<TITLE: Release and Neuromodulatory Effects of Taurine in the Rodent Striatum
ACADEMIC DOMAIN: medicine
DISCIPLINE: internal medicine
EVENT TYPE: doctoral defence discussion
FILE ID: UDEFD130
NOTES: continuation of UDEFP130

RECORDING DURATION: 110 min 13 sec

RECORDING DATE: 14.12.2006

NUMBER OF PARTICIPANTS: unknown

NUMBER OF SPEAKERS: 2

S2: NATIVE-SPEAKER STATUS: Russian; ACADEMIC ROLE: research student; GENDER: female; AGE: 24-30

S3: NATIVE-SPEAKER STATUS: Finnish; ACADEMIC ROLE: senior staff; GENDER: male; AGE: unknown>



<PRESENTATION UDEFP130 by S2>

<S3> distinguished <FOREIGN> kustos </FOREIGN> disputant , er ladies and gentlemen colleagues and all the friends so this is a very ni- nice occasion because i see see many friendly faces here and usually when you are an opponent er you you er the faces look a little bit different but er anyway we are we are going to going to discuss on brain diso- er brain er brain disorders and brain research which is basically very important area er for the for the research and for the society because er the society is actually they they really want to have some results and and basically er very soon of the of the tremendous er er input that has been put into brain research so so there has been probably er hundreds of molecules that have been er proven efficient in animal models of neuro-degeneration or or ischemia models but unfortunately hardly anything is coming to the to the finish so there are still a lot of things that we ha- we have to we have to learn , so this is er this is basic research and it's about the taurine and and er and er glutamate which are two very important er er systems in the brain although in a way fairly very much different systems but anyway this interaction is now under study so there will be some some interesting findings and er we hope that we will discuss er all the things er in in such a detail that we can we can make some conclusions and er and as usually as everybody knows er this dissertation er occasion is er actually gives the opportunity for the disputant to explain everything what what er what disputant has been doing during the studies and and er i'm pretty sure that er that er today the disputant has many things that she wants to tell us about the experiments okay we will proceed now to the more detailed examination <GETTING SEATED, P:09> okay so this er i don't know whether my voice is is er everybody can hear it yeah without the microphone so so this er this s- book is is er is printed er very nicely and er it's er it's in a way easy to read and er the topic release and neuromodulatory effects of taurine in the rodent striatum is very exact what has been what has been done and er and release was studied er er in vivo mostly and then then this neuromodulation was studied mostly in in vitro systems so it's er it's exactly what was done , okay this er this whole er whole research er tradition on taurine actually has been long studied here in in in tampere and er and er i don't know how many students have been doing this kind of work but i think this this er again takes one step forward this whole whole study so if we first look at the page five list of er er original communications in the thesis we can immediately note that er that er <NAME S2>'s name is first in every single paper here and er that probably means that she has something serious to do with all these papers and er because i doubt that anybody would er could write five papers for somebody else so so i think that this is er this is a good acc- achievement nowadays because fairly often er er people are making these thesis projects in in much larger groups and then there will be like like er like s- joint first authorships or or something like that in some of the papers but here the contribution of the of the er disputant er seems to be very clear so the papers er have been published er and er also the last one is in press and er so they have all been proved the normal er international referee process and er and er in that sense they have been already found that they are they are they are acceptable in er with these international standards so er the journals er erm er the impact factors range er somewhere from two to five or something like that or or maybe a little bit less than five but anyway they are like like er like er good er medium class er er er international publications er in neurochemical and er brain research fields so this whole study is basically basically neurochemistry also neuropharmacology to some extent and er and in a way also neuroscience so it has many many of these er fields included . okay i was thinking that we will just discuss a little bit about the about this er this background information and and then we will go through the original publications where we spend most of the time and er after that we will go to the to the results and discussion and er and er and go in that way through the whole system , we already heard that the that taurine is actually used in the clinics and er and er but is there is there any dis- any human disease that has like er clear but er defects in taurine-related genes or in or or something like that er er in humans with cats there is but erm but what about humans </S3>
