mann-Whitney test: W = 162, P = 0.005; Fig. 2D), but did not differ in other passive membrane 128
properties (input resistance: Welch’s t20.8 = -1.4728, P = 0.1558; rheobase: t23.8 = 1.3593, P = 129
0.1868; Fig. 2E,F). Next, we conducted in vivo optrode recordings to isolate properties of 130
photoidentified CaMKII vs. GAD1 single units (Fig. 2G). Spike widths were broader in CaMKII 131
units than GAD1 units (action potential width (peak-to-peak): W = 318, P = 2.7e-05; Fig. 2H; 132
width of action potential at quarter height (i.e., spike quarter-width): W = 342, P = 9.9e-07; Fig. 133
2I; no difference in non-light evoked units; fig. S2). Light-evoked spike latencies were longer for 134
CaMKII compared to GAD1 single units (W = 273.5, P = 0.004; Fig. 2J), consistent with their 135
slower spike onset kinetics (fig. S3) and greater degree of network suppression. The sharp 136
physiological distinctions between CaMKII and GAD1 neurons in NCM therefore mirror those 137
of mammalian neocortical excitatory neurons and inhibitory interneurons, respectively. 138
139
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140
Fig. 2. CaMKII and GAD1 single units in NCM have distinct physiological properties. (A) 141 Transduced cells in whole-cell current clamp configuration (left) and reliable photopotentials to 142
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25 ms blue light pulses (top row = CaMKII cell; bottom row = GAD1 cell). (B) CaMKII cells 143 exhibit phasic responses (top) while GAD1 cells exhibit tonic responses (bottom) to current 144 steps. (C) Mean + SEM action potentials for CaMKII cells (green; n = 18) and GAD1 cells 145 (magenta; n = 11) in response to current steps. (D) Spike width, (E) Input resistance, and (F) 146 Rheobase of CaMKII cells vs. GAD1 cells in whole-cell recordings. (G) Raster plots and 147 histograms of exemplar in vivo transduced single units in electrophysiological response to blue 148 light pulses. (H) Action potential widths, (I) spike quarter-widths, and (J) latency to light-evoked 149 response peak in optically-identified single units in vivo. *P < 0.05 for Mann-Whitney U tests. 150 151
Auditory coding roles are distinct between cell types in mammalian neocortex, where 152
auditory stimuli elicit higher spiking activity from interneurons and quicker latencies compared to 153
sparse-firing excitatory cells53–55. We therefore tested whether in vivo responses of CaMKII vs. 154
GAD1 neurons segregated accordingly. Conspecific song drove GAD1 at higher rates 155
compared to CaMKII units (Fig. 3A,B; W = 106, P = 0.040). GAD1 single units had a quicker 156
response latency compared to CaMKII units (Fig. 3C; W = 207, P = 0.002). In rat auditory 157
cortex, inhibitory interneurons typically respond promiscuously to sensory stimuli, whereas 158
excitatory neurons in association layers have more selective representations55. Likewise in 159
associative auditory avian pallium, putative excitatory neurons are more stimulus-selective 160
compared to putative interneurons37. Single-unit spike trains fed to a custom pattern classifier 161
produced higher decoding accuracy values for GAD1 than for CaMKII units, indicating that 162
GAD1 units carried information about a variety of auditory stimuli (W = 84, P = 0.006825; Fig. 163
3D,E; fig. S4). By contrast, when examining stimulus selectivity (see Materials and Methods), 164
CaMKII single units tended to be more selective for a subset of conspecific song stimuli (W = 165
241, P = 0.057; Fig. 3D,F). These distinctions in auditory coding roles for CaMKII and GAD1 166
neurons precisely match the established sensory encoding roles of mammalian neocortical 167
excitatory neurons vs. inhibitory interneurons. 168
169
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170
Fig. 3. Auditory-response properties of CaMKII and GAD1 neurons. (A) Spectograms and 171 rasterplots of a CaMKII single unit (top) and a GAD1 single unit (bottom) in NCM responding to 172 conspecific song. (B) Song-evoked Z scores of optically-identified single units. (C) Latency in 173 seconds for optically-identified single units to respond to white noise stimuli. (D) Heat map of 174 single unit timing accuracy measures across auditory stimuli. Values closer to 1 represent 175 higher timing accuracy. Histogram insets show density distribution of accuracy metric across 176 stimuli for CaMKII (left) and GAD1 single units (right). (E) Pattern classifier timing accuracy 177 averaged across auditory stimuli for optically-identified single units displayed in D. (F) Measure 178 of transduced single unit selectivity for subsets of conspecific stimuli (see Materials and 179 Methods). *P < 0.05 and #P = 0.057 for Mann-Whitney U tests. 180 181
One well-described feature of mammalian neocortical microcircuits is feedforward 182
suppression, in which incoming excitation drives inhibitory interneurons followed by excitatory 183
neurons in tandem, yielding temporally-precise excitation quenched rapidly by inhibition53,56,57. 184
Consistent with this prediction, GAD1 neurons had faster auditory onset latency than CaMKII 185
neurons (Fig. 3C). Furthermore, anesthetized optrode recordings showed that GAD1-ChR2 186
