Innervation of taste buds revealed with Brainbow‐labeling in mouse

Abstract

Nerve fibers that surround and innervate the taste bud were visualized with inherent fluorescence using Brainbow transgenic mice that were generated by mating the founder line L with nestin‐cre mice. Multicolor fluorescence revealed perigemmal fibers as branched within the non‐taste epithelium and ending in clusters of multiple rounded swellings surrounding the taste pore. Brainbow‐labeling also revealed the morphology and branching pattern of single intragemmal fibers. These taste bud fibers frequently innervated both the peripheral bud, where immature gemmal cells are located, and the central bud, where mature, differentiated cells are located. The fibers typically bore preterminal and terminal swellings, growth cones with filopodia, swellings, and rounded retraction bulbs. These results establish an anatomical substrate for taste nerve fibers to contact and remodel among receptor cells at all stages of their differentiation, an interpretation that was supported by staining with GAP‐43, a marker for growing fibers and growth cones.

Introduction

Intra‐oral taste buds are innervated by facial nerve fibers (anterior tongue fungiform buds and palatal buds) and glossopharyngeal nerve fibers (posterior tongue circumvallate and foliate buds). Data on the morphology and intragemmal distribution of taste bud nerve fibers derived from these cranial nerves are incomplete owing to technical limitations of the methods used to date. Data based on the transport of neuroanatomical tracers, e.g. WGA‐HRP (Bradley et al. 1986) and DiI (Finger & Bottger, 1990), immunocytochemical labeling based on fiber neurochemical expression (Silverman & Kruger, 1989; Finger et al. 1990), and histochemical staining (Muller, 1996a,b; Muller & Jastrow, 1998) have established some general features of taste bud innervation, but do not show the detailed morphology of individual fibers. Morphological information is necessary to establish where, in the bud, fibers distribute, the extent of branching of individual gemmal fibers, and structural evidence for fiber growth and remodeling that likely attend taste cell turnover. Electron microscopic reconstruction of single fibers has demonstrated their synaptic contacts (Kinnamon et al. 1988, 1993) but only small portions of relatively few fibers have been assessed, preventing a characterization of their full branching patterns or relationship to various bud regions. The Golgi stain, in the single publication that employed the method on taste buds, did prove informative on the detailed structure of gemmal nerve fibers, many of which were morphologically quite elaborate (Whitehead & Kachele, 1994, in hamster), but the technique is successful only in developing animals (Ramon y Cajal, 1960). Thus, whether the morphology of previously described Golgi‐stained fibers is similar to that of mature taste bud fibers is not known. Since descriptions of taste bud fibers in the literature are incomplete, we pursued the study of fibers in mice genetically labeled with the Brainbow method (Livet et al. 2007; Lichtman & Sanes, 2008; Weissman et al, 2011). This genetic labeling method, in our hands, revealed the entire surface morphology of randomly labeled, inherently fluorescence‐filled single gustatory fibers, thus permitting a closer examination of the innervation of taste buds than previously possible.

Taste receptor cells are generated on the periphery of the taste bud and migrate centrally as they differentiate (Beidler & Smallman, 1965; Farbman, 1980; Oliver & Whitehead, 1992; Perea‐Martinez et al. 2013). Over a brief period of days or weeks, they mature, die, and are replaced from the periphery. Since individual mouse fungiform buds are innervated by only three to five ganglionic nerve fibers (Zaidi & Whitehead, 2006), it is possible to trace single fibers throughout the bud and relative to central and peripheral regions. Thus, with confocal microscopic mapping of Brainbow‐labeled fibers in mouse, the relationship of taste nerve fibers to the bud regions engaged in gemmal cellular dynamism was explored.

Materials and methods

The Brainbow‐Nestin labeled mice (n = 10) were obtained by mating homozygous mice from the founder line L, strain #7910 [B6;CBA‐Tg(Thy1‐Brainbow1.0)LLich/J] with hemizygous Nes‐Cre mice, strain #3771 [B6.Cg‐Tg(Nes‐cre)1Kln/J]. Both nestin‐Cre and synapsin‐cre transgenic mice when mated with Brainbow mice yield labeling of a random subset of sensory ganglion cells (J. Sanes pers. comm.). In the present application of the method, results were better for Nestin‐Cre, Brainbow animals. F1 progeny were genotyped by tail clipping for the presence of Brainbow‐Nestin alleles. Animals were deeply anesthetized, perfused with 4% paraformaldehyde, and the tongues infiltrated with 30% sucrose. Transverse sections of the tongue (a plane resulting in longitudinal sections of most taste buds) were cut at 20 μm on a cryostat and the sections affixed to slides and coverslipped with Vectashield Mounting Medium (Vector Laboratories, H‐1000). Some sections, prior to coverslipping, were immunostained with affinity‐purified goat anti‐KCNQ1 (Santa Cruz, SC‐10646, raised against a synthetic peptide of human KCNQ1) in order to define the borders of the taste bud. Specificity of the antibody was confirmed by the manufacturer via Western blotting and verified by other researchers (Rasmussen et al. 2004). These sections were rinsed, then immunoreacted with donkey anti‐goat Alexa 647 to precisely demonstrate the extent and peripheral edges of the taste bud (Wang et al. 2009). Additionally, the trigeminal, geniculate, and glossopharyngeal ganglia were harvested from the fixed animals and imaged with confocal microscopy as whole mounts on slides.

Imaging of Brainbow fluorescence was performed with a Leica TCS SP5 II confocal microscope with settings for CFP (excitation at 405 nm, emission 300–450 nm), YFP (excitation at 515 nm, emission 800–960 nm), and RFP (excitation at 488 nm, emission 400–500 nm), fluorescence. All photomicrographic images in Figs 2, 3, 4, 5, 6 were obtained as merged z stacks of 20‐μm sections with line averages of 5–8 and frame average accumulations of 2. Each 20‐μm section was typically imaged as 40 optical z slices.