<S2> as to my knowledge erm i can't say any diseases er that evoke the er changes in gene expression of a synthetiser ta- er enzymes or taurine transporters as i understand it's , in some cases there is a deficiency of taurine maybe due to the lack of er supply of food or due to a lower synthesis rate however er in the usual case the synthesis rate plus dietary taurine is usually enough to keep the quantity of taurine er in a normal conditions in human body </S2>
<S3> yeah what ha- what happens in the body when you when you change this taurine system when you remove the system in the for example in the in the mice </S3>
<P:08>
<S2> i think that the most er profound effect would be on changes of the blood pressure but since taurine have so wide physiological actions it's hard to say what component will (be in) first so maybe er what's more easiest to detect is the changes of the blood pressure and after that there may be some problems with the er osmolarity of the cells their (xx) osmolarity of the cells there are two maybe quite er general er physiological functions </S2>
<S3> so how do you how do you get this kind of modification done with mice how can you how can you <S2> mhm </S2> remove or block the taurine [effects] </S3>
<S2> [it's] it's quite hard nowad- nowadays we still don't have very good model for inhibiting some actions of taurine er the first er approach is to apply a lot of er er inhibitor of taurine uptake for example beta-alanine or cloning DNA (xx) er however these er substances have a number of side effects and er er nowadays er you can't be sure if you if the changes you can observe in animals (are) related to er er reduced concentration of taurine inside the cells or some side effects of the substances and now the approach is er er mice na- or the lock out o- on er taurine transport and these mice are generally they are alive they can survive in er homozygotes er however they have er serious er eye diseases and they have retinal degeneration of the eye </S2>
<S3> yeah how how w- what are their taurine levels if this transporter is is knocked out </S3>
<S2> i don't really know (xx) [(@@)] </S2>
<S3> [yeah] and maybe maybe ten per cent of the of the normal o- or maybe even less [and and er] </S3>
<S2> [maybe yes] </S2>
<S3> they have the- they have like serious problems in in in development the brain development is is er is abnormal and er retinal development is abnormal the kidney function is abnormal and probably many other things also so this means that taurine is really needed <S2> yeah </S2> so that's the that's the idea so you describe here on page er er ten you describe er the the erm metabolism of taurine in mammalian systems , so how much taurine is produced by this metabolism as as er compared to the to the diet- dietary supply </S3>
<S2> in humans it's little bit less er compared to the dietary supply in rodents it's er (big deal) of taurines produced due to the synthesis and the main sources mainly erm (xx) of synthesis of taurine are liver and the brain so brain has it in enzymatic machinery to synthesise taurine for its own purposes </S2>
<S3> is it in the in the er neurons or in the (glial cells) in the brain </S3>
<S2> i think both both types of cell </S2>
<S3> okay so so this input and output function it it it's dependent very much on the species and er and with humans you need also also this dietary <S2> yes </S2> er <S2> that's right </S2> to some extent but you can you can get along rather well also with the with the metabolic (xx) , okay so you mentioned that there are some some inhibitors of this taurine uptake so how are they working </S3>
<S2> they are quantitative inhibitors so they bind to the same er site as taurine does and they are transferred to to the cell however they compete with taurine for the (xx) so they by this competing they reduce the intake of taurine to the cell </S2>
<S3> can they compensate the loss of taurine in these in these er in these mouse models for example </S3>
<S2> mhm it's it's not discussed in (xx) so but i think [they can't] </S2>
<S3> [yes they cannot because] their [(different form)] <S2> [yeah] yeah </S2> okay so so then there is discussion about taurine release from nervous system and er it has been long time (quite er quite) a problem i think think because er this has been difficult to demonstrate the the calcium dependency of the of the release which is usually required for the for the neur- neurotransmitters for their release so can you summarise what is known about the calcium dependency of the taurine release </S3>
