Mouse Taste Receptor Cells Biology Essay

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The peripheral gustatory system carries out two primary functions: nutrient detection and toxin avoidance. To accomplish either of these tasks, ingested substances must mix with saliva during the mastication process. This process allows for the efficient break-down of these substances into molecules that can be recognized by taste receptor cells (TRCs). These molecules can be relatively simple, small molecules and ions or complex, organic molecules {Gilbertson, 1996}. Therefore, taste stimuli are always in solution and the osmolarity of these solutions can vary widely compared to the intracellular osmolarity of taste receptor cells {Feldman, 1995}. These extremes in solution osmolarity can range from very low (hypoosmotic) as in the case of drinking water to very high (hyperosmotic) as is the case while consuming salty potato chips. It has been hypothesized that the osmolarity of these solutions may be an important factor in determining the overall response of the gustatory system and recent studies have shown direct effects of osmolarity on taste receptor activity {Gilbertson, 2002; Lyall, 1999}.

Lyall et al. (1999) examined the effects of hyperosmotic stimuli on responses to salt by recording from the chorda tympani nerve in rat. They found that increasing extracellular osmolarity by adding mannitol or cellobiose to an isotonic NaCl solution significantly increased responses in the chorda tympani. This response was reversible upon removal of these compounds. In addition, when mannitol or cellobiose solutions were perfused onto isolated fungiform TRCs, there was a rapid decrease in cell volume that was also reversible.

In another study, Gilbertson (2002) used whole-cell patch clamp recording to examine the effects of hypoosmotic stimuli on TRCs in rat. He found that approximately two thirds of the cells showed an increase in conductance and an increase in cell capacitance, a response indicative of an increase in cell volume. Again, these responses were reversible and the conductance was identified as due to opening of a Cl- channel, similar to the volume-regulated anion channels identified in a number of epithelia {Jentsch, 2002}. The results of these studies indicate that TRCs can and do respond to changes in osmolarity and these responses are associated with changes in cell volume. Therefore, rapid water influx and efflux has a significant impact on cell signaling in the peripheral taste system. The most likely means by which water could be moved across the cell membrane in such a manner is by aquaporin channels.

Aquaporins (AQP) are small, intrinsic membrane proteins {Agre, 2002}. The functional channel is tetramer of identical subunits forming a structure similar to that of potassium channels or cyclic nucleotide-gated channels {Boassa, 2006; Yool, 2002}. Unlike potassium channels where the subunits come together to form a single ion transporting pore, each individual subunit of the AQP channel contains a pore for water movement. Water moves bi-directionally through these channels and is driven by osmotic gradients. There are currently 13 members of the mammalian AQP family, AQP0-12.

We have recently characterized the expression of 3 members of the AQP family expressed in rat TRCs, AQP1, 2 and 5 {Watson, 2007}. Immunocytochemical labeling for these channels showed different expression patterns for AQP1 and 2 compared to AQP5. AQP1 and 2 were localized to the basolateral membrane of TRCs while AQP5 was predominantly expressed on the apical membrane. It is not unusual to find different AQP channels expressed on different regions of the cell membrane in transporting epithelia. For example, in the collecting duct of the kidney, AQP3 and 4 are localized to the basolateral membrane, while AQP2 is expressed in the apical membrane of principal cells [ref]. This arrangement serves an important function because it permits regulation of water movement in the collecting duct. When vasopressin is released from the posterior pituitary gland in response to low blood volume or increases in plasma osmolarity, AQP2 is inserted into the apical membrane of collecting duct principal cells to allow water to be reabsorbed [ref]. However, the functional relevance of the differential expression of AQP channels in TRCs is currently unknown. We hypothesize that water movement through the apically localized AQP5 channel would be important with regard to the osmolarity of ingested substances within the oral cavity while water movement through the basolaterally expressed AQP1 and 2 would be affected primarily by the osmolarity of interstitial fluids.

In addition to identifying AQP channels expressed in rat TRCs, we also found functional evidence that water entry through these channels leads to changes in conductances observed in response to hypoosmotic stimulation. Electrophysiological experiments showed that blocking AQP channels with TEA significantly reduced hypoosmotically-induced increases in conductances in TRCs and this effect was reversible (Watson et al., 2007). Therefore, water entry through AQP channels is necessary for inducing conductances in response to hypoosmotic stimulation.