Figure 2.

(A) Brainbow‐labeling of geniculate ganglion neurons demonstrating their multicoloration resulting from random recombination; they constitute a subset of all ganglion cells (unlabeled cells are not evident in the images). (B–D) Labeled ganglion cells viewed with each of the three fluorescence settings that are merged in (A): GFP/green (B); RFP/red (C); CFP/blue (D). Note that some cells express mainly green fluorescence (arrowheads), other cells express mainly red (thick arrows), and other cells express mainly blue (thin arrows). Additionally, some cells express significant mixtures of fluorescence: green + red (arrowhead + thick arrow); red + blue (thick arrow + thin arrow); green + blue (arrowhead + thin arrow); green + red + blue (asterisks). Thick labeled fibers in cross‐section, upper right, are axons of the motor facial nerve. Thin multicolored profiles between the labeled somata are fibers of the geniculate neurons. Scale bar: 10 μm.

Figure 3.

Brainbow‐labeling of intragemmal and perigemmal innervation of a fungiform taste bud and papilla.demonstrating random multicoloration of the fibers. (A) Merged fluorescence illustrates perigemmal fibers and endings expressing predominantly GFP (green) (arrowheads), perigemmal fibers expressing predominantly CFP (blue) (thin arrows), and perigemmal endings expressing a mixture of green (GFP), blue (CFP), and red (RFP) (asterisk). Two intragemmal fibers expressing predominantly green fluorescence (double arrowheads) enter the base of the bud. One intragemmal fiber ends as a blunt ending in the bud periphery (triple arrowheads) and the other expands with filopodia at its ending in the central bud; the expansion expresses green and blue fluorescence (arrowhead + thin arrow) (compare the coloration of this structure in A, B, C, D). (B–D) Labeling viewed with each of the three fluorescence settings that are merged in (A). Scale bar: 10 μm.

Figure 4.

Brainbow‐labeled intragemmal and perigemmal innervation of a fungiform papilla, the taste bud of which also contains Brainbow‐fluorescent cells. (A) A thin intragemmal fiber that expresses green and red fluorescence only (double arrowheads + thick arrow) ascends the papillary core and enters the base of the taste bud. Fluorescent perigemmal fibers include a thick one ascending the papillary core and perigemmal endings, both expressing predominantly green and blue (arrowhead + thin arrow), and perigemmal fibers and endings expressing predominantly green and red (arrowhead + thick arrow). Two adjoined, spindle‐shaped Brainbow‐fluorescent gemmal cells with non‐fluorescent nuclei express green, red and blue in the central bud (asterisks). Immunostaining for KCNQ1 delineates the bud (pink) (dashed lines). (B–D) Labeling viewed with each of the three fluorescence settings that are merged in (A). Scale bar: 10 μm.

Figure 5.

Intragemmal and perigemmal fibers labeled with Brainbow fluorescence in merged images and corresponding drawings (fiber drawings were arbitrarily colored digitally for clarity). (A) Thin intragemmal fibers (1,2) ascend to the taste bud and become expanded within the bud (dashed lines). Fiber 1 (see also drawing in B) is unbranched, bears preterminal swellings (arrowheads), and ends bluntly. Fiber 2 (see also drawing in B), by contrast, is irregularly shaped, branches widely in the basal half of the bud and bears thin terminal filaments and swellings (e.g. at arrowheads), one of which appears as a retraction bulb (small double arrows) below the bud. A thick perigemmal fiber (5) (see also drawing in B) ascends the papillary core and crosses over the intragemmal fibers to terminate as lumpy enlargements in the epithelium surrounding the bud. Red fluorescence within the bud appears to indicate faintly labeled gemmal cells. Small yellow fluorescent blobs (see text) are seen in and below the bud (inset: higher magnification). (C) Intragemmal fibers (3,4) ascending to the taste bud (dashed lines) become expanded as elaborately branched endings within the bud (see also drawings in D). Fiber 3 enters the central bud at its base, then branches abruptly, entering the peripheral bud before recurving centrally to end in the apical half of the bud; its changes of direction are marked by swellings (arrowheads). Fiber 4 thickens after entering the peripheral bud before trifurcating into apical and centrally directed branches; the branch point bears a large swelling (arrowhead). The endings of fiber 4 appear, morphologically, as growth cones (small arrows). Fibers 5 and 6 are perigemmal fibers; they bear lumpy endings in the non‐taste epithelium. Scale bars: 10 μm (3 μm for inset).

Figure 6.

Brainbow‐labeling of intragemmal and perigemmal fibers associated with fungiform and circumvallate taste buds. (A) A single intragemmal fiber, seen in its entirety in the plane of this oblique section, exhibits shape irregularities immediately below and within the base of the bud (the basal lamina is indicated by dashed lines) where it branches to reach a broad region within the central bud. The endings are expanded (small arrows). (B) Drawing of the fiber in A. (C) Perigemmal fibers (arrows) ascend in the epithelium adjacent to the sides of the taste bud (TB), ending as rounded swellings in the non‐taste surface epithelium (asterisks). Intragemmal fibers (arrowheads) enter and terminate, often with swellings (small arrow) or ball‐on‐stalk endings (double small arrows) within the taste bud. Inset: small yellow/white fluorescent blobs (see text) are present in the central bud. (D) Vallate taste buds contain intragemmal fibers that reach all regions of each bud. The fibers are predominantly oriented vertically, toward the surface of the epithelium. They are branched and bear preterminal and terminal swellings. Preterminal gustatory fibers course parallel to the epithelial basal lamina and turn at right angles to enter the buds. Brainbow‐fluorescent gemmal cells (arrows) within vallate buds; the apical process of one terminates at the taste pore (P). Inset: There are few perigemmal fibers (asterisk). Scale bars: (A–C) 10 μm, (inset to A–C) 3 μm, (D) 20 μm, (inset to D) 40 μm.