optical stimulation drove short-latency suppression in ~25% of non-transduced single units, 187
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compared to 0% for CaMKII -ChR2 experiments (fig. S5). To test this further in awake animals, 188
we directed a 32-channel opto-microdrive at the NCM of mDlx-ChR2-transduced birds. At sites 189
with photo-identified inhibitory neurons, a separate population of cells were photo-suppressed 190
and quickly rebounded at light-pulse offset (Fig. 4A). We also identified units that rebounded at 191
light-pulse offset using a GAD1-ChR2 construct in an awake animal (fig. S6A). mDlx and GAD1 192
interneurons therefore suppress local synaptic targets in avian NCM, in a manner similar to 193
mammalian neocortex. 194
Inhibitory interneurons in mammalian neocortex also coordinate broad-scale spike 195
synchrony and oscillations in the gamma frequency band58,59. With mDlx-ChR2, optical 196
stimulation of NCM increased synchrony of single units with narrow waveforms (narrow single 197
units, W = 4184, P = 2e-05; broad single units, W = 146, P = 0.124; Fig. 4B). Optical stimulation 198
of mDlx cells in NCM also increased the amplitude of the gamma frequency band at 30-34 and 199
36-43 Hz (All P < 1e-05; Fig. 4C,D). Optodrive recordings with a GAD1-ChR2 construct showed 200
similar results (fig. S6B,C). Conversely, with a GAD1-archaerhodopsin construct that 201
hyperpolarized avian cells in vitro (fig. S7), we observed that optical suppression of GAD1-202
archaerhodopsin cells decreased synchrony of narrow but not broad cells (narrow: W = 5288.5, 203
P = 6e-04; broad: W = 206, P = 0.95; Fig. 4B) and attenuated the gamma band specifically at 204
39-41 Hz (All P < 0.001; Fig. 4C,D). Rapid shifts in NCM inhibitory network activation therefore 205
drove tuning of cortical network synchrony and gamma oscillations. 206
Finally, a balance of excitation and inhibition is thought to be critical for neocortical 207
function60. In rodent auditory neocortex, disrupting inhibition alters excitatory neuron auditory 208
responses, including firing rates, and stimulus selectivity61. To test whether this was the case in 209
zebra finch auditory pallium, we examined optodrive recordings using GAD1-archaerhodopsin in 210
NCM. Specifically, we recorded the responses of broad waveforms to conspecific songs with 211
and without green laser stimulation. Firing rates during stimulus presentations in single units 212
with broad waveforms were not different as a group between laser off and laser on conditions 213
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(Fig. 4E; Wilcoxon signed rank test, V = 55; p = 0.80; Chi-square test for increased/decreased 214
firing, X1 = 0.8, p = 0.371). However, all single units with broad waveforms had decreased 215
stimulus selectivity during laser on vs. laser off conditions (Fig. 4F; Wilcoxon signed rank test, V 216
= 15; p = 0.063; Chi-square test for increased/decreased selectivity, X1 = 5, p = 0.025). This 217
suggests that disruption of inhibition in NCM has functional consequences for coding of 218
ethologically-relevant auditory stimuli. 219
220
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221
Fig. 4. Inhibitory neurons in NCM drive suppression, synchrony, gamma oscillations, and 222 functional auditory response properties. (A) Instantaneous firing rates of light-evoked single 223 units (magenta; n = 5) and light-suppressed single units (orange; n = 5) from optical stimulation 224 (50 ms blue pulse) of mDlx-ChR2-transduced cells drawn from n = 36 total units isolated in 225 NCM in vivo. (B) Violin plots show change in cross-correlation of waveforms when NCM was 226 stimulated with blue light in mDlx-ChR2 experiments (top row) and when stimulated with green 227 light for GAD1-archaerhodopsin experiments (bottom). Red line denotes zero change. (C) Heat 228 map (% max LFP power) showing LFP change over time in an mDlX-ChR2 optodrive 229 experiment. Cartoon lasers above represent bins with blue light pulses. (D) LFP power spectra 230 before and during stimulation of NCM with blue light for mDlx-ChR2 experiments (top) and with 231
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green light for GAD1-archaerhodopsin experiments (bottom). Gray shading represents gamma 232 frequency range for which LFP power is significantly different from baseline according to 233 predictions from mammalian cortex (P < 0.05); $ is range for which LFP power is significantly 234 different from baseline against predicted direction (P < 0.05). (E) Single units with broad 235 waveforms are plotted with respect to their stimulus firing rates in hertz to various conspecific 236 songs when GAD1-archaerhodopsin was stimulated with green laser (y-axis) compared to when 237 there was no laser (x-axis). Each point represents a single unit’s response to one conspecific 238 song, and single units are grouped by a unique shape and color. (F) The same single units as E 239 are plotted with respect to their selectivity across all conspecific stimuli when green laser was on 240 (y-axis) compared to when green laser was off. For E & F, points deviating from the red line 241 denote a non-zero difference between laser off and laser on conditions. (G) Summary 242 schematic showing features of avian excitatory principal cell-like (EN) and inhibitory interneuron-243 like (IN) neurons in NCM in reference to predictions from mammalian cortex. Arrows imply 244 effects of neuron stimulation and do not imply nature of synaptic connectivity. 245 246