Unfortunately, there are currently no blockers available for specific AQP channels so using pharmacological manipulations to examine the role of AQP5 in rat TRCs is not possible at this time. Additionally, the use of siRNA to knock down AQP channel expression in native cells is currently very difficult, if not impossible. However, there are knockout mouse models currently available for several AQP channels which could be utilized to examine the role of AQP channels in taste. One of these AQP knockout mouse models is of particular interest, the AQP5 knockout mouse created by Anil Menon (University of Cincinnati Medical Center). As mentioned previously, a closer examination of this apically localized AQP channel which we hypothesize contributes to responses associated with the ingestion of foods and fluids rather than systemic fluid balance, would provide important insights into the role of this channel in taste. However, all data collected thus far on the effects of osmolarity on taste and expression of AQP channels were in rat. Therefore, the main goal of these experiments was to determine whether using a mouse model to study the role of aquaporin channels in taste was a feasible route to pursue.

For these experiments, we chose to examine 2 commonly used inbred mouse strains, C57BL/6ByJ (B6) and 129X1/SvJ (129). These strains are often used as background strains for transgenic mice. In addition, these strains tend to show differences in taste preferences which might affect the taste phenotype of a transgenic mouse model [refs].

Another important consideration which has not yet been investigated involves the use of mannitol in electrophysiological experiments performed on rat. Mannitol was used to alter solution osmolarity while maintaining ionic concentrations. This sugar alcohol is very effective for this purpose due to its relative impermeability to cell membranes. However, it is not known whether rats or mice behaviorally respond to ingesting varying concentrations of mannitol. Therefore, in the current study, we initially evaluated whether or not B6 and 129 mice show behavioral responses to mannitol using a taste preference test procedure and then used cellular techniques to examine expression of AQP5.

Materials and Methods

Subjects. Adult, male C57BL/6ByJ and 129X1/SvJ mice were used in these experiments (The Jackson Laboratory, Bar Harbor, ME). All mice were maintained on a 12 h:12 h light/dark cycle with normal rodent chow and water available ad libitum. All procedures involving animals were carried out with approval of the Institutional Animal Care and Use Committee of Utah State University and in accordance with American Veterinary Medical Association guidelines.

Behavior: 24-h 3-Bottle Preference Test. Mice were not water restricted for these tests prior to introducing solutions of interest and were individually housed during testing. Three modified 25 ml pipettes with sipper tubes and rubber stoppers were placed on the mouse's home cage (see Monell Mouse Taste Phenotyping Project - http://www.monell.org/MMTPP) and measurements recorded for each tube. Preference scores were calculated as follows:

Preference score = (ml solution/total intake) x 100.

For mannitol, which was found to be avoided at higher concentrations in pilot studies, the test solutions were placed in outside tubes while distilled water was placed in the center tube. This allowed for an avoidance range of 0 - 67%. Eight concentrations were tested ranging from 55 mM to 440 mM. For saccharin, a preferred taste, distilled water was placed in the outer tubes and the test solution in the center thus giving a preference range of 33 - 100 %. Three concentrations of saccharin were tested in this experiment: 1.5, 3 and 15 mM. Test solutions were presented in ascending concentration order. Preference scores (dependent variable) were analyzed separately for mannitol and saccharin using a Strain by Concentration factorial ANOVA.

We tested mannitol to assess behavioral responses to osmolarity, however, mannitol has a transiently, mildly sweet taste. Therefore, we decided to test another sweet tasting solution, saccharin, for comparison purposes and because previous work (see Bachmanov et al., 2001) has shown a different pattern of responses between B6 and 129 (P3/J, not X1/SvJ) mice.

