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Rapid structural remodeling of peripheral taste neurons is independent of taste cell turnover [1]

['Zachary D. Whiddon', 'Department Of Anatomical Sciences', 'Neurobiology', 'University Of Louisville School Of Medicine', 'Louisville', 'Kentucky', 'United States Of America', 'Jaleia B. Marshall', 'David C. Alston', 'Aaron W. Mcgee']

Date: 2023-09

Taste bud cells are constantly replaced in taste buds as old cells die and new cells migrate into the bud. The perception of taste relies on new taste bud cells integrating with existing neural circuitry, yet how these new cells connect with a taste ganglion neuron is unknown. Do taste ganglion neurons remodel to accommodate taste bud cell renewal? If so, how much of the structure of taste axons is fixed and how much remodels? Here, we measured the motility and branching of individual taste arbors (the portion of the axon innervating taste buds) in mice over time with two-photon in vivo microscopy. Terminal branches of taste arbors continuously and rapidly remodel within the taste bud. This remodeling is faster than predicted by taste bud cell renewal, with terminal branches added and lost concurrently. Surprisingly, blocking entry of new taste bud cells with chemotherapeutic agents revealed that remodeling of the terminal branches on taste arbors does not rely on the renewal of taste bud cells. Although terminal branch remodeling was fast and intrinsically controlled, no new arbors were added to taste buds, and few were lost over 100 days. Taste ganglion neurons maintain a stable number of arbors that are each capable of high-speed remodeling. We propose that terminal branch plasticity permits arbors to locate new taste bud cells, while stability of arbor number supports constancy in the degree of connectivity and function for each neuron over time.

Data Availability: The data for each Figure is available here: https://data.mendeley.com/datasets/d58vz7wfrf/1 The raw image data set for taste arbors imaged every 12 hours for 10 days is available here: 10.6084/m9.figshare.23589351 . Arbor reconstructions from which convex hull and arbor length were measured are available here: https://neuromorpho.org/dableFiles/whiddon_krimm/Supplementary/swc_files.zip Custom MATLAB software (ArborTools) used in this study is available for free download here: https://github.com/Dalston817/ArborTools .

Here, we examined the structure of peripheral taste axons using in vivo two-photon microscopy to determine how the structure of taste ganglion neurons changes over time. We discovered that peripheral axons of taste ganglion neurons have regions that rapidly remodel and regions that are stable throughout adulthood. Terminal branches (branch produced by the most peripheral branch point in the arbor) remodel rapidly, faster than expected based on the rates of taste cell turnover. This structural plasticity appears to be an inherent feature of the neurons and is not regulated by cell turnover. The total number of arbors (the portion of the axon that innervates the taste bud) for each taste ganglion neuron is stable. We conclude that taste ganglion neurons maintain their peripheral circuitry by having a stable number of arbors, with each arbor continuously remodeling to connect with new taste bud cells over time. We propose this combination of stability and plasticity supports consistency in the total number of taste-transducing cells providing input to an individual taste ganglion neuron and provides flexibility for forming connections with a continuously renewing population of taste cells.

In other parts of the nervous system, longitudinal in vivo imaging has been used to study the structural plasticity of axons and dendrites over time. Structural remodeling has been shown to occur during development and following injury or disease [ 15 – 21 ]. Under these conditions, axonal degeneration is the process that has been most extensively described [ 22 , 23 ]. In adult neurons, dendritic branches are stable and remodeling is restricted to dendritic spine turnover [ 24 – 26 ]. Axons may show a greater capacity to remodel than dendrites, but the magnitude of this remodeling varies between types of neurons [ 27 ]. It has not been determined if peripheral taste axons have the capacity to remodel and, if so, to what extent remodeling occurs.

Progress toward understanding the cellular movements occurring during taste bud cell turnover has been hindered by analysis of fixed-tissue [ 1 , 2 , 10 , 11 ]. This approach fails to capture the dynamic processes within the taste bud, such as taste cell movements and axon remodeling [ 1 , 2 , 12 ]. Fixed-tissue studies also fail to describe what happens to axon remodeling when cell turnover is disrupted (e.g., chemotherapies) [ 6 , 8 , 13 ]. Additionally, because it is unknown whether taste ganglion neurons undergo structural remodeling, it is unclear why there are morphological differences between individual taste ganglion neurons [ 14 ]. Morphological differences between individual taste ganglion neurons could be inherent features of the neuron that dictate differences in functional properties or could be snapshots in time of a continuously remodeling circuit that lacks functional stability. Understanding how taste ganglion neuron structure changes over time is required to relate taste ganglion neuron morphology to function in the taste system.