247
Discussion 248
Our findings demonstrate compelling computational and physiological similarities 249
between corresponding excitatory and inhibitory neuronal cell types of avian association pallium 250
and mammalian neocortex (Fig. 4G). Viruses designed to target excitatory neurons and 251
inhibitory interneurons in mammalian neocortex segregate similar populations in avian 252
association pallium with predicted intrinsic physiology, auditory coding roles, and network 253
organization that drives local suppression and synchronizes large-scale gamma oscillations, all 254
despite a non-laminar organization. 255
The mapping of physiological roles to molecular identity of excitatory and inhibitory 256
neurons in pallium is not a generalized feature of the vertebrate or even the mammalian brain. 257
For example, in contrast to pallium, GABAergic medium spiny neurons in the striatum express 258
high levels of CaMKII , and are the major source of efferent projections from the region62,63, and 259
both projection and interneurons in the striatum release GABA63,64. In the lateral hypothalamus, 260
parvalbumin-positive, glutamatergic neurons are fast-spiking and send long-range projections in 261
the brain65 Relative proportions of glutamatergic vs. GABAergic cell populations also vary 262
between brain regions, including but not limited to the amygdala, bed nucleus of the stria 263
terminalis, and ventral tegmental area, where glutamatergic neurons are the minority66–68. 264
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Neurons in some brain regions can also co-release GABA and glutamate69,70. Therefore, there 265
are many ways the brain builds functional circuits using all combinations of projection cells, 266
interneurons, excitatory and inhibitory neurotransmission, and various molecular machineries. 267
Our data demonstrate that CaMKII and GAD1 cell populations are distinct in avian pallium, and 268
that they mirror several physiological features of excitatory and inhibitory neocortical neurons. 269
These observations strongly suggest that 1) this set of shared features are core physiological 270
properties that are critical for pallial function, and 2) a shared ancestral origin of cell types and 271
circuit elements constrains the evolution of divergent physiological phenotypes between bird 272
and mammalian pallium. 273
In vitro, mammalian neocortical excitatory neurons can be distinguished from inhibitory 274
interneurons by the electrical phenotype of continuous accommodation to depolarizing current 275
injections71. In the present study in finches, CaMKII neurons exhibit very strong adaptation by 276
firing 1-2 phasic action potentials throughout the current step protocol, whereas GAD1 neurons 277
exhibit a non-accomodating electrical phenotype. In vivo physiological recordings showed that 278
CaMKII and GAD1 neurons can also be distinguished as broad-spiking and narrow-spiking, 279
respectively, as in mammalian neocortex. We extend previous findings in songbirds 280
demonstrating that auditory coding roles of broad-spiking and narrow-spiking neurons show 281
strong parallels with the role of excitatory neurons and inhibitory interneurons in mammalian 282
auditory cortex7,28,35–37,39,40, and attribute those properties to CaMKII vs. GAD1 neurons 283
specifically. Additionally, our findings suggest that in avian auditory pallium, GAD1 neurons may 284
tune stimulus selectivity of excitatory neurons, a functional outcome predicted directly from 285
recent data in rodent auditory cortex61. These data strongly imply that in birds as in mammals, 286
the molecular identity of excitatory vs. inhibitory neurons in pallium is tied to a fundamental set 287
of physiological and sensory coding properties. The relationship between molecular identities 288
and physiological roles exist irrespective of their anatomical organization, such as the denser, 289
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clustered organization of pallial neurons in birds compared to less dense, layered mammalian 290
neocortex72,73. 291
Our data also highlight similarities between CaMKII and GAD1 cells in birds and 292
subclasses of excitatory and inhibitory cell types in mammalian pallium. CaMKII neurons were 293
largely non-pyramidal in morphology, and previous work has not identified projections from 294
NCM that exit pallium (though see 74). NCM instead makes reciprocal connections with other 295
pallial domains, including the mesopallium, a large territory with neurons that recent 296
developmental and genetic data demonstrate to have similar molecular markers to mammalian 297
intra-telencephalic (IT) cells12,22. A majority of CaMKII cells in this study could conceivably 298
represent a class of conserved IT pallial cells75, though this requires further study. Our GAD1 299
population exhibits a fair degree of variance in auditory coding properties (Fig. 3), suggesting 300