Taste Bud Isolation. This procedure was a slightly modified version of the one used in this lab for work in rat. Approximately 0.1 ml of enzyme cocktail was injected between the epithelium and underlying muscle of the anterior tongue. Much smaller amounts of enzyme were injected for foliate and circumvallate papillae. The enzyme cocktail was made in Tyrode and contained 0.5 mg/ml collagenase A, 2.9 mg/ml dispase, and 1.0 mg/ml trypsin inhibitor. Following the injection, the tissue was incubated for 40 min in Ca2+/Mg2+ free Tyrode and bubbled with O2. Following incubation, the lingual epithelium was pealed from the underlying tissue and pinned out in Ca2+/Mg2+ free Tyrode in a Sylgard-lined petri dish with the mucosal side facing down. Taste buds were removed from the epithelium using a large bore (~150-200 µm) pipette and applying gentle suction then expelled into a microfuge tube containing 200 µl of RNAlater (Ambion, Austin, TX) for RNA extraction.

RNA Isolation. After isolating the taste receptor cells, the microfuge tubes containing these cells were centrifuged for 7 minutes at 6000 rpm (3300 x g) at 8° C. The pellet was resuspended in a lysis buffer from the RNeasy Mini Kit from QIAGEN (Valencia, CA) and extraction of RNA was done using the manufacturer's recommended procedures including treatment with DNase I (RNase-free, Gibco, Grand Island, NY). RNA for use as positive controls was obtained from 129 mouse lung where AQP5 is highly expressed. Approximately 100 mg of tissue was obtained for RNA extraction using Tri-reagent (MRC, Inc., Cincinnati, OH) and following manufacturer's instructions. Quantity and quality of RNA were determined using the Agilent Technologies 2100 bioanalyzer (Santa Clara, CA) according to manufacturer's instructions.

RT-PCR. The Omniscript RT kit (QIAGEN Inc.) was used to synthesize first-strand cDNA. Total TRC RNA or 50 ng of an appropriate control RNA was used in this reaction with a total volume of 20 μl. DNA contamination was evaluated by setting up a reaction where the reverse transcriptase was omitted. Once cDNA synthesis was complete, 1 μl cDNA was added to the PCR mixture. The final concentration for this reaction was 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.0 mM Mg2+, 1X MasterTaq, 200 μM dNTPs, ~500 nM forward and reverse primers and 1.25 U Taq polymerase. PCR amplification of AQP5 involved a 5-minute denaturation step, which was followed by 40 cycles of a 3-step PCR. This included 30-second denaturation at 95°C, 30-second annealing at 55°C and 45-second extension at 72°C. A 7-minute final extension step completed the process. The forward primer (5' CCC TCT CAC TGG GTC TTC TG 3') and reverse primer (5' CCT TTT CTC CAG TGG TCC AG 3') corresponded to nucleotide sequences 1094-1113 and 1324-1343 of the mouse AQP5 sequence (accession no.: NM_009701), respectively. Visualization of the amplified sequences was done by electrophoresis in 2% agarose gels poured using 1X TAE buffer (40 mM Tris-Acetate and 1 mM EDTA). The size of the expected PCR product was 250 bp. Purification for sequencing involved directly purifying following PCR using the QIAquick PCR purification kit (QIAGEN Inc.). An ABI Model 3100 Automatic Sequencer (Foster City, CA) determined the sequence using the dye-terminator method. Partial sequences for AQP5 were examined using the BLAST 2.0 search engine (NCBI; http://www.ncbi.nlm.nih.gov/BLAST/).

Real Time qPCR. For real-time quantitation of RT-PCR, the RT reaction was the same as described previously. However, the PCR reaction was performed in a real-time thermalcycler (SmartCyclerâ„¢, Cepheid, Sunnyvale CA). The PCR reaction mix used was the same as described above, except the magnesium acetate concentration was increased from 2.0 mM to 3.5 mM and a 2-step PCR (15 s denaturation, 60 s annealing and extension) was used rather than a 3-step PCR. We used a TaqMan (ABI) detection system and primer pairs for channel-specific sequences were multiplexed with the primer pairs for the housekeeping gene, GAPDH, for comparison of expression levels in the 3 types of taste buds. AQP5 and GAPDH were detected using dual-labeled fluorogenic probes. Initially, the probes were designed using the Oligo 6.0 Primer Analysis Software (Molecular Biology Insights, Inc., Cascade, CO), then the primers were designed. The AQP5 probe was labeled at the 5'-end with FAM as the reporter dye and TAMRA at the 3'-end as the quencher dye. The GAPDH probe was labeled with ROX as the reporter and 3BHQ-2 as the quencher. The probes were obtained from Integrated DNA Technologies (Coralville, IA). To quantify expression, the cycle threshold (point at which the growth curve crosses 30 fluorescent units - user defined, to occur during the log-linear phase of the growth curve) was obtained for GAPDH and AQP5.

