The Cellular and Mechanical Basis for Response Characteristics of Identified Primary Afferents in the Rat Vibrissal System

Abstract

Compared to our understanding of the response properties of receptors in the auditory and visual systems, we have only a limited understanding of the mechanoreceptor responses that underlie tactile sensation. Here, we exploit the stereotyped morphology of the rat vibrissal (whisker) array to investigate coding and transduction properties of identified primary tactile afferents. We performed in vivo intra-axonal recording and labeling experiments to quantify response characteristics of four different types of identified mechanoreceptors in the vibrissal follicle: ring-sinus Merkel; lanceolate; clublike; and rete-ridge collar Merkel. Of these types, only ring-sinus Merkel endings exhibited slowly adapting properties. A weak inverse relationship between response magnitude and onset response latency was found across all types. All afferents exhibited strong "angular tuning," i.e., their response magnitude and latency depended on the whisker's deflection angle. Although previous studies suggested that this tuning should be aligned with the angular location of the mechanoreceptor in the follicle, such alignment was observed only for Merkel afferents; angular tuning of the other afferent types showed no clear alignment with mechanoreceptor location. Biomechanical modeling suggested that this tuning difference might be explained by mechanoreceptors' differential sensitivity to the force directed along the whisker length. Electron microscopic investigations of Merkel endings and lanceolate endings at the level of the ring sinus revealed unique anatomical features that may promote these differential sensitivities. The present study systematically integrates biomechanical principles with the anatomical and morphological characterization of primary afferent endings to describe the physical and cellular processing that shapes the neural representation of touch.

Keywords: 3D reconstruction; active sensing; firing properties; ganglion; in vivo recording; peripheral system; piezoelectric stimulator; rodent; single-cell labeling; trigeminal.

Figure 1.. Representative Primary Afferents and Endings: RS-Merkel, RRC-Merkel, Lanceolate, and Club-like

(A) Morphologies labeled via intra-axonal injection. Arrows indicate trunks of labeled afferents. Arrowheads indicate peripheral endings. (B) Example 3D reconstructions of follicles that contained recorded axons. Axons and terminals are yellow. Skin, follicle capsule, vibrissal shaft, and Ringwulst are blue, cyan, gray, and green, respectively. Axon thickness and terminal size are exaggerated for clarity. Expanded views for each reconstruction (right column) show semiquantitative renderings of mechanoreceptor terminal shapes. The scale for the longest dimension of the mechanoreceptor is quantitatively accurate, although the scale for the other two dimensions is approximate, due to limitations of the microscope and 3D Neurolucida tracing system. RW, Ringwulst; SG, sebaceous gland; VS, vibrissal shaft. See also Figures S1, S2, and S3 and Table S1.

Figure 2.. Response Properties of Primary Afferents to Whisker Deflection in the Afferent’s Best Direction

All histograms use bin width = 1 ms; error bars in (B) and (C) indicate standard deviations. (A) Representative raster plots (20 trials) and post-stimulus time histograms (PSTHs) for each mechanoreceptor type. Gray lines represent piezoelectric stimulator motion (5° amplitude). ON/(OFF) responses were defined to occur during the 10-ms onset/(offset) ramp. (B) For each receptor type, spike timing variability is quantified as the standard deviation of the latency to first spike across 20 trials. Error bars indicate SD across all afferents in each type. (C) Inverse relationship between onset latency (ON responses) and spiking magnitude for each recorded afferent. Error bars indicate SD across 20 trials in each afferent. (D) Histograms of ON latencies and ON magnitudes for all trials from all afferents, categorized by afferent type. n, number of afferents. (E) Population PSTHs constructed by averaging all best-direction responses for all endings of the same type. See also Figure S2.

Figure 3.. Identifying the 3D Location of the Mechanoreceptor in the Follicle

(A) Vibrissa orientation relative to mystacial pad. (B) Locations of peripheral endings were projected into a plane normal to the vibrissal length. (C) Representative 3D reconstruction of a vibrissal follicle. Peripheral endings are yellow. (D) Reconstructed data are observed from a direction along the vibrissal length. See also Figures S2 and S3.