we may be capturing multiple subclasses of inhibitory interneurons with this viral construct. 301
Previous work has demonstrated there is immunoreactivity in NCM and other pallial regions for 302
parvalbumin (PV) as well as calbindin, and that these cells invade pallium during development 303
in a tangential migration similar to mammals15,72,76,77. In the present study we observed that 304
GAD1-positive cells (and mDlx cells) co-localize with PV (Fig. 1D,E). The physiological roles of 305
GAD1 neurons in NCM that we observed, including a fast-spiking phenotype, faster response 306
latencies, broader selectivity for auditory stimuli, and control over gamma oscillations, are 307
consistent with the role of PV cells in mammalian neocortex53,78,79. Future studies matching 308
molecular identities to physiological properties will be necessary to determine the extent to 309
which subclasses of pallial cell types have diverged or retained intrinsic physiological properties 310
across amniotes. Additionally, future studies of projection patterns within and outside the 311
telencephalon, combined with dendritic and axonal morphology of CaMKII and GAD1 cell 312
types, will further clarify the distinctions and similarities between avian and mammalian pallial 313
cell types. 314
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At the network level, we find several similarities in birds compared to mammals in the 315
organization of cell types. Driving GAD1 and mDlx neurons induces local suppression and 316
synchronizes networks of narrow-spiking cells at the level of single units and the broad-scale 317
gamma oscillations in the surrounding pallium. Suppressing GAD1 neurons has the opposite 318
effect on pallial synchrony. Suppressing GAD1 neurons also altered stimulus selectivity of 319
broad-spiking single units, suggesting inhibitory control of downstream excitatory sensory 320
coding. However, contrary to prediction, suppression of GAD1 cells did not alter firing rate of 321
broad-spiking units. This suggests a species-level difference in the role of interneurons in 322
constraining firing rate, but more precise cell-type manipulations could reveal specific effects of 323
PV+ SOM+ or VIP+ neurons on firing rate that are obfuscated in the present study by 324
simultaneous activation of multiple interneuron types with opposing network roles 80–82. Pallial 325
circuits in birds may therefore exhibit further similarities to mammalian cortical microcircuits, 326
including feedforward suppression56,57, though our data do not preclude alternative circuit 327
mechanisms for local signal processing. 328
Our current findings highlight both similarities and differences that reveal core intrinsic 329
and network features of excitatory neurons and inhibitory neurons that either evolved 330
independently or are conserved features of pallium in amniotes83. In either case, the retention- 331
or convergent evolution- of certain core physiological and network features of pallial cell types in 332
birds and mammals clarifies candidate features of pallium essential for advanced cognition. 333
334
Materials and Methods 335
Animals 336
Adult zebra finches used in this study were housed in unisex aviaries prior to 337
experiments under a photoperiod of 14-h light:10-h dark. Birds were provided with food and 338
water ad libitum, as well as several forms of weekly dietary enrichment (e.g., egg food, fresh 339
millet branches, cuttlebone). All procedures and protocols adhered to the guidelines of the 340
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National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were 341
approved by the University of Massachusetts, Amherst Institutional Animal Care and Use 342
Committee. 343
Virus injection surgeries 344
4-6 weeks prior to optogenetic experiments, we injected viruses bilaterally into the 345
caudomedial nidopallium (NCM; the avian auditory association pallium) of male and female 346
zebra finches. Birds were fasted ~30 min prior to surgery, anesthetized with 2% isoflurane in 2 347
L/min O2, fixed to a custom stereotax (Herb Adams Engineering) equipped with a heating pad 348
(DC Neurocraft) at a 45o head angle, and maintained on 1.5% isoflurane, 1 L/min O2 for the 349
duration of the surgery. Points overlying NCM were marked by scoring the skull lateral and 350
rostral to our coordinates (i.e., a crosshair), and a craniotomy exposed the brain surface (NCM 351
coordinates = 1.1 mm rostral, 0.7 mm lateral of stereotaxic zero, defined as the caudal edge of 352
the bifurcation of the midsaggital sinus). A glass pipette (tip diameter: 20-50 µm) filled with 353
mineral oil was loaded with virus (for constructs and titers see below), attached to a Nanoject 354
(Drummond Scientific Company, Broomall, PA), and lowered into the brain at a depth of 1.5 mm 355
from the surface. We injected 625 nL of virus, 2 nL/sec, 125 nL/cycle, 5 cycles, with a 60 sec 356
wait between cycles. Following injections, we waited 10 min before slowly retracting the 357
injection pipette from the brain. After injections were made in both hemispheres, crainiotomies 358
were filled using Kwik-Cast (World Precision Instruments, Sarasota, FL), and the scalp was 359
fixed around the crainiotomy using cyanoacrylate adhesive. 360