All laboratory procedures were approved by the University of California San Diego Laboratory Animal Care and Use Committee and followed the NIH ‘Guide for the Care and Use of Laboratory Animals’.

Image processing and analysis

Fluorescently labeled taste buds were mapped and quantified (for the extent of fluorescence labeling) using the java‐based imagej software (freely available from NIH; http://imagej.nih.gov/ij/). Multicolored confocal images were first converted to 8‐bit gray‐scale images, adjusted for optimum contrast and brightness, converted into binary image with two pixel intensities (black = 0 and white = 255), before tracing the outer bud boundary and superimposing, in a standardized fashion, a template on each bud image dividing it into four intra‐gemmal bud regions, namely: apical central (a), basal central (b), peripheral 1 (p1) and peripheral 2 (p2) (Fig. 1). The apical region (yellow triangle ‘a’ in Fig. 1) was established by connecting three reference point coordinates located at 12 o'clock (usually the taste pore), +r (r = radius of the traced circle closely enclosing the bud), −r coordinates (at 3–9 o'clock line; as shown in Fig. 1). Similarly, the basal region (cyan quadrilateral ‘b’; Fig. 1) was drawn by connecting the baseline of the apical triangle to 5½ and 6½ o'clock coordinates (at the bottom of the taste bud; usually including the entry point of the nerve fibers; see Fig. 1). Two flanking regions, p1 and p2, were drawn by tracing the outer margins of the taste bud between coordinates, 12 o'clock, 9 o'clock, 6½ o'clock, and connecting it to – ½r (highlighted in pink in Fig. 1) and 12 o'clock (for p1; highlighted in pink in Fig. 1), or 12 o'clock, 3 o'clock, 5 ½ o'clock and +r (for p2; highlighted in green in Fig. 1), respectively. A line connecting ½r and ‐r (equal to the base of the apical triangle), is drawn using a ‘line’ tool, calibrated for the known distance/size by setting the ‘scale line’ ‘set scale’ to 25 μm. ‘ROI Manager’ tool was used to measure areas (μm²) of ‘b’, ‘a’, ‘p1’ & ‘p2’ bud regions. Similarly, ‘Analyze Particle’ tool was used to measure integrated density of fluorescently labeled pixels of the nerve fibers, selectively, within each region. More detailed methodology can be found at http://imagej.nih.gov/ij/docs/index.html.

Figure 1.

Illustration of the regions within each bud section for which the degree of nerve fiber fluorescence was quantified (see data in Fig. 7). Numbers refer to locations around the bud as if viewing a superimposed clock: 12 o'clock at the taste pore, 3 and 9 o'clock at the bud sides, etc. Letters refer to subregions within the bud: central apical region (a, yellow); central basal region (b, cyan); peripheral regions (p1, red) and (p2, green). See Materials and methods for full description.

Nerve fiber drawings

Drawings were made of representative single nerve fibers, both intragemmal and perigemmal. The fibers selected for drawing were studied in the z series and determined to have all branches and endings entirely contained within the 20‐μm thickness of the histological sections. This was verified by examining each of the 40 ‘optical z slices’ and noting that Brainbow‐labeled processes terminated within the section and were not truncated by cryostat microtomy. The drawings were reconstructed from tracings on clear plastic film of each segment of the depicted nerve fiber evident in each of the ‘optical z slices’. Shading of the fibers and their branches by stippling was employed to indicate the depth of the processes within the section and to show the relative depth of one fiber to another in cases where several fibers were depicted within one taste bud. Thus, light stippling indicates elements close to the surface of the section and darker stippling indicates elements more deeply located. Drawings of some fibers (e.g. in Fig. 5B,D) were arbitrarily colored to differentiate one fiber from another in single sections.

Assessing GAP‐43 immunoreactivity

To assess for possible growth and remodeling of nerve fibers suggested by the morphology and distribution of labeled fluorescent cellular elements, we analyzed the distribution of GAP‐43 immunoreactivity, an established measure of developing fibers and growth cones (e.g. Allegra Mascaro et al. 2013). For immunohistochemistry of this marker, slides with 20‐μm, thick frozen, longitudinal sections of taste buds from wild‐type mice were rinsed three times in phosphate‐buffered saline (PBS) and treated with a ‘primary’ solution of 0.3% Triton X‐100, 5% normal goat serum, 1× PBS and rabbit anti‐GAP‐43 antibody (1 : 250; Millipore) at 4 °C for 12 h. The antibody as supplied had been purified from GAP‐43 protein isolated from cat brain. The specificity of this polyclonal antibody was previously established by immunoblots of proteins from cat, monkey, human and bovine neural tissue showing specific staining of a single protein in both soluble and membrane fractions (McIntosh & Parkinson, 1990). The slides were then rinsed three times with PBS and treated with a ‘secondary’ solution of 0.3% Triton X‐100, 1× PBS, and Alexa Fluor 488 goat anti‐rabbit antibodies (1 : 750; Invitrogen). Slides were shaken for an hour in a light‐tight box.

