UVM Theses and Dissertations
Format:
Print
Author:
Yang, Chun
Dept./Program:
Biology
Year:
2010
Degree:
PhD
Abstract:
The vomeronasal organ (VNO) present in many vertebrate animals detects pheromones and some volatile odorants that are important in regulating animal behavior, such as reproduction, hunting and aggression. Vomeronasal sensory neurons (VSNs), located in the VNO, are the initial site for transducing these chemical signals into electrical signals. The pheromone/odorant receptors on microvilli of VSNs are G-protein coupled receptors. Their activation stimulates the phospholipase C (PLC) pathway, increasing diacylglycerol (DAG) that activates a transient receptor potential channel (TRPC2). This induces an influx of Na and Ca²⁺. However, under normal physiological conditions, non-selective cation channels can be partially blocked by extracellular Ca²⁺. Thus, we asked if there is any mechanism for amplifying the primary response and if there is mechanism for modulating the pheromone/odorant response.
Using perforated patch clamp, we found that ~80% of the urine-induced inward current in mouse VSNs was carried by chloride. This was confirmed by whole-cell patch clamp where the reversal potential of urine responses showed no change when extracellular Na was lowered but had a positive shift when extracellular cr was lowered. Removing extracellular Na⁺ and Ca²⁺ resulted in a complete elimination of urine responses. The chloride channel blocker had no effect on urine responses in zero extracellular Ca²⁺, suggesting the chloride current was dependent on extracellular Ca²⁺. Moreover, we recorded channel activity in membrane patches taken from the dendritic tips of VSNs with only chloride as the permeable ion. Our data showed the presence of Ca²⁺-activated C1⁻ channels in VSNs. Several possible candidates for the Ca²⁺ -activated cr channel were detected in VNOs with RT-PCR These data indicate that during urine stimulation, the influx of Ca²⁺ activates chloride channels and the efflux of chloride dramatically increases the response to urine.
Moreover, using TRPC2⁻/⁻ mice, we showed some interesting preliminary data that suggest the presence and biological function of an arachidonic acid (AA)-activated Ca² -permeable channels other than TRPC2 in VSNs. During urine stimulation, Ca²⁺ influx through these AA-activated channels also activates the chloride channel and causes the efflux of chloride.
We also studied the hormonal modulation on VSNs and found that oxytocin modulated the firing frequency and urine responses of VSNs. By using calcium imaging, we recorded oxytocin-induced [Ca²]i increases and oscillations mainly in the soma. This [Ca²⁺]i increase and oscillation required the presence of extemal Ca²⁺ and activation of second messengers. By using whole cell recording, we also recorded the increased voltage-dependent Ca²⁺ current with oxytocin stimulation. Further, we detected the expression of oxytocin receptor in VSNs. Taken together, we propose a model for oxytocin modulation: oxytocin induces the calcium entry through the OXTRG protein-PLC pathway and the increase of intracellular calcium modulates the neural excitability as well as the responsiveness to pheromones/odorants.
Using perforated patch clamp, we found that ~80% of the urine-induced inward current in mouse VSNs was carried by chloride. This was confirmed by whole-cell patch clamp where the reversal potential of urine responses showed no change when extracellular Na was lowered but had a positive shift when extracellular cr was lowered. Removing extracellular Na⁺ and Ca²⁺ resulted in a complete elimination of urine responses. The chloride channel blocker had no effect on urine responses in zero extracellular Ca²⁺, suggesting the chloride current was dependent on extracellular Ca²⁺. Moreover, we recorded channel activity in membrane patches taken from the dendritic tips of VSNs with only chloride as the permeable ion. Our data showed the presence of Ca²⁺-activated C1⁻ channels in VSNs. Several possible candidates for the Ca²⁺ -activated cr channel were detected in VNOs with RT-PCR These data indicate that during urine stimulation, the influx of Ca²⁺ activates chloride channels and the efflux of chloride dramatically increases the response to urine.
Moreover, using TRPC2⁻/⁻ mice, we showed some interesting preliminary data that suggest the presence and biological function of an arachidonic acid (AA)-activated Ca² -permeable channels other than TRPC2 in VSNs. During urine stimulation, Ca²⁺ influx through these AA-activated channels also activates the chloride channel and causes the efflux of chloride.
We also studied the hormonal modulation on VSNs and found that oxytocin modulated the firing frequency and urine responses of VSNs. By using calcium imaging, we recorded oxytocin-induced [Ca²]i increases and oscillations mainly in the soma. This [Ca²⁺]i increase and oscillation required the presence of extemal Ca²⁺ and activation of second messengers. By using whole cell recording, we also recorded the increased voltage-dependent Ca²⁺ current with oxytocin stimulation. Further, we detected the expression of oxytocin receptor in VSNs. Taken together, we propose a model for oxytocin modulation: oxytocin induces the calcium entry through the OXTRG protein-PLC pathway and the increase of intracellular calcium modulates the neural excitability as well as the responsiveness to pheromones/odorants.