Genetically identified neurons in avian auditory pallium mirror core principles of their mammalian counterparts

Abstract

In vertebrates, advanced cognitive abilities are typically associated with the telencephalic pallium. In mammals, the pallium is a layered mixture of excitatory and inhibitory neuronal populations with distinct molecular, physiological, and network phenotypes. This cortical architecture is proposed to support efficient, high-level information processing. Comparative perspectives across vertebrates provide a lens to understand the common features of pallium that are important for advanced cognition. Studies in songbirds have established strikingly parallel features of neuronal types between mammalian and avian pallium. However, lack of genetic access to defined pallial cell types in non-mammalian vertebrates has hindered progress in resolving connections between molecular and physiological phenotypes. A definitive mapping of the physiology of pallial cells onto their molecular identities in birds is critical for understanding how synaptic and computational properties depend on underlying molecular phenotypes. Using viral tools to target excitatory versus inhibitory neurons in the zebra finch auditory association pallium (calmodulin-dependent kinase alpha [CaMKIIα] and glutamate decarboxylase 1 [GAD1] promoters, respectively), we systematically tested predictions derived from mammalian pallium. We identified two genetically distinct neuronal populations that exhibit profound physiological and computational similarities with mammalian excitatory and inhibitory pallial cells, definitively aligning putative cell types in avian caudal nidopallium with these molecular identities. Specifically, genetically identified CaMKIIα and GAD1 cell types in avian auditory association pallium exhibit distinct intrinsic physiological parameters, distinct auditory coding principles, and inhibitory-dependent pallial synchrony, gamma oscillations, and local suppression. The retention, or convergence, of these molecular and physiological features in both birds and mammals clarifies the characteristics of pallial circuits for advanced cognitive abilities.

Keywords: auditory; calcium/calmodulin-dependent kinase II alpha; cell type; evolution; gamma oscillation; glutamate decarboxylase 1; interneuron; pallium; principal cell; songbird.

Fig. 1.. Viruses targeting CaMKIIα and GAD1 promoters segregate cell types in avian auditory association pallium.

(A) Sagittal schematic of avian pallium (top left) showing the main thalamorecipient region of auditory pallium (Field L), and auditory association regions of pallium (caudomedial mesopallium, CMM; caudomedial nidopallium, NCM). Left is rostral, top is dorsal. 20x confocal image (top right) shows non-laminar clustered cytoarchitecture of NCM. Bottom: magnified from white box in top right (blue=DAPI; white=NeuN). (B) Non-overlapping cell types identified by viral expression of fluorophores (left), and 60x images of transduced CaMKIIα (green) and GAD1 (magenta) cells. (C) Cell soma area at largest cross-sectional diameter. (D) 60x images of viral and antibody co-expression. Top row: CaMKIIα viral expression (green) co-localizing with CaMKIIα immunolabeling (white). Bottom: GAD1 viral expression (magenta) co-localizing with GABA (gold) and parvalbumin (PV; white) immunolabeling. White arrowheads = co-localization. (E) 40x images of viral and antibody labeling. Top row: typical CaMKIIα viral expression (green) not co-localizing with GABA (gold) or PV (white); white asterisk shows example of weaker CaMKIIα viral expression (~8% of cells) that co-express GABA and PV. Bottom row: typical GAD1 viral expression (magenta) not co-localizing with CaMKIIα immunolabeling (white); asterisks show example of GAD1 viral expression (~8% of cells) that co-expresses CaMKIIα. White arrowheads in both rows show position of exemplar viral cell across each image. (F) Typical widefield view of viral injection site in NCM. Shown is mDlx-GFP expression (left), PV immunolabeling (middle), and overlaid images (right) showing specificity (>66% viral cells co-labeled) and efficiency (>88% PV cells co-labeled) of transduction. All scale bars = 50 microns. See also Figure S1.

Fig. 2.. CaMKIIα and GAD1 single units in NCM have distinct physiological properties.