P2X2 immunoreactivity, a taste nerve fiber marker (Finger et al. 2005), was assessed to determine whether GAP‐43 staining coincides with intragemmal innervation. For P2X2 immunohistochemisty, the primary solution contained 0.3% Triton X‐100, 5% normal donkey serum, 1× PBS rabbit anti‐GAP‐43 (1 : 250), and goat anti‐P2X2 (1 : 100). The P2X2 antibody is an affinity‐purified goat polyclonal antibody raised against a peptide mapping within an internal region of P2X2 of human origin (Santa Cruz Biotechnology). Slides were then treated with a secondary solution of 0.3% Triton X‐100, 1× PBS, Alexa Fluor 488 donkey anti‐rabbit (1 : 750), and Alexa Fluor 594 donkey anti‐goat (1 : 500). Control for specific immunoreactivity in the present experiments was the demonstration of no immunoreactivity when processing control tissue as above while eliminating the primary antibody. Slides were mounted with Aqua‐Mount (Lerner Laboratories) and refrigerated in the dark until analyzed.

Results

Sensory ganglion cell labeling

A key requirement for labeling of taste bud nerve fibers with Brainbow fluorescence is labeling of the gustatory ganglion cell somata that give rise to gemmal innervation. Therefore, we evaluated the geniculate and glossopharyngeal ganglia with fluorescence microscopy. Brainbow‐colored, fluorescent cell bodies were, in every case examined (n = 10), present as a subset of the total ganglionic somata (Fig. 2); many cells were unlabeled. Similarly, fluorescent somata were also seen in the glossopharyngeal ganglion (data not shown). In terms of coloration, the labeled geniculate somata expressed RFP (colored red in the images), YFP (colored green), and CFP (colored blue), variably expressed and often mixed together in single cells. Thus, some labeled ganglion cells contained red, green, and blue in approximately equal amounts (Fig. 2, asterisks). Other cells exhibited mostly red and green fluorescence (Fig. 2, thick arrow + arrowhead), green and blue fluorescence (Fig. 2, arrowhead + thin arrow) or red and blue fluorescence (Fig. 2, thick arrow + thin arrow). Finally, some cells were predominantly red (Fig. 2, thick arrow), predominantly green (Fig. 2, arrowhead) or predominantly blue (Fig. 2, thin arrow).

Counts of single mid‐sections (horizontal plane) of the geniculate ganglia from four animals yielded the following proportions of colored cells (n = 152): 39 cells (26% of total) were predominantly red, 46 cells (30%) were predominantly green, and 18 cells (12%) were predominantly blue. Cells with readily discernible mixtures were: six cells (4%) containing red, green, and blue; four cells (3%) containing mostly red and green, 16 cells (10%) containing mostly green and blue; and 23 cells (15%) containing mostly red and blue. Consistent with the coloration of the geniculate ganglion cell bodies, the fibers of these cells were multicolored at the level of the ganglion (small colored profiles scattered between the cells bodies in Fig. 2A).

Fungiform papilla fibers, trajectory and morphology: perigemmal nerve fibers

Two types of sensory fibers are known to innervate fungiform papillae. One type, ‘perigemmal’ fibers, is derived from the lingual branch of the trigeminal nerve and innervates the non‐sensory epithelium immediately flanking the bud (Nishimoto et al. 1982; Whitehead et al. 1985; Finger, 1986). The other type, ‘intragemmal’ fibers, is derived from the facial nerve and innervates the taste bud proper. Both types were evident as colored fluorescent fibers in most of the fungiform papillae examined that contained labeling (Figs 3, 4, 5). Only a minority, approximately 25%, of fungiform papillae examined contained labeled nerve fibers in the present material; the others were devoid of fluorescent fibers, consistent with the Brainbow fluorescence appearing randomly in a subset of neurons generally (Livet et al. 2007) and in the taste ganglia in the present material (Fig. 2).

Perigemmal fibers ascend from the connective tissue core of the papilla, i.e. from the region immediately below the bud, especially below the lower sides of the bud (Fig. 3, thin arrows; Fig. 4, arrowhead + thin arrows; Fig. 5A,B, fiber 5; Fig. 5C,D, fiber 6). The fibers then pass into the non‐taste epithelium and ascend alongside the bud to the outer layers of the squamous epithelium to end near the surface in the region surrounding the apex of the taste bud, i.e. within the epithelium flanking the taste pore. These extragemmal fibers form preterminal apically directed branches within the perigemmal epithelium and, despite their proximity to the taste bud, they generally skirt the bud, often very closely, but do not enter it. Their terminal morphology consists of multiple large rounded enlargements that, collectively, in the case of several fluorescent fibers, appear as grape‐like clusters or ‘strings‐of‐pearls’ surrounding the pore near the surface of the epithelium (Fig. 3, single arrowheads, asterisk; Fig. 4, intraepithelial thick arrow + arrowhead and arrowhead + thin arrow; Fig. 5, fiber 6; Fig. 6C, asterisk). Consistent with the identity of these extragemmal fibers as trigeminal, fluorescent cell bodies were seen in the mandibular division of the trigeminal ganglion (data not shown). Regarding Brainbow coloration, perigemmal fibers expressed either one color predominantly, e.g. green fluorescence (Fig. 3, arrowheads) or blue fluorescence (Fig. 3, thick arrows), or mixtures of colors, e.g. blue and red (Fig. 3, asterisk); green and blue (Fig. 4, arrowhead + thin arrow) or red and green (Fig. 4, thick arrow + arrowhead).