The viruses and titers used in the present study included: CaMKII , pAAV9-CaMKII -361
hChR2(E123A)-EYFP (titer: 1x1013 viral genomes/mL; Addgene: 35505); GAD1, pAAV9-362
hGAD1-GLAD-NLS-CRE (titer: 1.25x1012 viral genomes/mL; made in-house), pAAV9-mDlx-363
ChR2(H134R)-EYFP (titer: 1.58x1012 viral genomes/mL; made in-house), AAV9 pCAG-FLEX-364
tdTomato-WPRE (titer: 1.5x1012 viral genomes/mL; Addgene: 51503). 365
Immunofluorescence 366
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A subset of birds (N = 3; 2 males, 1 female) given injections with viruses targeting both 367
CaMKII and GAD1 promoters were transcardially perfused with 4 % paraformaldehyde 4-6 368
weeks following surgery. Brains were extracted, post-fixed overnight in 4 % paraformaldehyde, 369
then dehydrated in 30 % sucrose in 0.1 M phosphate-buffered saline (PBS). After dehydration, 370
brains were placed in 2 x 2 x 2 inch plastic blocks filled with cryo-embedding compound (Ted 371
Pella Inc., Redding, CA). Brains were sectioned coronally at 40 microns and sections were 372
stored in cryoprotectant solution (30 % sucrose, 1 % polyvinylpyrrolidone, 30 % ethylene glycol, 373
in 0.1 M phosphate buffer) at -20 °C. 374
We labeled tissue sections for aromatase, a reliable marker for NCM compared to 375
neighboring regions72,84. At all times during this procedure tissue was processed in a shaded 376
room away from direct light sources. Tissue sections were washed 5 times in 0.1 M PBS, 3 377
times in 0.1 M phosphate-buffered saline with 0.3% triton (0.3 % PBT), blocked using 10 % 378
normal goat serum, and incubated at 4 °C for two days in rabbit anti-aromatase diluted 1:2000 379
in blocking serum (aromatase antibody provided as a generous gift from Dr. Colin Saldanha). In 380
N = 1 bird, tissue was simultaneously incubated in mouse anti-NeuN (Millipore, Danvers, MA; 381
RRID: AB_2298772) diluted 1:2000 in blocking serum. 382
Sections were then washed 3 times in 0.1 % PBT and incubated in secondary antibodies 383
diluted 1:500 in 0.3 % PBT. Secondaries used were goat anti-mouse Alexa 405 (Abcam, 384
Cambridge, MA; for N = 1 bird, tissue that was incubated in mouse anti-NeuN above) and goat 385
anti-rabbit Alexa 647 (Invitrogen, Waltham, MA; for N = 3 birds). After 3 more washes in 0.1 % 386
PBT, sections were mounted onto slides, and coverslipped using Prolong antifade mounting 387
medium with DAPI stain in the 405 channel (for bird with tissue incubated in mouse anti-NeuN, 388
Prolong mounting medium contained no DAPI; Invitrogen, Waltham, MA). Slides were dried 389
overnight at room temperature, and stored at 4 °C until imaging. 390
Sections were imaged on a confocal microscope (A1SP; Nikon, Tokyo, Japan) at the 391
UMass light microscopy core facility. Images were acquired using NIS-Elements software 392
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(RRID: SCR_002776). We determined gain and laser intensity separately for each tissue 393
section to minimize background fluorescence. Injection sites were localized within NCM by 394
dense fluorescence in the aromatase-positive field of NCM. To ensure we were only capturing 395
images of transduced cells within NCM, pictures of ventral and dorsal NCM were taken only 396
within the aromatase-positive population of the region dorso-medial to the medial part of the 397
dorsal arcopallial lamina. 398
Images of tissue sections containing NCM were first taken at 10X to serve as a 399
reference for higher magnification imaging. All cells within an image in NCM were taken at 60X 400
using z-stacks of 1-2 m through the entire thickness of the tissue section. 401
Analysis 402
For all confocal images we quantified the number of EYFP-positive cells, the number of 403
tdTomato-positive cells, and the number of cells expressing both fluorophores (reflecting 404
transduction of the CaMKII and GAD1 promoter, or both, respectively) The total number of 405
transduced cells across all observed slices (n = 122) were tallied. Cells were counted only when 406
positive staining was associated with DAPI or NeuN. In slices that were stained with antibody 407
targeting NeuN, no labeled cells were observed that did not co-localize with NeuN labeling. 408
In vitro neuronal response properties 409
Birds were rapidly decapitated and dissected, then the caudal telencephalon was 410
bisected on a petri dish immersed in wet ice and each hemisphere was sectioned at 250 411
microns on a vibratome. A glycerin-based external cutting solution (substituting for NaCl & 412
sucrose) was used for sectioning to improve slice survival time 85, containing (in mM): 222 413
glycerin, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 3 MgCl2•6H2O, 25 dextrose, 0.4 414
ascorbic acid, 2 sodium pyruvate, 3 myo-inositol; 310 mOsm/kg H2O; pH 7.4 when saturated 415
with 95% O2/5% CO2. Following sectioning, the slices were warmed to ~ 40 °C in external 416
solution containing (in mM): 111 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 1 417
MgCl2•6H2O, 25 dextrose, 0.4 ascorbic acid, 2 sodium pyruvate, 3 myoinositol; 310 mOsm/kg 418
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H2O; pH 7.4 when saturated with 95% O2/5% CO2. Our potassium-based internal solution 419
