The Medler Lab

Kathryn Medler, PhD.

Kathryn Medler, PhD

Physiology of Neuronal Cell Signaling Pathways

Some stimuli interact with receptors that initiate second messenger cascades, while others interact directly with ion channels to cause a cellular response. As characterization of these cellular mechanisms continues, we can begin understanding how the brain gathers information about its surroundings. The long term goal of the lab is to begin understanding how signaling mechanisms are regulated within taste cells and how this regulation impacts the generation of the stimulus signal to the brain. We use molecular and physiological techniques, including live cell imaging to investigate how signaling mechanisms in taste cells function.

The Medler Lab Research Projects

Faculty Profile and Publications

Department of Biological Sciences
619 Cooke Hall, North Campus
State University of New York at Buffalo
Buffalo, NY 14260
Phone: (716) 645-4947
Email: kmedler@buffalo.edu

The Medler lab studies the physiology of signal transduction pathways and the regulation of these pathways in neuronal systems. We focus on peripheral sensory systems, primarily the taste system. Chemical sensory systems, which are comprised of olfaction and taste, play important roles in feeding, territorial recognition and social interactions. The taste sensory system is used to determine whether potential food items will be ingested or rejected while the olfactory system is used in a multitude of behaviors such as kin recognition and mate selection. The taste system is extremely heterogeneous and is made up of distinct cell types that depend on multiple signaling pathways to detect stimuli. 

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Current schematic of the known signaling pathways involved in transducing different taste stimuli. Ionic stimuli (salt and sour) activate ionotropic receptors that activate a classical chemical synapse (far left). More chemically complex stimuli (bitter, sweet and umami) activate a GPCR signaling pathway in two different cell types (middle and far right).

The Medler Lab Research Projects

The Medler Lab Research Projects

The Medler Lab studies the physiology of signal transduction pathways and the regulation of these pathways in neuronal systems. We focus on peripheral sensory systems, primarily the taste system. The peripheral taste system is heterogeneous and is made up of distinct cell types that depend on multiple signaling pathways to detect stimuli. Some stimuli interact with receptors that initiate second messenger cascades, while others interact directly with ion channels to cause a cellular response. We recently identified a new taste cell type that expresses a unique signaling pathway and is broadly responsive to multiple types of taste stimuli. We also identified a novel role for TRPM4 in peripheral taste cells. This channel is required for the detection of bitter, sweet and umami stimuli but how it functions during the transduction of these stimuli is unknown. As characterization of these cellular mechanisms continues, we can begin understanding how the brain gathers information about its surroundings.

Characterization of the Broadly Responsive taste cells

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BR taste cells respond to multiple taste stimuli independently of the IP3R3 protein expressed in Type II cells. 
Representative traces of BR taste cells that were stimulated with multiple bitter (A), sweet (B), or umami (C) stimuli as well as 50mM KCl (arrow, Hi K).   D) Chi square analysis with Yate’s correction for continuity was used to compare the response rate or frequency of evoked Ca2+ responses to different taste stimuli between wild type (WT) and IP3R3-KO (KO) mice (p=0.062).  E) Chi square analysis with Yate’s correction for continuity was used to compare the response rate or frequency of evoked Ca2+ responses by BR cells from wild type (WT) and IP3R3-KO (KO) mice for circumvallate (CV, p=0.09), foliate (Fol, p=0.89) and fungiform (Fun, p=0.7) taste cells. Number of responses per number of cells tested are presented in the graphs.

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Some Type III cells respond to bitter, sweet and umami stimuli.
A) Representative trace of taste cells from IP3R3-KO mice that responded to bitter (5mM denatonium, Den), sweet (20mM sucralose, Sucr), umami (10mM monopotassium glutamate, MPG), 50mM citric acid (CA), and 50mM KCl (Hi K).  B) Representative trace of a separate subset of Type III cells that responded to 250mM NaCl, 50mM CA and 50mM KCl but were not sensitive to the bitter, sweet, umami stimuli tested.  C) Summary of taste cells from IP3R3-KO mice that responded to 50mM KCl and CA with Ca2+ signals (n=65), 28% only responded to CA (n=18), 20% responded to CA and NaCl (n=13), and 52% responded to CA and bitter, sweet, and/or umami stimuli (B,U,S, n=34) while no cells responded to all taste stimuli.  D)  A summary of the response profiles for a larger group of BR Type III cells (n=127) that either responded to bitter (n=7), sweet (n=9), umami (n=7), bitter + sweet (n=13), bitter + umami (n=15), sweet + umami (n=21), or bitter, sweet + umami (n=55). 