The replacement of cells in the taste bud was discovered more than 50 years ago [ 1 ]. In fact, cells within a taste bud are one of the fastest renewing cell populations in the body, with an average life span of 10 days [ 1 – 4 ]. Taste bud cells transduce chemical stimuli in the oral cavity and transmit a signal to the axons of taste ganglion neurons, which carry this information to the brain. Peripheral taste neurons are like other pseudounipolar sensory neurons in that each has a single process (axon) projecting from the cell body with an axon hillock. The single axon bifurcates and has a central terminal and peripheral receptive process. Because the peripheral portion of the taste axon is connected to taste bud cells, each time a taste bud cell dies, the axon of a taste ganglion neuron must connect with a new taste bud cell. These continuous reconnections are required if the system is to maintain functional integrity over time [ 5 – 7 ]. How this process is orchestrated is still unclear but could involve structural remodeling of the peripheral taste axon [ 8 , 9 ]. Alternatively, taste bud cells could migrate to the peripheral axon of the taste ganglion neuron, making neuronal remodeling unnecessary.

Results

Acute in vivo imaging reveals axonal structural plasticity To determine whether the structure of the peripheral taste axon is fixed or remodels over time, we developed an in vivo preparation to image the portion of the axon of a taste ganglion neuron that terminates in the taste bud (S1 Fig, arbors). These arbors enter taste buds at the basement membrane of the lingual epithelium. On the anterior two-thirds of the mammalian tongue, taste buds are housed in epithelial structures called fungiform papillae, which are easily accessible for imaging in live animals [28]. Within taste buds, arbors form a dense plexus with many overlapping branches [11,29]. We used a sparse-labeling strategy to express tdTomato in a small number of taste ganglion neurons so that individual arbors could be distinguished [14]. In this preparation, fewer than 5 arbors were labeled with tdTomato per fungiform taste bud and many taste buds did not contain any labeled arbors. Individual arbors were imaged using two-photon laser scanning microscopy (Fig 1A). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Intravital imaging of taste neurons in fungiform papillae. An example of a fungiform papilla with a single tdTomato-labeled receptive arbor illustrating small-scale changes within 6 hours. (A) Top row shows the z-projection of an arbor imaged parallel to the dorsal tongue surface (X, Y). Bottom (XZ) shows a Y projection view of the same arbor within a fungiform papilla. This arbor was imaged every hour over a 6-hour period. Some keratinocytes were also labeled (magenta arrowhead; [33]). After T0, images for each consecutive time point are enlarged to show only the labeled arbor. At T0, the taste bud is outlined (green line), and the connective tissue core of the papilla is outlined (purple line). Background florescence levels permitted the regions of the papilla to be distinguished. (B) High-magnification view (XY projection) of the arbor within the papilla core (red box) and taste bud (yellow box). The portion branch ending inside the taste bud adds a small process (yellow arrowhead) after 1 hour and then retracts small process (green arrowhead) resulting in a change in branch end shape. Another branch grows into view at hour 5 (cyan arrowhead). The portion of the axon in the papilla core did not change across 6 hours. (C) Comparison of average and maximum fluorescence (mean ±SD), of the same receptive arbors at hour 0 and hour 6, from an experiment in which arbors were imaged every hour for 6 hours. Fluorescence levels between hour 0 and 6 were unchanged. (D) GFP expression in the anterior tongue epithelium illustrating a papillae map. Papillae with labeled arbors are indicated by solid boxes at T0 and dashed boxes 24 hours later. The tongue midline is indicated with a white dashed line, and rostral (R) and lateral (L) directions are indicated by white arrows. (E) High-magnification micrographs of TrkB-tdTomato-labeled nerve arbors in each of the 3 papillae indicated from the map. The first column shows the XY (dorsal) plane, which is parallel to the tongue surface, and the YZ plane is shown below XY. The papillae boxed in magenta and green had a single labeled arbor, and the papilla boxed in cyan had 2 separate labeled arbors, which are best visualized by counting the axons below the taste bud (yellow and red arrows). Micrographs outlined in dashed lines show high-magnification views of the same papillae 24 hours later. Some changes to each arbor can be seen during this time within the taste bud but not within the papillae core. (F) An example arbor is shown at the initial time point (T0) and 12, 24, and 240 hours later. Gray dashed lines in the z-plane images illustrate the approximate base of the taste bud defined by the background labeling produced by GFP expression, although GFP was not imaged separately to reduce laser exposure. The data underlying the graphs in the figure can be found in https://data.mendeley.com/datasets/d58vz7wfrf/1. https://doi.org/10.1371/journal.pbio.3002271.g001 We found that individual arbors could be imaged to a depth of 70 μm to capture the entire arbor, and a portion of the axon beneath the taste bud (S1 Fig). To estimate the frequency of structural plasticity for these arbors, first, we imaged the same arbors once per hour for a 6-hour period (n = 36). We found that the structure of the axon within the lamina propria (Fig 1A, purple line) was stable (Fig 1B, yellow box). Many of the terminal branches of arbors (Fig 1A, green line) displayed structural changes as quickly as within a few hours after the initial imaging session (Fig 1B, red box). Among the 36 arbors imaged, 24 arbors changed their number of terminal branches in a 6-hour window by adding and/or subtracting small terminal branches (Fig 1B, yellow box). To determine whether the low level of excitation power (see Methods) used for imaging induced evident photobleaching, we imaged a new group of arbors (n = 13 from 2 mice) using identical acquisition parameters every hour for 6 hours and compared the fluorescence intensity across image stacks (z-max intensity projections) for the first and last time points. We did not observe a significant decrease in average (t(24) = 1.34, p = 0.76) or maximum (t(24) = 0.27, p = 0.78) fluorescence intensity despite imaging these structures repeatedly (Fig 1C). Thus, this imaging approach was suitable for acute in vivo imaging of individual arbors and demonstrates that arbors of taste ganglion neurons display high-speed structural remodeling.