contained (in mM): 2.4 potassium gluconate, 0.4 KCl, 0.002 CaCl2, 0.1 HEPES, 0.1 EGTA, 0.06 420
Mg-ATP, 0.01 Na-GTP, 0.4 C4H8N3O5PNa2•4H2O; 290-305 mOsm/kg H2O; pH 7.4. 421
Tissue was imaged using a charge-coupled camera (QIClick; QImaging) mounted to a 422
fixed stage microscope (Eclipse FN1; Nikon) that was equipped with a water emersion objective 423
(CFI Fluor; 40X; NA = 0.8; WD = 2.0 mm; Nikon). Glass pipettes were pulled from borosilicate 424
glass capillary tubes (1B150F-4; World Precision Instruments) using a two-stage, vertical puller 425
(PC-10; Narishige International USA). Positive pressure was applied to each pipette while 426
moving through aCSF and tissue. Liquid junction potential was automatically subtracted. 427
Pipettes had a tip resistance of 4-8 M when backfilled with internal solution, which routinely 428
included neurobiotin tracer (Vector Laboratories) for identification post-recording. Once a whole-429
cell configuration was successfully achieved, series resistance and slow capacitive transients 430
were counterbalanced. Each recording configuration was allowed to stabilize for a minimum of 431
five min before beginning protocols 86. Experimental data were acquired at 40 kHz using multi-432
channel acquisition software (PATCHMASTER; HEKA Elektronik), digitized using a patch clamp 433
amplifier (EPC 10 USB; HEKA Elektronik), then exported to Igor Pro (WaveMetrics) and 434
processed using a 1 kHz low-pass filter. 435
Post-recording, tissue sections were drop fixed in 4% Paraformaldehyde dissolved in 436
0.025 M phosphate buffer (PB), then stored at -20 °C in a cryoprotectant solution composed of 437
30% sucrose, 30% ethylene glycol, and 1% polyvinylpyrrolidone in 0.1 M phosphate buffer (PB). 438
Analysis 439
Recordings were made from N = 5 birds (N = 3 males, N = 2 females), n = 18 cells 440
transduced with CaMKII -ChR2 and N = 4 birds N= 2 males, N = 2 females), n = 11 cells 441
transduced with GAD1-ChR2. Steady-state voltage (SSV), action potential kinetics, and action 442
potential width (duration at 50% of action potential depolarization peak from resting membrane 443
potential) were each calculated using custom scripts written in Python. 444
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Anesthetized extracellular recording 445
Extracellular recording was performed in vivo under urethane anesthesia as in previous 446
studies 87. On the day of recording, birds were injected in the pectoral muscle with 90-120 µL 447
20% urethane (30 µL every 45 min; specific amount depended on the mass of the bird). 448
Anesthetized birds were fixed to a custom stereotaxic apparatus as above and a stainless steel 449
headpost was fixed to the head using acrylic cement. Birds were then moved to a sound-450
attenuation booth (Industrial Acoustics) on an air table (TMC, Peabody, MA) for optotagging and 451
extracellular recording experiments. In the booth birds were fixed to a custom stereotax (Herb 452
Adams Engineering) at a 45o head angle using the attached headpost and the craniotomy 453
above NCM was exposed. We recorded preferentially from the left hemisphere but both 454
hemispheres are represented in our dataset. 455
An optrode consisting of a single tungsten electrode (A-M Systems, Sequim, WA) 456
epoxied to an optic fiber (diameter: ~200 µm; Thor Labs, Newton, NJ; distance between 457
electrode and fiber: 400-600 µm) was lowered into the brain at the NCM coordinates specified 458
above. The optrode was calibrated by inserting the optrode into a light meter (Thor Labs, 459
Newton, NJ) in a dark room, adjusting laser power and obtaining a calibration curve of laser 460
setting to output wattage. To elicit ChR2-induced spike activity locally around our electrode we 461
used a blue laser (447 nm) with an output wattage of ~2 mW. Recordings were made between 462
1.2 and 1.8 mm ventral to the brain surface to search for characteristic NCM baseline and 463
sound-evoked activity, as well as light-evoked activity. Light stimuli consisted of either 25 msec 464
pulses or 100 ms pulses, and pulses were separated by 4-10 sec. Following light-only trials 465
where light-evoked units were identified (see below), we immediately ran auditory trials in the 466
same location. Auditory trials consisted of seven stimuli: 6 conspecific songs and white noise, all 467
normalized to ~70 decibels. Stimuli were randomly presented during trials and each stimulus 468
was presented 15 times. At a given recording site, we randomly presented 3 of 6 conspecific 469
songs and white noise (i.e., 4 stimuli total). Interstimulus interval was 10+2 sec. Recordings 470
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were amplified, bandpass filtered (300 to 5000 Hz; A-M Systems), and digitized at 16.67 kHz 471
(Micro 1401, Spike2 software; Cambridge Electronic Design). Following recordings, birds were 472
transcardially perfused and brains were extracted as above for anatomical confirmation of 473
optrode sites. 474
Analysis 475
Data were processed in Spike2 (version 7.04) to identify light-evoked units. Recordings 476
from light-only trials were thresholded above the noise band and peristimulus histograms and 477
raster plots were generated to examine multiunit responses to light stimulation. We only 478