 

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PLCb3 is required for the detection of bitter, sweet and umami stimuli in BR cells.
A) A representative trace from PLCb3-KO taste cells that responded to 50mM citric acid (CA) and KCl (50mM, Hi K) but did not respond to bitter (5mM denatonium, Den), sweet (20mM sucralose, Sucr), or umami (10mM MPG) stimuli. B) A representative trace from a separate subset of cells that responded to 250mM NaCl, CA, and KCl but were not sensitive to the bitter, sweet, or umami stimuli tested.  C) Summary of responsive taste cells from PLCb3-KO mice (n=38): CA (n=18); CA +NaCl (n=3); CA and bitter, sweet, and/or umami stimuli (B, U, S) (n=0); and B, U, or S only (n=17). Representative taste-evoked responses for bitter (D), sweet (E), and umami (F) in the PLCb3-KO mice.  None of these cells responded to CA or KCl.  G) Chi square analysis with Yate’s correction for continuity was used to compare the response rate or frequency of BR cells in wild type (n=53) and PLCb3-KO (n=68) mice (***, p<0.001) for a larger number of cells.  H) The percentage of BR taste cells from the CV, Fol, and Fun papillae of the PLCb3-KO mice and WT mice were compared (***, p<0.001 for CV and Fol; **, p=0.011 for Fun). 

 

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PLCb3 and IP3R1 colocalize in taste receptor cells.
GFP fluorescence (green) is associated with IP3R3 expression. Anti-PLCb3 (red) and anti-IP3R1(blue) labeling of the same section. An overlay of anti-PLCb3 and anti-IP3R1 reveals heavy co-localization for the labeling of these two proteins. An overlay of anti-PLCb3, anti-IP3R1 and IP3R3-GFP expression indicates that IP3R3 is found primarily in a separate population of taste cells with very little overlap between PLCb3 and IP3R1. The bright field image of the taste buds is shown. Scale bar = 20 mM. 
 

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IP3R3-GFP and IP3R1 expression do not overlap in circumvallate taste cells.
IP3R3-GFP expressing taste cells (green) are shown in the left panel while IP3R1-IR is shown in red in the middle panel. The right panel shows the co-localization of these two proteins. No overlap in their expression was found, indicating they are expressed in separate taste cell populations. Bar = 20 mM

Defining the role of TRPM4 in taste cell signaling

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TRPM4 is expressed in Type II and III taste cells.
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Expression of TRPM4 (left panel), the Type II cell marker TRPM5-GFP (middle panel) and an overlay with DIC image (right panel) are shown. The asterisks identify TRPM4 expressing cells that do not express TRPM5-GFP. B. Expression of TRPM4 (left panel), the Type III cell marker SNAP-25 (middle panel) and an overlay with DIC image (right panel) are The asterisks identify TRPM4+ cells that do not express SNAP-25. Scale bar= 20μm.

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Taste evoked Na+ signals in isolated taste cells depend on TRPM4 and TRPM5.
Representative dual Ca2+ (black line) and Na+ (red line) imaging traces from wild type mice showing cytosolic Ca2+ and Na+ responses to different taste stimuli: A. bitter (10 mM denatonium benzoate, Den), B. sweet (20 mM sucralose), and C. umami (20 mM monopotassium glutamate [MPG]). D. A representative dual Ca2+ and Na+ imaging from a TRPM5-KO cell showing the evoked Ca2+ and Na+ responses to a taste stimulus (10mM Den). E. Representative dual Ca2+ and Na+ imaging from a TRPM4-KO cell showing evoked responses to a taste stimulus (10mM Den). F. TRPM4/5-DKO cells lack a Na+ response but did generate a Ca2+ response to 10mM Den. Chi-square analysis was used to compare the percentage of responsive taste receptor cells from the CV papillae (G) and fungiform papillae (H) for wild type and TRPM5-only, TRPM4-only, and TRPM4/5-DKO mice.
 