(A) Transduced cells in in vitro whole-cell current clamp configuration (left) and reliable photopotentials to 25 ms blue light pulses (top row = CaMKIIα cell; bottom row = GAD1 cell). (B) CaMKIIα cells exhibit phasic responses (top) while GAD1 cells exhibit tonic responses (bottom) to current steps. (C) Mean ± SEM action potentials for CaMKIIα cells (green; n = 18) and GAD1 cells (magenta; n = 11) in response to current steps. (D) Spike width of CaMKIIα cells vs. GAD1 cells in whole-cell recordings. *P < 0.05 for Mann-Whitney U test. (E) Afterhyperpolarization half-duration of CaMKIIα cells vs. GAD1 cells in whole-cell recordings. *P < 0.05 for Mann-Whitney U test. (F) Input resistance of CaMKIIα cells vs. GAD1 cells in whole-cell recordings. (G) Rheobase of CaMKIIα cells vs. GAD1 cells in whole-cell recordings. (H) Raster plots and histograms of exemplar in vivo transduced single units in electrophysiological response to blue light pulses. (I) Action potential widths of optically-identified CaMKIIα vs. GAD1 single units in vivo. *P < 0.05 for Mann-Whitney U test. (J) Spike quarter-widths of optically-identified CaMKIIα vs. GAD1 single units in vivo.. *P < 0.05 for Mann-Whitney U test. (K) Latency to light-evoked response peak of optically-identified CaMKIIα vs. GAD1 single units in vivo. *P < 0.05 for Mann-Whitney U test. See also Figure S2.

Fig. 3.. Auditory coding roles distinguish CaMKIIα and GAD1 neurons in NCM.

A) Spectograms and rasterplots of a CaMKIIα single unit (top) and a GAD1 single unit (bottom) in NCM responding to conspecific song. (B) Song-evoked Z scores of optically-identified single units. *P < 0.05 for Mann-Whitney U test. (C) Latency in seconds for optically-identified single units to respond to white noise stimuli. *P < 0.05 for Mann-Whitney U test. (D) Heat map of single unit timing accuracy measures across auditory stimuli. Values closer to 1 represent higher classifier timing accuracy. Histogram insets show density distribution of accuracy metric across stimuli for CaMKIIα (left) and GAD1 single units (right). (E) Pattern classifier timing accuracy averaged across auditory stimuli for optically-identified single units displayed in D. Accuracy is pattern classifier performance in correctly assigning spike trains to auditory playback stimuli. For details, see methods. *P < 0.05 for Mann-Whitney U test. (F) Measure of transduced single unit selectivity for subsets of conspecific stimuli (see Methods). #P = 0.057 for Mann-Whitney U test. See also Figure S3.

Fig. 4.. GAD1 and/or mDlx neurons in NCM drive suppression, synchrony, gamma oscillations, and functional auditory response properties.

(A) Instantaneous firing rates of light-evoked single units (magenta; n = 5) and light-suppressed single units (orange; n = 5) from mDlx-ChR2 optical stimulation drawn from n = 36 total units isolated in NCM in vivo. (B) Violin plots show change in cross-correlation of waveforms following mDlx-ChR2 optical stimulation experiments (top row) and GAD1-archaerhodopsin optical stimulation experiments (bottom). Red line denotes zero change. *P<0.001 for Mann-Whitney U tests. (C) Heat map (% max LFP power) showing LFP change over time in an mDlX-ChR2 optodrive experiment. Cartoon lasers above represent bins with blue light pulses. (D) LFP power spectra before and during mDlx-ChR2 optical stimulation experiments (top), and before and during GAD1-archaerhodopsin optical stimulation experiments (bottom). Gray shading represents gamma frequency range for which LFP power is significantly different from baseline according to predictions from mammalian pallium (P < 0.05); $ is range for which LFP power is significantly different from baseline against predicted direction (P < 0.05). (E) Single units with broad waveforms are plotted with respect to their stimulus-evoked firing rates to various conspecific songs following GAD1-archaerhodopsin optical stimulation (y-axis) compared to no optical stimulation (x-axis). Each point represents a single unit’s response to one conspecific song, and single units are grouped by a unique shape and color. (F) The same single units as E are plotted with respect to their selectivity across all conspecific stimuli when green laser was on (y-axis) compared to when green laser was off. For E & F, points deviating from the red line denote a non-zero difference between laser off and laser on conditions. (G) Summary schematic showing features of avian CaMKIIα neurons (green) and GAD1/mDlx neurons (magenta) in NCM in reference to predictions from mammalian pallium. Arrows imply effects of neuron stimulation and do not imply nature of synaptic connectivity. See also Figure S4.