Chronic in vivo imaging requires a fungiform papilla map Next, we examined axonal structural plasticity over days. Reliably identifying the same taste buds across imaging sessions was critical for chronic imaging experiments. To identify the same taste buds over days or weeks, we combined our approach for sparse labeling of taste ganglion neurons (Ntrk2CreER:tdTomato) with transgenic mice that express green fluorescent protein (GFP) in taste buds (Sox2GFP; [30]). When viewed at low magnification, GFP expression in the anterior tongue epithelium revealed a unique taste bud pattern for each mouse (Fig 1D) This provided a set of fiduciary marks used to locate the same taste buds and arbors repeatedly over time. Three taste buds in the example papillae map contained labeled arbors; they are shown at high magnification 24 hours apart (Fig 1E). No other fungiform taste buds viewed in this field contained labeled arbors due to the sparseness of genetic labeling of taste ganglion neurons (Fig 1D, taste buds with no box). Taste bud cells have an average life span of 10 days [1]. Given this rate of taste bud cell turnover, we investigated the structural plasticity of arbors every 12 hours for 10 days. In 5 mice, we observed the same 31 taste buds and 60 labeled arbors 21 times over 10 days. We have made these image stacks publicly available (10.6084/m9.figshare.23589351). Among these arbors, 4 were lost during this period, and none were added. One taste bud lost GFP expression in the epithelium, but the arbor remained, consistent with taste bud loss as the animal ages [31,32]. Examination of a single-labeled arbor at the beginning and end of the 10-day experiment illustrates that portion of the axon in the papillae core was stable, whereas the arbor structure in the taste bud was plastic (Fig 1F).

