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[Preprint]. 2024 Mar 30:2024.03.27.587042.
doi: 10.1101/2024.03.27.587042.

Lineage-tracing reveals an expanded population of NPY neurons in the inferior colliculus

Marina A Silveira  1   2 , Yoani N Herrera  1 , Nichole L Beebe  3 , Brett R Schofield  3 , Michael T Roberts  1   4
Affiliations

Lineage-tracing reveals an expanded population of NPY neurons in the inferior colliculus

Marina A Silveira et al. bioRxiv. .

Update in

Abstract

Growing evidence suggests that neuropeptide signaling shapes auditory computations. We previously showed that neuropeptide Y (NPY) is expressed in the inferior colliculus (IC) by a population of GABAergic stellate neurons and that NPY regulates the strength of local excitatory circuits in the IC. NPY neurons were initially characterized using the NPY-hrGFP reporter mouse, in which hrGFP expression indicates NPY expression at the time of assay, i.e., an expression-tracking approach. However, studies in other brain regions have shown that NPY expression can vary based on a range of factors, suggesting that the NPY-hrGFP mouse might miss NPY neurons not expressing NPY proximal to the experiment date. Here, we hypothesized that neurons with the ability to express NPY represent a larger population of IC GABAergic neurons than previously reported. To test this hypothesis, we used a lineage-tracing approach to irreversibly tag neurons that expressed NPY at any point prior to the experiment date. We then compared the physiological and anatomical features of neurons labeled with this lineage-tracing approach to our prior data set, revealing a larger population of NPY neurons than previously found. In addition, we used optogenetics to test the local connectivity of NPY neurons and found that NPY neurons routinely provide inhibitory synaptic input to other neurons in the ipsilateral IC. Together, our data expand the definition of NPY neurons in the IC, suggest that NPY expression might be dynamically regulated in the IC, and provide functional evidence that NPY neurons form local inhibitory circuits in the IC.

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Conflict of interest statement

