Breadth of tuning in taste afferent neurons varies with stimulus strength.

Animals

Adult transgenic mice (ages 10 weeks to 1 year) of both sexes that express GCaMP3 in sensory neurons were used for this study 27 . All procedures for surgery and euthanasia were reviewed and approved by the University of Miami IACUC committee.

Surgery

Mice were anaesthetized with ketamine and xylazine (intraperitonially 0.12 mg g −1 ketamine, 0.01 mg g −1 xylazine) and placed supine on a pad warmed by circulating water. The animal's core temperature was monitored with a rectal probe and the mouse was maintained between 36.5 and 37.5 o C. We monitored the level of anaesthesia by hind paw withdrawal reflex. A deep surgical plane of anaesthesia was maintained throughout the surgery and recording session by injections of ketamine (0.12 mg g −1 ketamine per h, or as required). The trachea was exposed and cannulated for respiration. The geniculate ganglion was reached by retracting muscles to expose the middle ear on one side, piercing the bulla, and removing a small piece of the thin temporal bone on the opposite wall 18 . As described by Sollars and Hill 18 , a small flexible tube was introduced into the esophagus and passed forward into the oral cavity to deliver taste stimuli uniformly to the palate and tongue, regions innervated by the geniculate ganglion cells. By attaching a small piece of flexible plastic mesh (4 × 7 mM) to the end of the delivery tube in the oral cavity, we optimized the even distribution of taste solutions across the palate and tongue. We monitored access of taste stimuli in preliminary surgeries by observing the spread of a dilute solution of methylene blue over the tongue and palate.

Functional imaging

The surgically prepared mouse was carefully transferred to the stage of an Olympus FV1000 confocal microscope equipped with a × 20-long working distance objective that allowed us to image most of the exposed ganglion. Confocal scans of GCaMP3-labelled ganglion neurons using 488-nm laser excitation with a 505–605 nm emitter filter were taken at ∼3 hz while taste stimuli were introduced into the oral cavity. Scans were digitized and stabilized using ImageJ 2.0.

Fluorescence intensity of regions of interest

(ROIs) drawn over individual ganglion neurons were quantified using imageJ and analysed with MatLab software. We used custom Matlab software code for analysing ROI data (see Supplementary Methods ). Responses were quantified as peak stimulus-evoked fluorescence change divided by baseline fluorescence (that is, Δ F / F 0 ). Criteria for analysing responses included that Δ F / F 0 exceeded three times the s.d. of the baseline and occurred at a consistent latency after stimulus onset. Further, two replicates were obtained for every stimulus. An alternative approach, measuring the area under the curve during the response yielded similar results but was more time consuming and difficult to automate. Latency of responses reflects the interval for stimuli to traverse the deadspace in the perfusion system, including the oesophageal tube.

Data analysis

To obtain concentration–response relations for taste-evoked signals in geniculate ganglion cells, a given stimulus was presented in an ascending series of concentrations. This was followed by a second replication of the same presentations. Data points for the two replications were averaged. EC 50 values were obtained from concentration–response relations fit with a variable slope model (Graphpad Prism v.6.02) that derives a Hill Slope from the data, making no a priori assumptions of its value. Moreover, for the detailed analyses of NaCl concentration–response relations ( Fig. 8 ), EC 50 values were only derived from neurons where there was a good correlation in the responses between the two sequential replications of NaCl concentrations ( r 2 >0.5) and the goodness-of-fit ( r 2 ) for the sigmoidal plot was >0.6. Entropy ( H ) for a neuron was calculated as follows 33 where n =number of taste qualities and K is a scaling constant to limit 0> H ≤1 ( K =1.43 for n =5). p i is the proportion of a neuron's response (Δ F / F 0 ) to the i th taste solution relative to the sum across all n responses for that neuron. In all cases, we took the mean of two responses (that is, from two separate trials) to calculate p i . H =0 indicates that the neuron responded only to one taste quality (highly tuned); H =1 indicates the neuron responded equally to all the taste stimuli (broadly tuned). Taste stimuli Stimuli were applied at room temperature for 5 s, preceded and followed by a 55 s rinse with artificial saline. Artificial saline consisted of: NaCl (14.8 mM); KCl (22.1 mM); CaCl 2 (3.1 mM); MgCl 2 (0.6 mM). Chemical stimuli for the basic tastes consisted of: sweet–sucrose, 30 mM to 1 M; salty–NaCl, 30 to 500 mM; sour–citric acid, 1 to 30 mM; bitter–a mixture of cycloheximide, 0.3 to 30 μM and quinine·HCl, 0.1 to 3 mM; umami–MSG, 30 to 1 M with 1 mM IMP. Chemical stimuli were delivered by gravity perfusion through a computer-controlled manifold at 3 ml min −1 . Responses to MSG/IMP were distinguished from responses to NaCl and assigned to the category ‘MSG/IMP' if the glutamate mixture elicited Δ F / F 0 >1.5 × that of equimolar NaCl. When 100 mM MSG and 250 mM NaCl were used as stimuli, responses were categorized as ‘MSG/IMP' if Δ F / F 0 >1 × that evoked by NaCl.

Electrical stimulation

To emulate taste-evoked responses in geniculate neurons using electrical stimuli, we applied 10 ms square wave pulses from a stimulator (Grass S9) via a 4 mm silver-silver chloride disk (A-M Systems) applied to the anterior tongue. The stimulus strength was adjusted to just below that which elicited visible muscle contractions. The stimulator was triggered by transistor-transistor logic (TTL) pulses that mirrored action potentials obtained from microelectrode recordings from single geniculate neurons in the rat 19 20 . That is, neuronal responses to oral stimultion with 30, 100 and 300 mM monosodium glutamate, applied for 5 s, were digitized and converted into TTL pulses. The microelectrode recordings were kindly provided by A. Nikonov and R. Contreras, Florida State University. Immunostaining Geniculate ganglia were dissected, fixed with 4% paraformaldehyde, washed with PBS, cryoprotected overnight with 30% sucrose, embedded in OCT, and cut at 25 μm. Sections were permeabilied with 1% Triton-X in PBS, treated with 4% normal goat serum in PBS followed by Avidin/Biotin Blocking kit, and immunostained with anti-NeuN (1:1,000, clone A60, biotin-conjugated, MAB377, EMD Millipore) and anti-GFP (1:1,000, GFP-1020, Aves Labs, Inc.). Secondary antibodies were Streptavidin, Alexa Fluor 594 conjugate (1:1,000, S-11227, Life Technologies) and Alexa Fluor 488 goat anti-chicken IgG (H+L) (1:1,000, A-11039, Life Technologies). Sections were mounted with Fluoromount-G (0100-01, Southern Biotech).

Supplementary Material Supplementary Information Supplementary figure 1 and Supplementary Methods Supplementary Movie 1 Responses (dF) of geniculate ganglion cells in vivo in GCaMP3 mice to a panel of 5 tastants presented consecutively in the oral cavity. The movie was collected as one continuous recording but for clarity, has been subdivided into 5 separate episodes. Each episode represents the presentation of one taste stimulus, identified along the bottom. Onset of the stimulus (t=2 sec, duration 5 sec) for each episode is indicated at the top of the left episode. Several neurons respond to the tastants. An example of a neuron that responds best to sucrose (and somewhat to MSG/IMP), S, is identified by a white dot. An example of a neuron that responds to multiple taste stimuli,G, is shown as a magenta dot. Stimuli were 500 mM sucrose, 300 mM MSG (with 1mM IMP), 500 mM NaCl, 50mM citric acid, or 30 ìM cycloheximide + 5 mM quinine.HCl.