Gain/loss of terminal branches is not regulated by taste bud cell turnover If arbors add new terminal branches to synapse with new taste bud cells, then altering the rate of new cell entry would be expected to alter terminal branch structural plasticity. To test the hypothesis that taste ganglion neuron remodeling is dependent on the addition of new taste bud cells, we prevented new cells from entering taste buds by blocking Hh-signaling. Administering LDE225 (trade name Sonidegib) stops new taste cells from differentiating and entering taste buds [6,13,38–41] (S2 Fig). This chemotherapeutic agent is used to treat basal cell carcinoma and is associated with loss of taste by patients receiving treatment [13,42,43]. Over time, old taste cells continue to die and taste bud volume decreases [39] (S2 Fig). We treated mice with LDE225 (n = 4) or vehicle (n = 2) for 10 days and imaged taste arbors every 4 hours for 12 hours on days 6, 8, and 10; this was required since a longer imaging window would fail to capture all the terminal branch additions and losses (Fig 2E). Despite the prevention of new cell entry into the taste bud, arbors remained in the taste buds of mice treated with LDE225 and appeared similar to arbors from vehicle-treated taste buds (Fig 3A). Because arbors had similar rates of terminal branch gain/loss on each day of imaging (H(30) = 29.5, p = 0.44), the data were combined across days. We found that the rate of terminal branch gain/loss was similar between the LDE225 and vehicle groups (U = 312, p = 0.61) (Fig 3B). Thus, we conclude that preventing new cells from entering taste buds does not alter the rate of terminal branch remodeling. We also examined how taste buds recover following treatment with LDE255. Of 33 imaged taste buds, 11 recovered normal taste bud morphology; however, 22 failed to recover and lacked Sox2 expression up to 40 days after daily LDE225 treatment for 10 days (Fig 3C). Nonrecovered papillae (no GFP expression) retained arbors even in the absence of taste buds (Fig 3D). Surprisingly, we observed at least some remodeling of terminal branches in all papillae that lacked taste buds over 20 days, although remodeling over 24 hours was limited (Fig 3E). We conclude from these findings that the taste bud is not required for terminal branch remodeling of taste arbors, although the rate of remodeling may be slowed.

Growth and retraction occur concurrently within the arbor Some terminal branches displayed distinct morphologies such as swellings on the tip of the terminal branch, as well as punctate swellings along the axon (Fig 4A). We suspected that these features could be associated with remodeling, as both retraction bulbs and growth cones have been described in taste arbors [9]. To determine if terminal branch swellings and beading were associated with gain or loss of terminal branches, we examined 12 arbors from 3 mice that were imaged every 12 hours for 10 days (n = 252 total time points). We observed 61 instances of swellings at the tip of the terminal branch and/or swelling along the branch. These terminal swellings along the axon were followed by retraction of the branch 90% of the time, whereas only 10% of terminal branches displayed terminal branch swelling without retracting (Fig 4B). The amount of retraction that occurred varied (Fig 4C) and took anywhere from 12 to 108 hours. Therefore, we conclude that terminal end swellings are often associated with retracting terminal branches (Fig 4A). PPT PowerPoint slide