<S2> so if to summarise in some cases it's possible to show er the evoked release of taurine to be in part calcium-dependent in other cases it was not shown this calcium dependency of er evoked release has not been shown and however er when measuring the basal release of taurine the (xx) of calcium (xx) for <COUGH> from the medium evoked taurine release so from these studies it's quite hard to get general conclusion if the release of taurine is calcium-dependent or not so results depend on the model used basically </S2>
<S3> yeah and the main problem is that er this er decreasing of calcium is producing changes in the <S2> [yes] </S2> [taurine release] okay we we will come back to that then later yeah er what about the what about tau- taurine in and er and er receptors , er is that a clear story that taurine acts on some some receptors like neurotransmitters and er and what kind of receptors are they what would w- do they do to the neurons </S3>
<S2> er that's not a clear story because er as i understand in a historical approach it was first proposed that taurine can bind to some specific taurine receptor however when er during the studies of this possible receptor of taurine it was found that binding of taurine to the cell membranes er can be er inhibited by application of GABA and glycine so it means that er they can bind to the binding site and after that it was shown that er several inhibitors of GABA receptors and glycine receptors may er inhibit some bia- biological effects of taurine so nowadays er finally there are some studies showing that taurine is a partial agonist at GABA or glycine receptors however er the question about the specific taurine receptor is not yet closed as still some groups propose the er existence of this receptor showing that in some er models experimental models taurine binding cannot be inhibited by a GABA or glycine agonist or antagonists so nowadays it seems that taurine can act on GABA receptors or glycine receptors and probably to some specific taurine receptor which is not shown </S2>
<S3> okay so what is the affinity of taurine to its (xx) </S3>
<S2> i can't say you the numbers but it's less th- compared to the native agonist GABA and glycine so it's a partial agonist at that receptors </S2>
<S3> so do partial agonists al- always have like reduced affinity </S3>
<S2> yeah i think so @@ </S2>
<S3> so wh- what kind of er what kind of (liken) we can say that it is a partial agonist in a receptor </S3>
<S2> yes i understand the agonist which has a lower affinity compared to the full agonist if </S2>
<S3> lower affinity </S3>
<S2> yeah </S2>
<S3> what about the efficacy </S3>
<P:05>
<S2> i can't say that i think we- i think that it's little bit easier to measure the affinity compared to efficiency </S2>
<S3> but if you use ecophysiology you can you can you can just use er use er neurons and er and er apply GABA or or taurine and er see their interaction and er if <S2> [yes] </S2> [the] taurine is partial agonist <S2> [yes] </S2> [then] then it should should produce less effect than GABA no matter how or what concentration you (call) it doesn't it's it's nothing to do with the affinity and er and if you add like GABA and taurine together taurine should then if it is a if it is a partial agonist at the same site it should th- then reduce the actions of GABA </S3>
<S2> yes [that's right] </S2>
<S3> [so] has that been shown </S3>
<S2> yes it's shown er i maybe i'm mistaken in this case but i suggest that er the most clear approach to characterise a receptor is to express it in some artificial system for example er (xx) to all sites and see the clear characterisation of this receptor when er somebody is measuring the characteristics of erm for example GABA and taurine evoked occurrence on the neurons there may be some additional regulatory mechanisms so that's @why i@ said that maybe the affinity is more er reliable characteristic er compared to efficiency </S2>
<S3> yeah <S2> [but @@] </S2> [but the i- the the affinity] doesn't doesn't matter if you if you are speaking about partial agonist <S2>  mhm </S2> it is the efficacy </S3>
<S2> okay </S2>
<S3> and er so this means that er that er er if there is more taurine in the brain than GABA then basically taurine can then limit the actions of GABA reduce them </S3>
<S2> yes that's right </S2>
<S3> so is is taurine then i- is it is it like an excitatory substance does it does does it er is it er (proconvulsant) produce anxiety and er </S3>
<S2> i- in this aspect is it has not been shown <S3> yeah </S3> i- i- it has been shown that in (xx) (long-term application) of taurine may evoke er the mhm reduced er expression of GABA receptors , so it means that maybe in a normal condition taurine interacts with GABA receptors so when we apply the high dosages and get reduced GABA receptors and er then er there will be a shift to the more exi- excitatory er systems and (xx) systems so but er i- i- it can't be said that in normal conditions taurine can be excitatory due to the reduction of (xx) or GABA (xx) receptors </S2>