In order to compare relative expression of AQP5, delta CT (ΔCT) was calculated by subtracting the cycle threshold (CT) for GAPDH from the CT of AQP5. These values allowed for the comparison of relative transcript abundance between samples. Therefore, smaller ΔCT values indicate higher expression of AQP5. To obtain relative quantification of the samples, the following formula was used: 2- ΔΔCT. This formula takes into account the amount of target which was normalized to an endogenous reference (GAPDH) and relative to a calibrator. The calibrator was defined as the AQP5 sample with the highest expression (lowest ΔCT) for a given set of pooled TRCs. The ΔΔCT was the difference score between the ΔCT for each sample and the ΔCT for the calibrator. Therefore, the relative expression for AQP5 was calculated in the following manner [34]:

ΔCT1 = CTAQP5 - CTGAPDH

ΔCT2 = CTCAL - CTGAPDH

ΔΔCT = ΔCT2 - ΔCT1

Relative Expression = 1/(2- ΔΔCT)

CT for AQP5 and GAPDH was obtained empirically and CTCAL was the cycle threshold for the most highly expressed sample in the assay. Strain by Tongue Region ANOVA followed by t-tests was performed to identify significant differences in relative expression of AQP5 in fungiform, foliate and circumvallate TRCs from B6 and 129 mice.

Additionally, an RNA dilution assay was performed to determine whether AQP5 and GAPDH amplification were consistent across different starting RNA concentrations. For the set of AQP5 and GAPDH primers, ΔCT values were evaluated in 3 separate multiplexed reactions. For each reaction, equal amplification efficiency for the different starting RNA concentrations were defined by the absolute value of the slope of the log input RNA versus ΔCT being less than 0.1.

Immunocytochemistry. Mouse tongues were removed and fixed in 4% paraformaldehyde for 2 h at room temperature. 50 μm thick slices were taken from blocks of tissue containing the circumvallate papillae using a vibrating microtome (Vibratome 3000, Vibratome Company, St. Louis, MO). Standard immunostaining procedures were followed to visualize AQP5 label. Briefly, tissue sections were washed in PBS followed by incubation in 3% normal goat serum. Slices were then incubated in AQP5 polyclonal antibody (1:100; Alpha Diagnostic International, San Antonio, TX) for 72 h at 4°C. After rinses in PBS, sections were incubated in biotin-conjugated anti-rabbit IgG (1:200) for 1 h. Slices were again rinsed with PBS and then incubated with Alexa-fluor 594-conjugated avidin (1:200) for 2.5 h at 4°C. Label was visualized using a BioRad laser confocal microscope.

Results

We used taste preference tests to characterize behavioral responses of B6 and 129 mice to changes in solution osmolarity by presenting several concentrations of mannitol which varied in osmolarity from hypoosmotic (55 mOsm) to hyperosmotic (440 mOsm). Previous studies examining electrophysiological responses of rat TRCs to non-isoosmotic solutions showed alterations in cell volume and conductances in response to this type of stimulation [refs]. For these experiments mannitol was used to alter solution osmolarity, however, it is unclear whether altering solution osmolarity with mannitol is a behaviorally relevant stimulus to the animal.

We then examined expression of AQP5 using immunocytochemical and molecular biological techniques. Of the 3 AQP channels identified in rat, only AQP5 was localized predominately on the apical membranes of TRCs [ref]. Based on the apical expression of AQP5, we hypothesize that this channel may be important for responses to changes in osmolarity within the oral cavity. Therefore, determining whether AQP5 is expressed and if this channel is localized to the apical membranes of TRCs in mice is necessary before any further investigation of its role in taste can be pursued.