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TIFF original image Download: Fig 4. Retraction bulbs predict terminal branch retraction, and arbor number is stable over 100 days. (A) Two example arbors shown across a 36-hour window. In the top row, a terminal branch retracted after displaying a bulb at the tip of the ending (green arrowhead) and swellings within the branch. A separate terminal branch on this same arbor extended (magenta arrow) in conjunction with terminal branch retraction. In the bottom row, a terminal branch displayed an end-bulb and swellings but did not retract (magenta arrowhead); instead, it extended, indicating that the bulb may have been a growth cone. (B) Most terminal branches that displayed end-bulbs and swellings retracted at later time points (green); however, a small portion did not retract (magenta) (n = 12 arbors from 3 mice). (C) Change in terminal branch length. (D) Change in convex hull after each terminal branch retraction. (E) Net change in total arbor length after each terminal branch retraction. (F) Sankey diagram showing arbors grouped based on total number of terminal branches (1 to 3 = dark blue, 4 to 6 = light blue, 7+ = orange). Most arbors did not change groups at 5 (t120) or 10 (t240) days of imaging, demonstrating some stability in arbor complexity over time. (G) Two papillae maps of the same regions shown 100 days apart. Purple taste buds contained labeled arbors (number of arbors labeled in white), and blue taste buds did not contain labeled arbors. One papilla in this map lost an arbor during the 100-day window (red label). (H) Example arbor at days 0, 10, 50, and 100. The taste bud as defined by GFP is outlined in a green dashed line and papillae core in magenta. (I) Taste buds were categorized by number of labeled arbors and plotted in a Sankey diagram. No taste buds acquired new arbors, and 2 taste buds lost an arbor during the first 60 days (n = 3). One mouse was not imaged past day 60, so fewer taste buds were imaged between days 60 and 100 (n = 2); however, there was no loss of taste buds. (J) Violin plots of terminal branch number for 10 arbors imaged every 10 days for 100 days. Median indicated with gray bars. The data underlying the graphs in the figure can be found in https://data.mendeley.com/datasets/d58vz7wfrf/1. https://doi.org/10.1371/journal.pbio.3002271.g004 Since not all branch loss is associated with a retraction bulb, one possibility is that only those retractions with a retraction bulb occur when taste bud cells are lost. To determine if this was the case, we calculated the frequency of retractions that were associated with retraction bulbs. Retractions with retraction bulbs occurred once every 2 days on average for each arbor, which was also faster than the rate predicted by cell turnover (10 days). This finding indicates that branches retract for reasons other than loss of taste bud cells. We next investigated whether retractions associated with retraction bulbs contributed to reduced arbor size using 2 measures of size before and after terminal branch retraction. The first measure was the smallest amount of 3D space enclosing the segmented arbor, which is the space occupied by the arbor within the taste bud (convex hull; Fig 4D). Physiologically, it is indicative of how many taste bud cells the arbor can reach. The second is the total length of the arbor (Fig 4E). Retraction of a single terminal branch did not alter total arbor size (Student unpaired t test, t = 0.4515, df = 74, p = 0.65). These lost branches may represent a small percentage of the total branch length. Consistently, the average length of retraction for a single branch identified using end bulbs and beading was 14.1 ± 11.9 μm (Fig 4C), which was a small portion of the total length of these arbors (mean = 79.2 ± 40.8 μm). In addition, branch formation/elongation commonly occurred in the arbor at different locations concurrently with terminal branch retraction. Consistently, 21 of 38 arbors increased in total length while retracting a terminal branch. These opposing changes in terminal branch length maintain arbor size over time. Given that arbors add and lose terminal branches concurrently, arbor complexity over time may remain constant. To examine this possibility, we grouped arbors based on their complexity and examined their extent of change over 10 days. Because the simplest arbors typically showed a range of 3 terminal branches (Fig 2C), we grouped them accordingly. We observed that most arbors maintained a limited range of complexity over 10 days (Fig 4F), such that the minimum and maximum number of terminal branches for each arbor was correlated (R2 = 0.698, p < 0.001). Arbors from the simple group (1 to 3 branches) never remodeled to become complex arbors (more than 6 branches) within a 10-day imaging window. Although branch retractions occurred frequently within 10 days, they occurred simultaneously with terminal branch formation, which limited the range of complexity for most arbors.

Variability in arbor size over 10 days differs across arbors Because the volume occupied by the arbor within a taste bud (convex hull) was not heavily impacted by the retraction of single terminal branches, we hypothesized that arbor size is maintained despite constant terminal branch remodeling. To determine if this was the case, we examined whether arbor size (convex hull and total arbor length) changed over 10 days. To perform comparisons across all time points within a 10-day period, we developed a semiautomated image analysis pipeline in MATLAB, called ArborTools, to segment arbors and quantify convex hull and total arbor length (S3A Fig). Reconstructions of each arbor were verified visually to confirm that the arbor and the reconstruction matched (S3A Fig, cyan versus white). Arbors with too much background for automatic segmentation were removed from analysis. The volume occupied by the arbor (convex hull) changed in the taste bud across 10 days. In general, convex hull changed along with total length (S3A Fig). These changes in size could be due to changes in height, x or y width, or combinations of these changes (S3C Fig). For example, in the illustrated arbor (S3A-S3C Fig), increases in size occur due to an increase in Y width from 0 to 12 hours, and, dramatically, increases in size between hour 228 and 240 occur due to concurrent increases in X, Y width and height. When examining the extent to which individual arbors displayed changes in size, we noticed that some arbors appear to be more variable than others (S3D and S3E Fig).

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