Fungiform taste bud fibers, trajectory and morphology: intragemmal nerve fibers

Intragemmal fibers enter the base of the bud after ascending in the connective tissue of the papillary core in close association with the extragemmal fibers; indeed the extragemmal‐bound fibers surround the intragemmal‐bound ones; the latter are typically central within the connective tissue core of the papilla, i.e. directly below the center of the bud (cf. Fig. 3, double arrowheads with Fig. 3, thin arrows; cf. Fig. 4, double arrowheads + thick arrow with Fig. 4, arrowhead + thin arrow; cf. Fig. 5, fibers 1–4 with Fig. 5, fibers 5,6; cf. Fig. 6, arrowheads with Fig. 6, large arrows). By contrast with extragemmal fibers that skirt the edges of the bud, intragemmal fibers can be traced directly into the bud, typically entering its base or basolateral edges. Determination of the boundaries of the bud was aided by counterstaining with immunocytochemistry for KCNQ1 (e.g. Fig. 4A, pink staining, dashed outlines). Such staining was also performed on serial sections (data not shown) adjacent to the ones depicted (e.g. in Figs 3, 5 and 6) as a check on the border between the taste bud and the non‐taste epithelium. Once within the bud, the intragemmal fibers often branch in a variety of directions. Some fibers enter the base of the bud at its center and ascend (Fig. 3, double arrowheads; Fig. 4, double arrowheads + thick arrows; Fig. 5, fiber 1). Other fibers enter the base of the bud near its sides, then bifurcate immediately, sending one major branch apically within the periphery of the bud, i.e. near and parallel to its sides, and one or more branches centrally (Fig. 5, fibers 2,4). Some terminal branches turn abruptly and are directed recursively, back toward the basal region of the bud (Fig. 5, fiber 4, thick arrow; Fig. 6B, thick arrow); many extend apically to terminate below the taste pore region (Fig. 5, fiber 4, thin arrows). The ends of the branches are typically expanded as ruffled lamellae (Fig. 3, arrowhead + small arrow) or as rounded swellings (Fig. 6C, small single arrow). Some bear small, thin protuberances resembling filopodia (Fig. 5B,D thin profiles extending from nerve fibers); others exhibit rounded ends terminating thinned preterminal process, and appear as ball‐on‐stalk ‘retraction bulbs’ (Morest, 1968; Whitehead & Kachele, 1994) (Fig. 5, fiber 2, double arrows; Fig. 6C, double arrows). Swellings are also common at the branch points of intragemmal fibers (Fig. 5, fibers 1,3,4, arrowheads). At the point of entry into the bud from the subjacent lamina propria, i.e. at the basal lamina, the fiber diameters frequently change abruptly from thin below, to expanded above (e.g. Fig. 5, fiber 4).

The detailed morphology of individual intragemmal fibers was documented in three dimensions by tracing the segments of single fibers in each of the 40 ‘optical z slices’ of the histological section, then uniting the segments and reconstructing the fibers as drawings with arbitrary coloration and shading indicating the depth of the various fiber elements within the section. Figures 5B,D (fibers 1–4) and 6B depict intragemmal fibers that, from close examination and tracing though all optical z sections, had branches and endings entirely contained within the 20‐μm histological sections images by confocal microscopy, i.e. the fibers drawn were not truncated by cryostat sectioning. The fibers drawn were the same as the ones appearing in the merged images of Figs 5A,C and 6A, respectively. The fibers all enter the bud at its base. Fibers 2, 3, and 4 (Fig. 5) then branch to reach all regions of the bud: central basal, central apical, and peripheral. The fibers are highly irregularly shaped with thick and thin segments; they bear swellings, end‐bulbs, and terminal shapes resembling growth cones (e.g. Fig. 5, fiber 4, small arrows). Fiber 1 (Fig. 5B), the simplest depicted, is unbranched and bears swellings (arrowheads) and a blunt tip; it terminates in the basal half of the bud. Fiber 2 (Fig. 5B) branches basolaterally and forms widely spaced irregular endings with extensions tipped by swellings. Fiber 3 (Fig. 5D) enters the bud basally, turns abruptly, first entering the periphery of the bud, then extending to the central bud; its direction changes occur at sites with swellings (arrowheads). Fiber 4 (Fig. 5D) enters basolaterally, ascends in the periphery of the bud before trifurcating, sending branches apically and central basally; the branch point is swollen (arrowhead), the endings appear as growth cones (small arrows). The fiber in Fig. 6A,B enters the bud basolaterally where it branches, sending a curved branch recursively to the basal central edge of the bud (thick arrow before ending centrally). Fibers 5 and 6 in Fig. 5 are perigemmal and end in lumpy ‘string‐of‐pearls’ swellings in the non‐taste keratinizing epithelium.

Innervation of different taste bud regions

The following quantitative summary of the distribution of labeled fibers in fungiform buds is based on six animals, 17 taste bud profiles, one to 10 buds per animal, including five instances of profiles comprising two serial sections. The most heavily innervated taste bud region is the ‘basal zone’ (defined in Materials and methods) (Fig. 7). This region obtains its innervation primarily from the nerve fibers in the subgemmal connective tissue, since that is the location from which most fibers, regardless of where they terminate, enter the bud. Additionally, some fluorescent processes in the basal zone derive from fibers in other zones that turn basally to enter or re‐enter this region of dense innervation. The ‘apical zone’ is slightly less heavily innervated than the basal zone, its fluorescent fibers and endings typically derived as apical extensions of more basal fibers, some reaching close to the taste pore. The ‘peripheral zone’ bordering the sides of the bud also contained labeled fibers, but, in terms of degree of fluorescence, to a lesser extent than the central bud. This fluorescence consists of fibers that were extensions of ones entering the bud alongside but lateral to the central basal region, or extensions or branches of fibers that first entered the central basal region. While the peripheral regions contained less fluorescence than the two central regions, the bud periphery in most cases contained at least some innervation from intragemmal fibers. Only three of 17 bud profiles quantified were devoid of peripherally located fluorescent fibers. The differences in the degree of fiber fluorescence in the central (a + b; Fig. 7) and the peripheral (p1 + p2; Fig. 7) regions were consistent. The central region contained a significantly greater amount of fiber fluorescence than the peripheral region (t‐test, P < 0.0001).