Figure 4.. Axons Exhibit a Diversity of Angular Tuning Profiles Relative to Location of Endings and Preferred Angle

(A) Responses of 11 identified Merkel afferents to deflections in four directions with respect to the laboratory frame (up, forward, back, and down). All subplots are rotated to place the mechanoreceptor (black dot) at the same angular location, permitting results from all afferents to be overlaid. Pink and cyan vectors indicate ON and OFF response magnitudes for the four deflection directions. For afferent 2, one pair of overlapping pink and cyan vectors is rotated approximately ±5° from nominal for visual clarity. (B) Angular tuning curves for six unidentified, slowly adapting primary afferents during whisker deflections in either 32 or 16 different directions. Pink/cyan polar plots indicate ON/OFF response magnitudes, respectively. (C) For Merkel neurons, mechanoreceptor angular locations were well predicted by a cosine function. (D)Responses of ten identified lanceolate and club-like neurons to deflections in four directions. Conventions are as in (A). For afferents 12 and 16, one pair of overlapping pink and cyan vectors is rotated approximately ±5° from nominal for visual clarity. Two pairs are similarly rotated for afferent 19. (E)For lanceolate and club-like neurons, mechanoreceptor angular locations were not well predicted by cosine fits. (F)Relationships between response latency and magnitude (number of spikes/10-ms ON or OFF window) are variable. See also Figure S4.

Figure 5.. Mechanical Modeling Suggests Differential Mechanical Sensitivities across Afferent Types

(A) Experimental ON and OFF responses for Merkel neurons. (B) Experimental ON and OFF responses for lanceolate neurons. (A and B) Column 1 (shaded) shows experimental data. The remaining columns show results of models based on specific mechanical signals, as labeled. F_X , axial force; F_X d, derivative of axial force; M_B, bending moment; M_Bd, derivative of bending moment. ON (pink) and OFF (cyan) response magnitudes for each direction are plotted as vectors and sorted by mechanoreceptor type. (C and D) Confusion matrices and magnitude error and latency error histograms for Merkel (C) and lanceolate and club-like endings (D). Each column shows results for a single modeling choice; abbreviations are as in (A) and (B). Each 2 × 2 array shows the number of true positives (TPs), false positives (FPs), false negatives (FNs), and true negatives (TNs) of predicted spiking responses compared to observed spiking responses. F1 scores (see STAR Methods) are indicated below each array. Histograms quantify error between experimental and modeled stimulus responses. Latency error histograms have 1-ms bins. Magnitude error histograms are binned at 0.1 units, where maximum response magnitude for each afferent has been normalized to 1. See also Figures S5 and S6.

Figure 6.. Ultrastructure of RS-Merkel and Lanceolate Endings

GM, glassy membrane (basement membrane); IRS, inner root sheath; IZ, intermediary zone; LE, lanceolate ending; MC, Merkel cell; ME, Merkel ending; MS, mesenchymal sheath; ORS, outer root sheath; 3D-EM, three-dimensional electron microscopy. (A) Semi-thin section parallel to the axis of the vibrissa. (B and C) Magnified views of the rectangle in (A) obtained from two sequential semi-thin sections. Arrowheads indicate an axon branch piercing the glassy membrane to enter the ORS. (D)RS-Merkel endings are distributed in the most lateral layer of the ORS. (E) Ultrastructure of a lanceolate ending. (F) Three orthogonal planes of stack data obtained with a scanning electron microscopic system. (G) Sequential EM images of nerve endings at the ring sinus level were reconstructed in 3D (gray, glassy membrane; yellow, Merkel ending; green, lanceolate ending). RS-Merkel endings are located in the epithelial sheath although lanceolate endings are localized in the loose space between the glassy membrane and the mesenchymal tissue. (H) Schematic representation of Merkel and lanceolate ending innervation at the level of the ring sinus.

Figure 7.. An Explanation for Response Variations across Mechanoreceptor Type Based on Differential Sensitivity to Mechanical Signals at the Whisker Base

(A) Mechanical input near the vibrissa tip is transduced by the vibrissa, which has an intrinsic curvature. The mechanical signal is decomposed into mechanical components that include bending moment (M_B) and axial force (F_X). (B) Each mechanoreceptor’s sensitivity will be influenced by its location and the surrounding structure of the follicle complex.