<S3> okay so maybe maybe taurine has many other effects also also not only these er these effects on the neurotransmitter receptors although they have been people have been most interested on those <S2> yeah </S2> because er they might er make it possible to directly influence the firing of the neurons , okay maybe we come back to those er those er things when we go to the publications so what does taurine do intracellularly , so this was apparently an extracellular effect </S3>
<S2> yes for most well-described intracellular effect of taurine is related to its regulation of er calcium storage in cells er , however er it can't be (ruled) out from the studies er that taurine enhance the uptake of calcium by mitochondria or it can stop the release of calcium through mitochondria the studies are little bit mhm opposite in the results however it's clear that taurine does something related to the calcium storage er in inside the mitochondria so it may prevent the excitatory death of the neurons er by preventing er the calcium release from the mitochondria but in some other studies it was shown that it can promote the accumulation of calcium in mitochondria so the to le- two little bit opposite er approaches however i- it's clear that it does something and the second (section) is not known it's it was shown that it's not related to the er , m- mitochondrial membrane transition pore so maybe it's mhm related to the calcium uptake system in mitochondria </S2>
<S3> yeah is the reason that it is not known is the reason that it has so poor poor affinity or sensitivity to these systems that it is difficult to find this kind of er molecular interaction with any any of these transporters or channels </S3>
<S2> <COUGH> i think no i think that er at first to my knowledge no-one has tried that i mean to search for a certain protein which can bind down in in the mitochondria and express its effect on the calcium regulation and er the next reason is that this whole calcium mitochondrial story is not clear yet at that time so it it's known that mitochondria are the one of the main calcium storage (xx) cell and for example under some damaging conditions er in ischemia or (xx) er mitochondria can accumulate er excessive calcium concentration from the (xx) to (xx) mitochondria er however when er the certain level of calcium or concentration is reached mitochondria can collapse and then then release the calcium inside the (cell) so this these are two opposite approaches s- accumulation of calcium to the mitochondria and after that release of calcium from the mitochondria and they have two opposite meanings for the cell survival or death and i think er that's quite complicated story without taurine so with the taurine it's quite i think we still know little about that </S2>
<S3> yeah so we don't know the findings are (sort of) [(xx)] </S3>
<S2> [yes] that's right </S2>
<S3> okay may might be an indirect effect also </S3>
<S2> yes it may be so </S2>
<S3> okay then er at the end of this er review of literature you are you are describing er neuroprotective effects of taurine and and er it seems that again you have the same kind of er of er ideas here . er actions on the on the receptors and er and er then actions on on glutamate release and er and er calcium and er and that basically then , then summarises what is known , okay so then we arrive to page 20 and th- there is aims of the study and it seems that er that these have been very carefully constructed so that s- so that you can get good answers to these er aims and er and er it's mostly so that one paper is then answering one question and er and that makes it much easier much easier to read and we will go to the aims then we go to the to the to the publications and act- actually that's what we are going to do now so we go to the first first paper , which is er which is <NAME S2> oja and saransaari published in amino acids characteristics of basal taurine release in the rat striatum measured by microdialysis so i would like you now fo- to to explain the aims the main methods and the results of this of this er study so this gives you the good opportunity to explain what you have been doing </S3>