Figure 7.

Degree of fluorescence‐nerve fiber labeling within the subregions of the fungiform taste bud (a = apical region, b = basal region, p1 and p2 = peripheral regions) (see Fig. 1). The central basal region (b) has the highest percentage of total bud fluorescence (36.9 ± 15.4%), and the central apical region (a) has an intermediate percentage (27.6 ± 15.3%). Left and right peripheral regions (p1 and p2) contain the least amount of labeling (21.3 ± 15.0%, 16.1 ± 13.7 SD). Total central (a + b) labeling (64.5 ± 17.3 SD) is ~ twofold higher than total peripheral (p1 + p2; 35.5 ± 17.3% SD) labeling (P < 0.0001).

Distribution of single intragemmal fungiform fibers

The above quantification was performed on the overall fluorescence of intragemmal fibers that varied in number from one to five labeled fibers at the entry zone of the bud. To characterize the regional distribution of single fibers, the confocal z series of the bud profiles (i.e. of the 20‐μm mid‐longitudinal sections) were analyzed for single fibers that could be followed from their entry into the bud and to their terminals (see Figs 5B,D and 6B) (or to their cut ends, for fibers leaving the plane of the section). In the sections examined, 28 single fibers from five animals were tracked from their entry into the bud throughout the three gemmal regions (‘zones a, b, and p’ as defined above and in Materials and methods, and depicted in Fig. 1) into which the fluorescent profiles, including any branches, extended. Three types of fibers in equal proportions were documented. Ten of 28 total fibers innervated both the basal and apical zones but did not enter the peripheral zone (e.g. fiber 2 in Fig. 5D). By contrast, eight of 28 fibers innervated the basal zone and peripheral zone, but not the apical zone. An additional nine fibers innervated all three zones (e.g. fibers 3 and 4 in Fig. 5D). The remaining single fiber innervated the peripheral and apical zones only.

Brainbow‐labeling of fungiform taste bud cells

Surprisingly, despite reports of neuronal specificity of Brainbow‐labeling (Livet et al. 2007; Lichtman & Sanes, 2008), fluorescence in the bud was not limited to nerve fibers. Labeling was also frequently seen in one or several cells within each bud (Fig. 4, asterisks). Cells were identified based on their spindle shape, and their confinement to the bud with a tapered apical end reaching the taste pore and a basal end foot at the basal lamina. Nuclei of the labeled cells were seen at the swollen mid‐points of the cells (Fig. 4, asterisks). Importantly for differentiating innervation from gemmal cells, the fluorescent cells were frequently colored differently from the nearby nerve fibers. Indeed, the cells in Fig. 4 clearly express all three fluorophores, red, green, and blue, unlike any of the fibers. In addition to recognizable gemmal cells, the central bud and the subgemmal connective tissue core of the papilla also frequently contained differentially colored, amorphous clusters of small intensely fluorescent blobs or small dots (Figs 5B and 6C, insets). These clusters of fluorescent blobs resemble degenerating cellular material or phagosomes. Fluorescent blobs were never seen in broad areas of the non‐taste epithelium of the anterior tongue but were a frequent finding in fungiform papillae within their connective tissue cores, and within the taste buds proper.

Circumvallate taste bud innervation

Sections of circumvallate taste buds were also examined for fluorescent nerve fibers. Nerve fibers were seen in most of the many buds lining the circumvallate trench, in contrast to the minority of fungiform buds containing labeled fibers. Nevertheless, morphologically, circumvallate bud innervation bore many similarities to fungiform bud innervation. Nerve fibers innervated all regions of the bud, basally, apically, and peripherally (Fig. 6D). The preterminal fibers traveled parallel to and immediately below the circumvallate epithelium before turning at right angles to enter the buds on both sides of the trench. The intragemmal fibers extended predominantly apically to terminate, although there were branches that extended in all directions. Individual fibers varied in diameter and bore swellings and irregular morphologies similar to those of fungiform taste buds. Circumvallate buds also occasionally contained Brainbow‐fluorescent gemmal cells (Fig. 6D, arrows). In contrast to fungiform papillae, extragemmal fluorescent fibers were few. The rare extragemmal fluorescent fibers terminated in the non‐taste epithelium between the circumvallate buds (Fig. 6D inset, asterisk).

Evidence for ‘growth’ of mature taste bud fibers

To obtain a measure of the nerve fiber plasticity suggested by the growth‐associated morphological features of intragemmal fibers [growth cone‐like expansions and filopodia, and features resembling degenerating or remodeling cellular elements, i.e. the fluorescent clusters of blobs (see above)], we analyzed the distribution of GAP‐43 immunoreactivity, indicative of developing fibers and growth cones (Benowitz & Routtenberg, 1997; Dent & Meiri, 1998; Frey et al. 2000). We found that GAP‐43 immunoreactivity was invariably detected as small clusters of immunoreactivity centrally in the taste bud proper and in the connective tissue core of the fungiform papilla below taste buds and superficial to the muscle layer (Fig. 8A). Similarly, GAP‐43 immunoreactivity in the anterior tongue was intense and specifically located exclusively within and below developing fungiform taste buds in newborn mice (Fig. 8B), a period when buds are first forming and have newly acquired innervation from ingrowing geniculate ganglion cell fibers during the late prenatal and early postnatal periods (e.g. Ma et al. 2009). This latter immunoreactivity in developing bud innervation is a positive control for the interpretation that GAP‐43 immunostaining in mature taste papillae represents growth or remodeling of preterminal fibers and fiber endings, and the presence of growth cones (see also Benowitz & Routtenberg, 1997; Berman et al. 1998; Dent and Meiri, 1998; Frey, et al. 2000; Inoue et al. 2006).

