Phys 173 BGGN 266 LPA Induced Cl Oscillations in Xenopus Oocytes Nini Huynh David Marciano Chisa Suzuki If only we hadn t poked these oocytes how cute would it be INTRODUCTION Electrophysiology in the Xenopus oocyte began in 1982 when scientists realized that they could inject mRNA into oocytes and express functional ion channels and receptors Since this time scientists have utilized this important technique to study several aspects of the structure and function of ion channels and receptors These include 1 Analyzing the properties of mutated channels as a means to understand the structure function relationships in ion channels 2 Studying the post translational processing and the assembly of multisubunit channels and receptors 3 Comparing the properties of channels from various tissues expressed in a common environment 4 Examining the modulation of channel and receptor function by various second messenger systems 5 Analyzing various aspects of receptor effector coupling 6 Functional screening of cloned genes that encode channels and receptors Source www axon com Most of these experiments require electrophysiological recording from oocytes It is therefore important to have a basic understanding of the oocytes and the techniques used to record from them The Xenopus Oocytes Xenopus oocytes are the precursor to frog eggs the difference being that they have not undergone the proper hormonal stimulation to mature The oocyte is a large cell with a diameter of 1 1 2mm making them ideal for electrophysiological recordings The spherical structure fig 1 of the oocyte is characterized by two distinct hemispheres the animal pole dark and the vegetal pole light the animal pole being where the nucleus is located Fig 1 www axon com Another important structural component of the oocyte is the follicle cell layer The follicle cell layer is a source of many potential problems the first being one of practicality It is harder to poke an oocyte when it has its follicle cell layer attached Another potential problem is that there is electrical coupling between cells in the follicle cell layer which can be mistaken as electrical events occurring inside the oocyte For these reasons it is important to remove the follicle cell layer before poking the oocyte see lab manual AMENDED PROCEDURE We closely followed the procedure describe in the 2001 Lab Manual for Oocyte Biophysics making only a few modifications Our goal was to introduce goat serum containing LPA into the oocytes bath and observe Cl oscillations A detailed explanation of the cascade that produces these oscillations is to follow in the next section One departure from the given procedure was the concentration of goat serum that we exposed the oocytes to Our dilution ratio was 1 10 while last years procedure called for 1 1000 A detailed discussion of the effect this change in serum concentration has on the Cl oscillations is to follow Another change to the given procedure was the technique used to introduce the serum The lab manual called for the oocytes to be perfused with a serum saline mixture Instead we setup a 3rd micro injector with a blunt tipped pipette We then aimed the pipette full of serum a few diameters upstream of the oocytes keeping the perfusion on A blast of 100 mseconds seemed to be an adequate amount of serum to illicit an oscillatory response After the oocyte was exposed to the serum we recorded for 800 seconds getting oscillations after 400 seconds BACKGROUND Ca2 mobilization system due to intracellular chemical reaction cascade in Xenopus Oocytes can be induced by acetylcholine ACh or serum Fernhout Dijcks Moonlenaar and Ruigt 1992 Both ACh and Lysophosphatidic acid LPA found in serum causes signaling pathways which increases intracellular concentrations of inositol 1 4 5 triphosphate IP3 IP3 binds to its surface receptor on the endoplasmic reticulum ER which results in a rapid release of Ca2 from the ER explained in more detail in 2001 Lab Manual for Oocyte Biophysics This is then followed by an influx of extracellular Ca2 via a plasma membrane signal activated by Ca2 depletion in the ER Callamaras and Parker 2000 This increase in intracellular Ca2 opens Ca 2 dependant Cl and K ion channels Dascal Gillo and Lass 1984 Further increase of Ca2 inhibits Ca2 release from the ER Superposition of Cl and K influx and Ca2 feedback causes current oscillation which can be observed using the voltage clamping technique Despite the considerable variation of oocyte response among donors and batch of collagenase used Dascal Landau and Lass 1983 there is some similarity in the character of oscillation that can be observed The shape of response to ACh can be analyzed as four components D1 D2 H and F D1and H found less variant with various conditions in oocyte than D2 and F Early fast inward current D1 and late slow inward current D2 both result from increase in Cl conductance due to Ca2 release and influx The fluctuations F are a result of individual Cl channel events Dascal Landau and Lass 1983 H is a long lasting outward current due to an increase in K conductance Dascal Gillo and Lass 1984 Fig 2 Four muscarinic responses to ACh in a voltage clamped Xenopus oocyte Dascal Landau and Lass 1984 D1 is the fast inward Cl current due to Ca2 release from the ER The Ca2 will then bind to the Cl channel activating it by opening the channel and thus allowing Cl to enter The stored Ca2 by the ER when released also induces extracellular Ca2 to enter the cell This influx of extracellular Ca2 raises the intracellular Ca2 high enough that it will bind to more chloride channels and so more Cl will enter the cell This gives the second peak D2 Since there are multiple types of Ca2 dependent Cl channels with different affinities for Ca2 Tigyi Dyer Matute and Miledi 1989 it makes sense that we see fluctuations indicated by the F component Also since influx of extracellular Ca2 is coupled to the ER depletion of Ca2 stores this may be further explanation of the fluctuations When intracellular Ca2 reaches a threshold IP3 is inhibited from binding to the Ca2 channel located on the ER which controls the release of Ca2 from storage As intracellular Ca2 decreases it triggers IP3 to bind to the ER releasing Ca2 from the stores again This continuous cycle explains the oscillation Finally the smooth H component is comprised of the slow long lasting outward K current The increase in intracellular Ca2 opens Ca2 dependant Cl channels as well as K channels This allows for K