performed auditory playback trials if there was a peak in the histogram during the light stimulus 479
that was consistent across trials (as shown by the raster plot; Fig. 3A). Recordings were made 480
from N = 3 birds (1 male, 2 females), n = 22 cells transduced with CaMKII -ChR2 and N = 3 481
birds (3 females), n = 16 cells transduced with GAD1-ChR2. 482
Templates for single units in light-only trials were isolated in Spike2 by their waveform 483
characteristics and filtered so that no units had an interspike interval > 1 ms as in previous 484
studies 87. Individual spikes were assigned to generated templates with an accuracy range of 485
60%-100%. Principal component analyses were used to confirm well-isolated units (i.e., non-486
overlapping clusters in 3-dimensional space). We could reliably obtain 2-4 units from each 487
recording site. We used isolated single unit waveforms from light-only trials to sort multiunit 488
activity in their paired auditory trials. At this point we again checked that none of the units had 489
interspike intervals > 1 ms and that principal component analyses still demonstrated non-490
overlapping clusters. 491
For waveform analyses, we compared physiological properties of light-responsive cells 492
between birds with virus targeting the CaMKII promoter and birds with virus targeting the 493
GAD1 promoter. We calculated action potential width as the time between the first (depolarized) 494
and second (hyperpolarized) peaks of the average waveform. Spike quarter-width was 495
calculated as the width of the waveform at a quarter of the height of the action potential. 496
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Z scores were calculated for each single unit’s response to conspecific song and white 497
noise to provide a normalized change in firing during stimulus presentation ( ) compared to 498
baseline ( ), using the following formula as in previous studies 88: 499
Z scores for conspecific songs for each single unit are reported as average single unit
500
responses across conspecific stimuli presented. 501
We calculated single unit response latency to white noise by adapting a previously 502
described method40. We calculated the mean and standard deviation of the baseline period (2 s 503
before stimulus onset). Then, peri-stimulus time histograms were created for each single unit’s 504
response to white noise, divided into 5 ms bins and smoothed with a 5-point boxcar filter. We 505
identified the first bin within 400 ms of white noise onset in which firing rate passed a threshold 506
of 3 standard deviations from the baseline mean. 507
To examine auditory coding properties of isolated single units, we used a custom pattern 508
classifier 39,89. The classifier uses timing accuracy of stimulus-evoked firing responses of single 509
units to discriminate amongst the different stimulus types, providing a measure of how well 510
stimuli can be distinguished by consistency of evoked firing across trials. 511
Specifically, for each single unit the classifier pseudorandomly picked one response per 512
stimulus to serve as templates. The classifier compared the selected templates with all other 513
stimulus-evoked responses of that single unit. Based on values of a distance metric (detailed 514
below) between responses and the templates, the algorithm would assign a template identity 515
(e.g. conspecific song 1) to the template that was most similar to a given response. The 516
classifier repeated this procedure 1000 times and then determined the mean accuracy of 517
assigning stimulus-evoked responses to their correct associated stimuli. 518
For the timing accuracy measure, data were convolved with Gaussian filters prior to 519
template comparison. The optimal standard deviation of the filter for each cell was picked based 520
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on which value gave the highest accuracy (values that were used: 1, 2, 4, 8, 16, 32, 64, 128, 521
256 ms). Comparisons between templates and responses used the Rcorr distance metric 39,90. 522
Where s represents the vectors of the trial and the template responses after filtering, dot
523
multiplied and divided by the product of their lengths. For the timing accuracy measure the 524
classifier matched responses (i.e., trials) to templates based on which template yielded the 525
highest Rcorr value. 526
To statistically test whether the accuracy of the classifier was greater than random 527
chance, we generated confusion matrices and used a trial shuffling approach (modified from 528
39,89). Classifier assignments of stimulus-evoked responses in the confusion matrix were shuffled 529
and randomly assigned to stimuli 1000 times. The distribution of the classifier accuracies across 530
the 1000 runs was compared to the randomly shuffled assignments. Accuracies were 531
considered significantly greater than random when Cohen’s d was > 0.2 39,91. All single unit 532
accuracies in this study had a Cohen’s d > 0.2. Analyses for conspecific song responses are 533
presented as single unit responses averaged across conspecific stimuli presented to a given 534
unit. 535
Temporal stimulus selectivity was measured by comparing the mean Rcorr values across 536