Figure 8.

(A) GAP‐43 immunoreactivity in developing and mature fungiform taste buds. GAP‐43 associated with fibers is present subepithelially, below the taste bud, in the fungiform core, between the base of the bud and the muscle layer, and within the basal taste bud proper (arrow). (B) Intense GAP‐43 immunoreactivity in a developing fungiform taste bud in a 1‐day‐old mouse, associated with growing GG cell fibers, the timing and nature of which are established for the mouse (Ma et al. 2009). Scale bars: 20 μm. (C) Confocal micrograph demonstrating co‐localization of GAP‐43 and P2X2 immunoreactivity. GAP‐43 (green) entirely overlaps P2X2 nerve fiber staining (red) in this merged image (yellow). Immunoreactive double staining is particularly dense at the base of the fungiform papilla (boxed area). Double staining of punctate profiles is also present in the taste bud (TB) proper (arrowheads). Some fibers located subepithelially or ascending to the taste bud are single‐stained for P2X2 (arrows). Scale bars: 20 μm.

The localization of GAP‐43 to the cores of normal fungiform papillae directly below the bud, and to the taste bud, is consistent with the location of gustatory fibers of the chorda tympani (Whitehead & Kachele, 1994; Lopez & Krimm, 2006). Double labeling for GAP‐43 and P2X2, a marker for chorda tympani and gustatory nerve fibers (Bo et al. 1999; Finger et al. 2005) showed a close overlap between the two markers (Fig. 8C). All green GAP‐43 immunoreactivity coincided with red P2X2 fibers; some P2X2 immunoreactive fibers and fiber segments were devoid of GAP‐43 immunoreactivity. The double‐labeled fibers were especially prominent in concentrations at the bases of fungiform papillae near the muscle layer of the tongue. There was also double immunostaining of fibers in the fungiform core, consistent with the single GAP‐43 labeling above, and in the taste bud itself, where the double‐labeled profiles were punctate, suggestive of nerve fiber swellings en passant or endings.

Discussion

Brainbow‐fluorescent labeling enabled discoveries about the nature of taste bud innervation. Intragemmal nerve fibers were found to innervate all regions of fungiform taste buds. Individual fibers branched within the bud, distributing terminals to both the periphery, where immature gemmal cells are located, and to central regions, apically and basally, where mature cells are located. The fibers are irregularly shaped with structural features resembling those of growing and remodeling fibers previously documented for the developing nervous system (e.g. Morest, 1968; Whitehead & Morest, 1985a,b). In addition to nerve fibers, gemmal cells were sometimes Brainbow‐fluorescent, as were scattered small amorphous elements within and below the bud, findings suggestive of degenerating elements of a neural or taste bud cellular origin.

Brainbow‐labeling of perigemmal and intragemmal nerve fibers

In the present application of the Brainbow method we obtained labeling of both perigemmal and intragemmal nerve fibers. Methodologically, this result is unprecedented; there are no reports of the Brainbow method being used previously to study peripheral sensory nerve fibers. Indeed, in general, neuroanatomical methods that have labeled peripheral fibers of sensory neurons are few. Those that have been used to label taste bud fibers obtained results consistent with those presented here. Perigemmal fibers surrounding the taste bud and ending around the taste bud pore region have been identified with immunocytochemistry for calcitonin‐gene‐related peptide (CGRP) (Finger, 1986; Silverman & Kruger, 1989), Substance P (Nishimoto et al. 1982; Finger, 1986), synapsin I (Finger et al. 1990), and stained with methylene blue (Muller, 1996a,b) and the Golgi method (Whitehead & Kachele, 1994). Brainbow‐labeling of perigemmal fibers showed them as thicker than intragemmal‐destined fibers in the connective tissue core of fungiform papillae and ascending closely alongside the taste bud to end near the surface of the papilla in the non‐taste epithelium. Clear labeling of the endings in the present material revealed them as forming apically directed branches and ending as clustered rounded enlargements, consistent with previous reports (Finger, 1986; Whitehead & Kachele, 1994; Muller, 1996a,b).These enlargements have been described in electron micrographs of the taste pore region (Whitehead et al. 1985; Kinnamon et al. 1988); they persist after chorda tympani resection (Whitehead et al. 1985), thus establishing their origin from the lingual, trigeminal nerve.

The Brainbow method produces various colors in neurons by random, combinatorial, and differential expression levels of a few spectrally distinct fluorescent proteins (Livet et al. 2007; Weissman & Pan, 2015). The recombination resulting in multicoloring is generated in the progeny of Brainbow animals mated with cre animals; in the present approach, mating was with nestin‐cre mice. This particular mating resulted in clear, differential fluorescent coloring of geniculate, glossopharyngeal, and trigeminal ganglion cell bodies, similar to results previously published for central neurons (Livet et al. 2007). The peripheral fibers of these cells, however, presented less variety of multicolored labeling. CFP alone appeared to color most fibers and a minority were colored with YFP and RFP, either alone or in combination with CFP. This discrepancy between cell body and peripheral fiber labeling, with less color variety in the latter, probably relates to the small diameters of the fibers compared with their parent cell bodies, together with limitations of the confocal technology available for this study. With our technology, the ‘windowing’ for wavelengths of excitation and emission spectra were suboptimum for RFP and YFP due to limitations of the available microscope configuration, and therefore CFP usually dominated in color. Nevertheless, because single fungiform taste buds of mouse are innervated by only a small number of geniculate ganglion cells, usually four to six (Zaidi & Whitehead, 2006), the Brainbow‐labeled fibers, being few, could usually be traced unambiguously throughout their extent in the 20‐μm‐thick sections analyzed, even when all fibers were colored similarly within a bud.