<S2> so er this manuscript is more or less erm description of the main er characteristics of basal taurine release er the reasons for this study was that er in the present literature there were there was no general in- agreement about the mechanisms of taurine release so in different er experimental system er different mechanisms are shown so er here we (set out) to describe er b- basal characteristic of taurine release namely er how much taurine is how high concentration of taurine is present extracellularly er what factors can influence this concentration and if er this concentration make these levels of taurine may be supplied by er synaptic release of taurine and the beneficial side of this er study is that it was done in vivo so it was done on the anaesthetised rats erm by means of in vivo microdialysis so w- we were able to detect the changes in the taurine concentration is- in more or less (invasive) brain of rodent er <COUGH> and er first we measured the extracellular concentration of taurine and actually we showed that it's quite high especially compared to er the classical neurotransmitters like glutamate and then we confirmed that er <COUGH> these high amounts of taurine may be result of er taurine release through er volume-sensitive chloride channels and er , they maybe regulates by the uptake of taurine by the taurine transporter so it means that in extracellular space the concentration of taurine is quite high and it's continuously regulated er even in under (xx) conditions so it's like a er dynamic system which is quite constant however it's continuously regulated and after that we checked er if taurine release may be er may correspond to a s- synaptic release and it apparently er . it we showed that @er we@ can't get any good answer (after) that question is taurine has- released by synaptic mechanism or by non- not synaptic er , one er reason for that is that er we are working w- with the whole brain not like an isolated er structure but in a whole brain so when we apply a classical inhibitor er , classical one classical <SIC> criterium </SIC> for neurotransmitter release the inhibitor for sodium channels (xx) their reduction er we inhibited the cells in the striatum and the nerve endings which er finish to this on the striatal cells so er by this application we aff- affect the whole brain system and apparently we cut the release of glutamate from and we suggest that its release comes from the cortical impulse in the striatum because we changed the whole sys- brain system so er using this inhibitor we can't say that er something about the synaptic release because we apparently get s- get some side effects and er when we used another <SIC> criterium </SIC> for the neurotransmittory release er the lowering of the extracellular calcium concentration er we got a quite good response on release of glutamate which is like the more or less classical neurotransmitter and we use it as a reference , however we got the increased release of taurine under these conditions and er this increased release of taurine under er the emission of calcium ions was er erm aim the the study of this effect was the aim of our next manuscript actually so in this manuscript we characterise the basic er like content and regulation of extracellular pool of taurine and set up the question about the er mechanisms of release of taurine vesicular s- synaptic vesicular (in this) or not synaptic er non-synaptic release and we study it a little bit fo- further in the next paper </S2>
<S3> okay thank you very much so it was a good account of the of the of the paper did you do all these er er or set up all these methods yourself or or <S2> yes </S2> or were they were they ever- was everything ready here when you arrived to tampere </S3>
<S2> there was an excellent set-up for microdialysis studies so er i came here and started to work and i got everything working @@ so i did o- everything by myself [but then (xx)] </S2>
<S3> [yeah you you did] the experiments yourself but you didn't have to set up the methods [or so] </S3>
<S2> [no] not in this case </S2>
<S3> yeah okay good okay so so this er the actually the most interesting result there is that the the extracellular concentration of taurine is here 25 micromole </S3>
<S2> yes that's right , [actually] </S2>
<S3> [so so it] it's it's very high <S2> yes </S2> so how much is er the extracellular concentration of GABA for example </S3>
<S2> er may i start with the extracellular concentration of glutamate because i know for sure that in the striatum of rats the <S3> [yeah] </S3> [extracellular] concentration of glutamate is five micromoles [and concentration] </S2>
<S3> [but but we we want to know] the the extracellular concentration of GABA </S3>
<S2> it it's less than five , that what i can say it's [maybe] </S2>
<S3> [less than] five </S3>
<S2> yeah it's maybe close to three or in the [less] </S2>
<S3> [excuse me] </S3>
<S2> yeah le- less than five [micromoles] </S2>
<S3> [or less than] one </S3>
<S2> maybe </S2>
<S3> less than point five something like that <S2> yeah </S2> so so this is this may be like hundred times times higher than the GABA concentration which means that if it is a partial agonist it might well have some actions on <S2> [yes that's right] </S2> [on some some] GABA A receptors <S2> that's right </S2> which er which er depending whether it's (saturated) concentration or not whether it it then makes any difference or not , okay but anyway you measured er you you use this kind of ser- zero net flux method , so is that is that a good method to estimate the concentration why don't you just get the the fluid and measure concentration and that's it </S3>