Conflict of Interest Statement: The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.. Most tdTomato+ neurons in NPY-FlpO x Ai65F mice express Npy and are GABAergic.
A-E. Fluorescent in situ hybridization (RNAscope) was used to determine the expression patterns of tdTomato, Npy and Vgat mRNA in the IC of NPY-FlpO x Ai65F mice. A. Low magnification coronal IC section from an NPY-FlpO x Ai65F mouse shows the distribution of tdTomato+ (magenta), Npy+ (cyan) and Vgat+ (yellow) neurons in the IC. B-E. High magnification confocal images show that tdTomato+ neurons (B, magenta) typically co-labeled with Npy (C, cyan) and Vgat probes (D, yellow). Merged image shown in E. White arrows highlight examples of neurons that co-labeled. Scale bar in E applies to images in B-E. F-H. Immunofluorescence shows that 92.3% of tdTomato+ neurons are GABAergic. High magnification confocal images show tdTomato+ neurons (F, magenta) immunolabeled with antibodies against the GABA synthetic enzyme, GAD67 (G, cyan). Merge in H. Scale bar in H applies to images in F-H.
Figure 2.
Figure 2.. NPYflp and NPYgfp neurons have similar distributions but different densities in the IC.
A. Low-magnification photomicrographs showing NPYgfp (left column, green) or NPYflp (right column, magenta) fluorescence in the IC. For each type of mouse, sections from the caudal (top row), mid (middle row) or rostral (bottom row) IC are shown. Note that commissural fibers can be seen in both types of mice (arrows), while GABA modules from the IClc (arrowheads) are only visible in NPY-FlpO x Ai65F mice. Scale = 500 μm. B. High-magnification photomicrographs showing NPYgfp (left column, green) or NPYflp (right column magenta) fluorescence in each IC subdivision. Fluorescent cells (denoted with asterisks) are readily visible in each subdivision in both types of mice (top row: ICc, middle row: ICd, bottom row: IClc). However, cells were more numerous in a given field in each subdivision in NPY-FlpO x Ai65F mice. Note especially the presence of many NPYflp cells in a GABA module in the IClc. Fluorescent axons and boutons are also readily visible in each subdivision in both types of mice, and commissural axons are especially prominent in the ICd of both types of mice. Scale = 20 μm. C. Plots showing each fluorescent cell in four representative sections in one case from each type of mouse. One green circle denotes one NPYgfp cell and one magenta circle denotes one NPYflp cell. For both cases, sections are arranged from caudal (left) to rostral (right). D - dorsal; M - medial. D-E. Bar graphs showing the percentage of total population in each subdivision (D) and density of cells per mm2 in each subdivision and overall (E) for NPYgfp cells (green bars) and NPYflp cells (magenta). Bars show mean +/− SD. n = 24,609 NPYgfp cells across 9 NPY-hrGFP mice and 33,911 NPYflp cells across 3 NPY-FlpO x Ai65F mice.
Figure 3.
Figure 3.. NPYflp and NPYgfp neurons exhibit similar intrinsic physiological properties.
A. NPYflp (magenta) and NPYgfp (green) neurons exhibited sustained firing patterns. B-G. NPYflp and NPYgfp neurons exhibited similar spike frequency adaptation ratios (B), membrane time constants (C), voltage-dependent sag ratios (D), resting membrane potentials (E), input resistances measured from the peaks of a series of hyperpolarizing responses (F), and input resistances measured from the steady state portions of a series of hyperpolarizing responses (G). p values from Welch’s t-tests are shown atop each plot. The right side of each graph shows a paired mean difference plot (Gardner-Altman estimation plot), with the horizontal black line indicating the paired mean difference (hrGFP minus FlpO). The vertical black line indicates the 95% confidence interval for the paired mean difference as calculated from a bootstrap analysis, with the gray distribution showing the distribution of mean differences obtained from the bootstrap analysis (see Methods for details). Dashed gray lines represent the level of zero difference. Final statistical determinations were based on the Welch’s t-test, but paired mean difference plots are provided to give a better sense of the distribution of differences between the pairs of experimental groups. H. Principal component analysis showed that the distributions of NPYflp (magenta) and NPYgfp (green) neurons largely overlapped. I. k-means cluster analysis identified two clusters of neurons from the PCA results, represented by yellow and blue dots (dot positions are identical to H). Both clusters contained NPYflp and NPYgfp neurons, with the yellow cluster containing 86.4% of NPYflp neurons and 71.3% of NPYgfp neurons, showing that NPYflp and NPYgfp neurons were not separable based on their intrinsic physiology. The number of clusters used for analysis was defined using the elbow analysis method, which showed that the addition of a third cluster did little to improve the separation between cluster centroids (inset graph).
Figure 4.
Figure 4.. NPYflp neurons in the ICc have stellate morphology similar to NPYgfp neurons.
A. Representative reconstructions of the morphology of 15 NPYflp neurons that were filled with biocytin during recordings from the ICc. Gray lines represent the approximate orientation and width of ICc isofrequency lamina (~45° angle and 100 μm, respectively). B. 3D PCA analysis was performed to classify neuron morphology using the ratio of the longest and second-longest axes of each neuron (i.e., length and width), with a ratio <3 indicating stellate morphology. C. Angular orientation of the long axis of reconstructed neurons. Angles indicate counter-clockwise rotation of the long axis relative to the medial-lateral (horizontal) axis of the IC such that a 45° angle is parallel to the isofrequency plane. D. Length dendritic arbors of NPYflp neurons extended perpendicular to the isofrequency laminae plane.
Figure 5.
Figure 5.. NPYflp neurons provide inhibitory input to IC neurons in the ipsilateral IC.
A. Confocal image showing the typical expression pattern observed when using a FlpO-dependent channelrhodopsin virus to transfect IC neurons in an NPY-FlpO mouse. This example shows transfection with AAV1/EF1α1.1-FLPX-rc [Chronos-GFP]. B. Representative example of a whole-cell recording targeted to a GFP+ cell from a mouse injected in the IC with a FlpO-dependent Chronos-GFP virus (AAV1/EF1α1.1-FLPX-rc [Chronos-GFP]). Presentation of a blue light pulse elicited action potentials, confirming the ability to use optogenetics to drive NPY neuron firing. Black trace highlights a representative trial, and gray traces show additional individual trials. C. Representative example of optogenetically evoked IPSPs elicited by activating NPYflp neurons and/or terminals in the local IC. Brief flashes of blue light could be used to elicit single IPSPs or trains of IPSPs (20Hz light presentation shown). D. Distribution of IPSP amplitudes observed across all recorded cells that received input from NPYflp neurons. E. Representative example trace of optogenetically evoked IPSPs recorded from a neuron that received input from an NPYflp neuron (left). The response was abolished with application of GABAA and glycine receptor antagonists (right). F. Application of gabazine (5 μM) and strychnine (1 μM) abolished light-evoked IPSPs in all cells. In C,E, gray traces represent individual trials and black traces represent averages across trials.
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