Innervation of gemmal regions

Taste bud cells are renewed over a time course of days to weeks (Beidler & Smallman, 1965; Oliver & Whitehead, 1992). The cells are generated by division of germinative cells at the base or basal sides of the bud (Perea‐Martinez et al. 2013). Newly generated cells are located on the periphery of the bud; older, more differentiated cells are located centrally (Beidler & Smallman, 1965; Oliver & Whitehead, 1992; Perea‐Martinez et al. 2013). Mature cells eventually die, break up, and are cleared from the bud by cellular processes that have not been investigated. Nevertheless, this cellular dynamism requires that gemmal nerve fibers continuously form, lose, and then reform connections with receptor cells that change over time. The present finding that many intragemmal fibers innervate both peripheral and central regions of the bud suggests that single fibers can contact both mature and newly generated gemmal cells, perhaps as a mechanism for ensuring continuity of signaling taste responses to the brain during receptor cell turnover. The swellings on Brainbow‐labeled fibers at branch points and at endings, both in the periphery of the bud and centrally in the bud, resemble those seen in previous light microscope studies (Muller, 1996a,b; Muller & Jastrow, 1998) and identified as synaptic or other functional receptoneural contacts in electron microscope studies (Kinnamon et al. 1988, 1993). Whether single Brainbow‐labeled fibers, by means of these putative contacts, form close associations with taste cell receptors all of the same type (Kinnamon et al. 1988) would in future light microscope studies require combining Brainbow single fiber labeling with labeling of identified gemmal cell types.

Brainbow‐labeling of cells

Both fungiform and circumvallate buds contained Brainbow‐labeled cells. This unprecedented and unexpected finding may reflect the ‘neural’ nature of taste bud cells. Indeed, both intragemmal nerve fibers and taste bud cells express neuron‐specific enolase (Yamagishi et al. 1995), a glycolytic enzyme specific for central and peripheral neurons (Marangos et al. 1976). Additionally, labeled taste bud cells were previously seen in studies that applied neuroanatomical tracers to taste bud nerves (Bradley et al. 1986; Finger & Bottger, 1990; Nagai, 1993). The labeled cells in these tracer studies were interpreted as resulting from the molecular transfer of applied nerve fiber marker into gemmal cells that presumably contacted the marker‐containing fiber. On the other hand, labeling of gemmal cells and fibers separately was previously reported as based simply on a common affinity for certain dyes (Muller, 1996a,b; Muller & Jastrow, 1998). In the present study, the labeled cells were not necessarily in contact with Brainbow‐labeled fibers, they were often colored differently from the nearby labeled fibers, and they tended to be located centrally despite a more widespread distribution of the fibers. These results suggest the interpretation that the Brainbow‐labeling of gemmal cells reflects expression of the marker in mature receptor cells independent from that of labeled nerve fibers. The present labeled gemmal cells resulted from mating Brainbow with nestin‐cre animals. Nestin was recently shown to be expressed in basal cells associated with the taste bud (Mii et al. 2014). The present labeling may, therefore, relate to cre expression in these bud progenitor cells. Alternatively, the labeled cells might correspond to a specific mature gemmal cell type, e.g. Type III cells, which in comparison with the other taste bud cell types express the most neuronal characteristics. Future study combining Brainbow‐labeling with markers for Type III cells (e.g. NCAM, PKD) or Type II cells (e.g. gustducin, PLCb2) would address this.

Morphological features and marker expression of growing, remodeling fibers

An earlier study of the morphology of developing taste bud fibers with the Golgi method showed that individual intragemmal fibers were characterized by multiple branchings, preterminal and terminal swellings, growth cones with filopodia, swellings, and rounded retraction bulbs (Whitehead & Kachele, 1994; in perinatal hamster). These morphological features have been identified in many previous light microscopic studies of developing neurons throughout the central and peripheral nervous system with the Golgi method and are well established in the literature as neural growth features (e.g. Ramon y Cajal, 1960; Morest, 1968, 1969; Whitehead & Morest, 1985a,b). Growing axons in the periphery viewed with time lapse in culture, or filled with the neuroanatomical marker HRP (Tosney & Landmesser, 1985; Bovolenta & Mason, 1987), similarly exhibit at their advancing, retracting, and remodeling ends, growth cone enlargements with filopodia and retraction bulbs. The present finding of similar structures on Brainbow‐labeled fibers suggests that intragemmal fibers may be engaged in continuous growth and remodeling. This interpretation is supported by the presence of GAP‐43 in mature mouse taste buds and, to a greater extent, in and below developing mouse taste buds during the ingrowth of their nerves. GAP‐43 expression is indicative of growing fibers and their growth cones (Benowitz & Routtenberg, 1997), including swellings associated with sprouting and remodeling axons (Allegra Mascaro et al. 2013). Evidence for growth‐related morphological and neurochemical features associated with intragemmal fibers supports the possibility that taste bud innervation is plastic, with synapses forming and retracting continuously (Kinnamon et al. 1993), perhaps in concert with the turnover of receptor cells.

Author contributions

Faisal Zaidi and Mark Whitehead designed the study, conducted data analysis, and wrote the manuscript. Vanessa Cicchini, Daniel Kaufman, Elizabeth Ko, Abaraham Ko and Heather Van Tassel generated the transgenic animals and acquired the data.