Behavior: Taste Preference Tests in B6 and 129 Mice

Our interest in examining AQP channel function in native mammalian TRCs is currently limited by the lack of specific AQP channel blockers and the current difficulties in employing siRNA technology in native cells. However, knockout mouse models for various AQP channels are available. To determine the feasibility of using mouse instead of rat as our model organism, we began by examining behavioral responses to solution osmolarity in 2 inbred mouse strains used as background strains for generating knockout mice, the B6 and 129 strains. We first characterized behavioral responses to osmolarity by presenting several concentrations of mannitol in taste preference tests. Mannitol was used in previous electrophysiological studies to alter solution osmolarity because mannitol does not cross biological membranes [refs]. Mannitol is a sugar alcohol that has a transiently sweet quality which could contribute to the palatability of the solutions. For this reason, we also characterized taste preferences for another sweet compound, saccharin, for comparison purposes. We chose saccharin because it has been previously characterized in B6 mice and another 129 strain [ref].

Mannitol. Taste preferences for 8 concentrations of mannitol were tested in 24-h 3-bottle preference tests. These concentrations ranged from 55 mM to 440 mM and osmolarity of these solutions ranged from 55 mOsm to 440 mOsm. Overall, 129 mice had significantly higher preference scores for mannitol than B6 mice (Figure 1; Strain X Concentration ANOVA, main effect of Strain, p<.001). For the more hypoosmotic concentrations, 129 mice appeared to prefer the mannitol while B6 mice were indifferent. In the near isoosmotic to hyperosmotic range of mannitol concentrations beginning at 220 mM, B6 mice began to avoid the mannitol. 129 mice only showed avoidance of mannitol at the most hyperosmotic concentration tested, 440 mM. Additionally, both mouse strains showed a significant decrease in preference for mannitol as solution concentration increased (p<.001).

Saccharin. B6 and 129 mice were also tested for taste preferences using 3 concentrations of saccharin (1.5, 3 and 15 mM). We tested saccharin because it is also a sweet tasting compound and has been previously characterized in B6 and 129P3/J mice [ref]. In contrast to what we found for mannitol preference, B6 mice had significantly higher preference scores for saccharin compared to 129 mice (Figure 2; p<.001). In addition, 129 mice were indifferent to the lower concentrations of saccharin tested while B6 mice preferred the saccharin solutions at all concentrations tested. Both B6 and 129 mice showed increasing preference scores with increasing solution concentration (p<.001). While these results were quite similar to previously reported findings [ref], they were not consistent with what we observed for mannitol.

AQP5 Expression in B6 and 129 Mice

Previous work in rat showed expression of 3 different AQP channels, AQP1, 2 and 5. Of these, AQP5 was localized primarily to the apical membrane of the TRCs whereas the other 2 were predominately found on the basolateral membrane. This differential expression pattern may indicate different roles for these AQP channels in TRCs. We hypothesize that AQP5 would be more important for responses to the osmolarity of ingested substances present in the oral cavity whereas AQP1 and 2 may be more responsive to osmotic changes in the interstitial fluid. Therefore, we examined expression of AQP5 in B6 and 129 mouse taste receptor cells using RT-PCR, real time qPCR and immunocytochemistry to determine whether mice, like rats, express AQP5 on the apical membrane of TRCs.

RT-PCR. Our initial investigation into whether B6 and 129 mice express AQP5 in TRCs used RT-PCR on pooled sets of RNA collected from the 3 regions of the tongue: fungiform (FF), foliate (FOL), and circumvallate (CV). We examined 3 such sets from each strain and found expression of AQP5 in both B6 and 129 mice in FOL and CV samples but not FF. This differed from our observations in rat which showed expression of AQP5 in all 3 tongue regions [ref]. Figure 3 shows the results for 2 sets of pooled B6 RNA and 1 set of 129 RNA. To verify the identity of our product as mouse AQP5, we sequenced product obtained from 129 FOL using an ABI Model 3100 Automatic Sequencer (Foster City, CA). Partial sequences were 100% homologous to the published mouse AQP5 sequence.