stimuli repetitions (spike-timing correlation) among the 3 different conspecific stimuli. 537
Specifically, for each single unit, the 3 mean Rcorr values (Rx, Ry, Rz) were used to generate a 538
3-dimensional vector ( ). Then, a second vector was generated such that vector magnitude was 539
identical to but Rcorr values for stimuli were equalized in the 3D-space (Rx = Ry = Rz), i.e. the 540
equal-selectivity vector ( ). The angle (in radians) between each single unit’s 3D vector ( ) and 541
its associated equal-selectivity vector ( ) was calculated using the arccosine distance metric: 542
, where values close to 0 reflect non-selectivity and larger values reflect 543
greater selectivity. 544
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Optodrive awake recordings 545
For larger scale recordings, custom optodrives were made by coupling a fiber optic (200 546
µm diameter) to 8 tetrodes arranged in a single bundle (customized from EIB Open-Ephys drive; 547
Open Ephys Inc., MA). Tetrodes were made from 12.5 µm insulated NiCr wires (Sandvik, 548
Sandviken, Sweden). The optic fiber was placed ~600 µm above the wires. The horizontal 549
distance between the fiber and wire bundle was ~200 µm. 550
Virally transduced birds were anesthetized with isoflurane, after which bilateral 551
craniotomies were made over NCM (see above for coordinates; sealed with Kwik-Cast) and 552
headposts were secured to the dorsomedial rostral skull with acrylic cement. 2-4 days later, 553
birds were restrained and head-fixed. Optodrives were lowered into NCM 1.5-2.0 mm ventral of 554
the brain surface. After finding a site with characteristic NCM baseline and sound-evoked 555
activity, the optodrive was allowed to stabilize in the brain for ~1 h. Following stabilization, a 556
blue (447 nm) or green (532 nm) laser was used to test network responses to manipulation of 557
transduced cells. Recordings were obtained using the Open-Ephys GUI 92, and amplified and 558
digitized at 30 kHz using Intan Technologies amplifier and evaluation board (RHD2000; Intan 559
Technologies, Los Angeles, CA). Laser delivery was controlled by custom MatLab (MathWorks, 560
Natick, MA) scripts and an Arduino Uno integrated with the evaluation board. 561
For the GAD-1 archaerhodopsin experiment, after testing network responses to 562
manipulation of transduced cells by the green laser, we tested how inhibition of GAD-1 cells 563
would affect functional auditory response properties of the network. We randomized playback of 564
4 conspecific songs. Each song was heard 10 times total, 5 times without the green laser, and 5 565
times with the green laser turned on specifically for the duration of the stimulus. This resulted in 566
20 playback trials with laser turned off (and songs randomized within these 20 trials such that 567
each song was played 5 times), followed by 20 playback trials with laser turned on only during 568
the duration of each song playback (and songs randomized within these 20 trials such that each 569
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song was played 5 times). We allowed 5 seconds of silence in between playback of different 570
stimuli. 571
Analysis 572
Optodrive single unit sorting was done with Kilosort 93. Sorting results were manually 573
curated and only well-isolated units (high signal-to-noise ratio; low contamination; good 574
segregation in waveform principal component analysis space; low frequency in violations of 575
refractory period) were used for these analyses. Data were common median filtered and 300 Hz 576
high-pass filtered. 577
Sample sizes for optodrive experiments were: mDlx-ChR2: N = 1 female; n = 36 single 578
units. GAD1-ChR2: N = 1 male; n = 30 single units. GAD1-archaerhodopsin: N = 1 male; n = 52 579
single units. Recordings were made from both left and right hemispheres. 580
For local field potential (LFP) analyses, raw traces were 150 Hz low-pass filtered and 581
local field potential power spectra were generated in Python (smoothed and imported using Neo 582
94 and Elephant libraries; Welch’s power spectrum frequency resolution at 1 Hz). 583
We also calculated stimulus firing rate in Hertz and auditory selectivity (as described 584
above in the analysis section for anaesthetized optrode recording) for the GAD1-585
archaerhodopsin experiment to compare responses to conspecific song when laser was off 586
versus when the laser was on. 587
Statistics 588
Statistical analyses included two-tailed t-tests and nonparametric statistics (Mann 589
Whitney U tests) when log10 transformations did not correct violations of parametric 590
assumptions. Bonferroni corrections were used to correct for multiple comparisons. Wilcoxon 591
signed rank tests were used to test paired responses to song in the same cells when laser was 592
off compared to on. Chi-square tests were used to test whether the number of cells that 593
increased or decreased firing rates and auditory selectivity was greater than expected by 594
chance. 595
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Data availability 596
Data from this study will be made available on Dryad. 597
598
599
600
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