Characterization of calcium buffering mechanisms in taste cells

Signal regulation within cells is a critical part of generating an appropriate response to a stimulus. However, little is known about how signals are regulated within taste cells and how this regulation impacts the stimulus signal sent to the brain. We use molecular and physiological techniques, including live cell imaging to investigate how signaling mechanisms in taste cells are regulated to produce the appropriate output signal.

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Defining the relationship between diet-induced obesity and taste cell signaling

The taste system is used to determine whether potential food items will be ingested or rejected and has important, though poorly characterized, roles in appetite and food intake.  When the relationship between taste and appetite is damaged, eating problems such as malnutrition and obesity can occur.  We are currently interested in deciphering the relationship between obesity and taste.  We found that diet induced obesity causes a significant reduction in the responsiveness of taste cells to some stimuli, particularly sweet stimuli.  Follow up studies in our lab have revealed that diet and weight can independently affect responses to stimuli, but there is a selectivity in this effect.  Current studies are focused on understanding how this happens.
 
 
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Differential effects of diet and weight on sweet stimuli.
A combination of live cell imaging and unconditioned brief-access licking assays were used to assess the relative effects of DIO and HF diet on the ability of the taste system to respond to sweet stimuli.  The left column is the total number of taste cells that responded to each stimulus. Significant differences are indicated by different letters. The second column is the response amplitude of the taste-evoked calcium responses for cells isolated from mice under each experimental condition (*, p<0.05; **, p<0.01) and the third column is brief-access licking behavior for each stimulus. The dotted line in the third column indicates the stimulus concentration used in the live cell imaging experiments. 

 

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DIO affects the protein expression of α-Gustducin and PLCb2 expression.   A) Representative immunohistochemical images of α-gustducin expression (red) in the CV taste buds of HF+/- CAP and CTL+/- mice. 
B) Representative immunohistochemical images of PLCβ2 (red) in CV sections of HF and CTL mice +/- CAP. 

Scale bars=20µm.

Transcriptional regulation of peripheral taste cell development and maintenance

Taste cells are unique in that they have characteristics of both neurons and epithelial cells. Like neurons, they are excitable cells, form synapses and fire action potentials but like epithelial cells, they are routinely replaced throughout an organism’s life. Cell turnover and maintenance is not well understood and many factors likely contribute to this process.  We have found that a transcription factor, WT1, which is important in cell growth and differentiation and plays a role in pediatric kidney cancer, is expressed in taste cells. Our initial studies have found that WT1 is required for the normal development of the peripheral taste system.  We have also found that an important WT1 regulator, BASP1, is co-expressed with WT1 in adult taste cells where they are required to maintain taste cell differentiation. Further studies are focused on elucidating the role of the WT1-BASP1 complex in the maintenance of the peripheral taste system.
 
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Knockout of BASP1 in the adult CV leads to a disruption of type II and type III cells markers.
(A) Immunohistochemistry of CV taste buds using anti-BASP1 (red) in CTL and KO mice that had been treated with tamoxifen. Scale bar=50µm. (B) As in part A except that NTPDase2 (type I marker) was tested. A plot of signal intensity per taste bud is shown at right. Horizontal bars represent mean intensity. (C) As in part A except that anti-PLCb2 (type II marker) was used. A plot of signal intensity per taste bud is shown below. (D) As in part A except that NCAM labelling (type III marker) was measured. (E) The number of PLCb2- (above) and NCAM- (below) positive cells per taste bud in CTL and KO cells is shown (***, p<0.001).

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The WT1-BASP1 complex represses LEF1 and PTCH1 expression in the differentiated cells of the CV. 
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 Quantitative PCR (qPCR) was used to detect LEF1 (above) and PTCH1 (below) expression relative to GAPDH in mRNA isolated from taste cells of CTL and KO mice. Mean (of three independent experiments) with standard deviation are reported.  (*, p<0.05, Student’s t-test). (B) ChIP analysis of isolated taste cells from adult mice. WT1, BASP1 or control (IgG) antibodies were used. After normalization to input DNA, fold enrichment at the LEF1 (top) or PTCH1 (bottom) promoter regions over a control genomic region is shown in CTL and KO mice. Mean (of three independent experiments) with standard deviation are reported (*, p<0.05, ***, p<0.001). (C) Immunohistochemical analysis of LEF1 expression in the CV of CTL or KO mice (left panels). Expression levels of PTCH1 in the CV of CTL or KO mice (right panels). Scale bars = 20µm.

 

The Medler Lab Research Areas