Real Time qPCR. From our RT-PCR results, we discovered that the bands present in B6 FOL and CV samples were consistently much fainter in intensity than those for 129 mice or for our positive control sample (rat lung). While RT-PCR does not provide a quantitative measure of differences in expression level, real time quantitative PCR allowed us to determine whether there were indeed differences in AQP5 expression in B6 and 129 mice. We used a primer and probe set for AQP5 in a multiplexed reaction with a housekeeping gene, GAPDH, to look at relative expression of AQP5 in B6 and 129 TRC samples. Relative PCR efficiency was assessed for AQP5 and GAPDH by examining the slope of the regression line fit for the log input RNA concentration and ΔCT. The absolute value of the slope of this relationship was 0.05 which is within acceptable limits (absolute value <0.1). Figure 4 shows relative expression of AQP5 relative to the calibrator which was a 129 CV sample. Expression of AQP5 in 129 mice was significantly greater than B6 mice (p=.003) and there were significant differences in expression based on tongue region (p<.05). As was observed using RT-PCR, there was little to no expression of AQP5 in FF samples in either B6 or 129 mice. However, AQP5 expression in 129 FOL and CV samples was considerably higher compared to B6, on the order of 100-1000 times higher in 129 FOL and 10,000 to 100,000 times higher in 129 CV.

Immunocytochemistry. Results from our molecular assays revealed the expression of AQP5 in B6 and 129 mouse TRCs. However, these assays only provide expression data concerning the mRNA present, not protein expression or, more specifically, protein localization which is crucial to any future investigation of the role AQP5 plays in taste. Therefore, it was also necessary to examine expression of AQP5 protein using immunocytochemistry. Tissue sections through the circumvallate region of the tongue were labeled with antibodies for AQP5 (Figure 5). Similar to the expression pattern we observed in rat, Figure 6 clearly shows labeling evident on the apical membranes of TRCs in both B6 and 129 mice. Labeling for AQP5 is not restricted to the apical membranes as it is also present on the basolateral membranes of many of these cells. Again these data are consistent with our previous findings in rat.

Discussion

Taste transduction can be modulated by a number of non-taste factors. These non-taste factors include somatosensory characteristics of the substances we ingest such as temperature and texture, the presence of hormones such as aldosterone, and ionic components present in saliva as well as the osmolarity changes that occur in the oral cavity during the mastication process [Talavera et al., 2005; Kinammon 1996; Herness & Gilbertson 1999; Gilbertson 2002; Lyall et al., 1999]. Many of these factors have been shown to directly alter taste cell activity and/or afferent nerve responses to different tastes. As a consequence, sensory signals transmitted from taste cells to the central nervous system may be affected such that these non-taste factors could contribute to our perception of taste and, subsequently, our selection of which foods and fluids to consume.

Our current interest in understanding how one of these non-taste factors, osmolarity, affects taste arises primarily from the findings of two studies. One study examined the effects of hyperosmotic stimulation on the peripheral gustatory system (Lyall et al., 1999) while the other characterized the effect of hypoosmotic stimulation (Gilbertson 2002). Lyall et al. (1999) examined the effects of osmolarity on salt taste. This study recorded electrical activity from the chorda tymani nerve (CT) in addition to measuring cell volume changes in isolated fungiform taste cells in response to NaCl solutions containing mannitol, cellobiose, urea or DMSO. These results suggest that CT responses to 150 mM NaCl were increased by the addition of 300 mM mannitol or cellobiose but not 600 mM urea or DMSO. In addition, only hyperosmotic stimulation by mannitol and cellobiose resulted in a sustained reduction in cell size when applied to isolated FF cells, while urea and DMSO application did not. Thus, sustained alteration in cell volume affects how taste cells respond to taste stimuli, in this case, NaCl.

A few years later, Gilbertson (2002) examined responses to hypoosmotic stimulation in isolated taste cells using whole-cell patch clamp recording. Again, mannitol was used to alter solution osmolarity in these experiments. As opposed to the decrease in cell volume associated with application of hyperosmotic solutions, hypoosmotic solutions resulted in an increase in cell volume. Additionally, the application of hypoosmotic solutions resulted in an increase in whole-cell conductance which was correlated to the decrease in solution osmolarity.

What was evident from both studies was the ability of taste cells to respond to changes on solution osmolarity that lead to alterations in electrical activity of the individual cells and/or the subsequent activity recorded from the primary afferent nerves innervating the taste buds. However, the mechanism by which the taste cells were either shrinking or swelling is not well understood yet. The findings on both papers propose a possible role for water movement through aquaporin channels. These small, intrinsic membrane proteins are capable of moving water rapidly across the cell membrane in response to changes in osmotic gradient and many of these channels are blocked by mercurials and/or tetraethylammonium (TEA; refs). Therefore, expression of this type of channel in taste cells would provide an ideal route for water movement during non-isosomotic. We recently showed that water entry through AQP channels was necessary for the increase in conductance observed in response to hypoosmotic stimulation {Watson, 2007}. This is the first functional evidence that aquaporin channels play a crucial role in the response of taste cells to hypoosmotic solutions. The problem with this approach is that TEA does not block all members of this protein family nor does it block any specific AQP channel, thereby making it impossible to precisely determine which of these channels was responsible.

In addition to the identification of AQP channels in rat taste cells, immunocytochemical experiments showed differential expression of these channels such that AQP5 was predominately localized to the apical membrane while AQP1 and 2 were predominately expressed on the basolateral membrane {Watson, 2007}. We hypothesize that such an arrangement may serve to respond to osmolarity changes in the oral cavity as opposed to osmolarity changes in the interstitial spaces. Unfortunately, there are several limitations to examining just what the role of these channels is in taste, particularly in rat. As mentioned previously, these limitations include a lack of specific AQP channel blockers and transgenic models as well as technical difficulties in applying siRNA technology to native cells. Therefore, we found it necessary to evaluate whether mouse was an appropriate alternative model organism.

We began by examining the behavioral responses of B6 and 129 mice to changing osmolarity using different concentrations of mannitol in a 24-h 3-bottle preference test. This was not an experiment we had previously conducted in the rat, however, mannitol was used to alter solution osmolarity in previous electrophysiological studies and it was unclear whether the animals would show any measurable behavioral response to it. In other words, conductance in individual taste cells varied with mannitol concentration or solution osmolarity but does this translate to a change in the animal's response to it? We found significant strain differences in their preference scores for these solutions. 129 mice consistently showed higher preferences for mannitol compared to B6 mice for all but the highest concentration tested which both strains avoided. While we cannot rule out the effects of post-ingestive factors, attributing these results to the sweet taste component does not fit well with previously reported preference data for B6 and 129P3/J mice {Bachmanov, 2001}. For the majority of sweet solutions tested in those experiments, B6 mice had higher preference scores compared to the 129 strain and these solutions varied on factors other than sweetness.

We also examined another sweet-tasting compound, saccharin, for comparison purposes in these two mouse strains. In contrast to our observations for mannitol, preference scores for saccharin were reversed. B6 mice had significantly higher preference scores compared to 129 mice and both strains showed increasing preference for saccharin with increasing solution concentration. Additionally, preference for saccharin appeared to be very similar for these two mouse strains compared to the results previously reported for B6 and 129P3/J mice by Bachmanov et al. (2001) as well as results reported more recently by Tordoff (2007) for B6 and 129X1/SvJ mice for the concentration range we examined. Therefore, we feel confident that not only our testing was a valid method, but that the differences we observed for mannitol between these two mouse strains was accurately characterized.

The second major factor we needed to evaluate in mouse was whether AQP5 was expressed in a similar manner to what we previously observed in rat. In rat, AQP5 channel was apically localized which we hypothesize would play an important role in detecting osmolarity changes associated with ingested foods and fluids {Watson, 2007}. To accomplish this, we characterized expression of AQP5 in both strains of mice. Both strains of mice expressed AQP5 in the posterior regions of the tongue (FOL & CV). This finding differs somewhat from rat where mRNA for AQP5 was detected in FF taste cells in addition to FOL and CV. We also assessed relative expression of the channel using qPCR and found 129 mice had considerably higher levels of AQP5 compared to B6 mice. Localization of AQP5 protein using immunocytochemistry showed AQP5 was expressed on the apical regions of the taste receptors cells but not restricted to this area. This finding was similar to what we observed in rat.

In conclusion, our findings do not directly link differences in AQP5 expression to the strain differences we found for B6 and 129 mice in our behavioral experiment. However, we do believe there is sufficient evidence to warrant further investigation into the role of AQP5 in taste using a transgenic mouse model. If AQP5 channel expression is important for the response to changes in osmolarity in a similar manner to what we observed in B6 and 129 mice, then we would predict that mice lacking this gene would show lower preferences for mannitol compared to wildtype mice.

ACKNOWLEDGEMENTS

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