<S2> <COUGH> the reasons for that is that er in microdialysis study we use <SIC> especial </SIC> probes er by which can er cut some , they are in probes with the (semi-permeable er membrane) (xx) and by perfusing the liquid flow of the probe we can er estimate er the relative concentrations of er for example amino-acids in the extracellular fluid it was it was shown that maybe ten per cents of er sub- of substance which present extracellular actually got into the probe and we have er these concentrations in our samples and er this microdialysis approach is very suitable for measuring the relative changes of concentration for e- for example if you have some (xx) and after that you stimulate your system somehow and you can see the changes and you can compare the changes with the (xx) however you can't say for sure that er this amount of substance which you are able to measure in the sample is for example exactly ten per cents er of what is actually in the brain of course it's possible to characterise a certain probe in in vitro conditions however in in vivo conditions the mechanisms of the diffusion of substances inside the probe is little bit different compared to the what happened in the (xx) so in this case we should apply some a little bit more sophisticated approach and we actually did quite er this zero net flux approach is actually very simple we perfuse er the (solution) which contains certain amounts of taurine and after the pef- perfusion we measure the implement or r- reduction of this er concentrations of taurine which were present initially so we can say er if er if the concentration inside the perfusion system is equal to that of the brain er this concentration will remain the same but or for example if this concentration is higher than that of the brain it will be reduced because of the diffusion of taurine to the brain , on their concentration rate </S2>
<S3> yeah okay so so how does taurine taurine er come out er from this brain tissue in into the probe , so you described already some of the mechanisms like the like the transporter and the volume-sensitive channels and er stuff like that and er if you i- i- if if taurine is coming through tau- taurine transport , and you add some er extra taurine there <S2> mhm-hm </S2> doesn't that produce then this kind of exchange transport and and er and and er change the whole system </S3>
<S2> yes that's possible , in general that's possible however if it will be the case then our , we (have got) a different concentration of taurine in this perfusion (medium) and if er this er high concentrations of taurine will have some effect on er working of for example taurine transporter er this line will not be straight so it will be like a curve for example something like that or that but we have quite good linearity in this case so i suggest that er taurine transporter in this case does not have the major effect maybe there is some effect but it's not [(xx)] </S2>
<S3> [yeah maybe] its concentrations are too low [to (have an effect)] </S3>
<S2> [maybe] </S2>
<S3> so so what does this probe to do to the brain tissue </S3>
<S2> [of course there is some damage] </S2>
<S3> [or so can you say that] it is like intact the brain tissue </S3>
<S2> er it's intact in the case that we are working with the er whole brain system however of course when er employed during implantation of the probe there will be some brain damage and , to avoid some er effects of this brain damage is it's necessary to keep some period of time for wash-out of er , damaged parts of the tissue or some er substances which are at least in the response to this damage and we er used one point five hour er interval between the implantation of the probe and er sampling of and start of studying of the sample </S2>
<S3> okay so i guess that's that should be should be good enough in order to get reliable results unless unless these er cells that are surrounding the probe are are are damaged and er and er then because they are if they are damaged then taurine metabolism or or fluxes may be different and and after that we don't know </S3>
<S2> of course they are damaged however when er your , er <COUGH> i guess that if i would start the measurement just after the probe implantation i will have very high levels of taurine in ou- in my sample however er with time it will reduce and er reach the linear more or less linear view at some time point and on this linear part of er the sampling it's possible to do some experiments </S2>
<S3> okay so then you show here on on figure two that er anion channel blockers er they reduce this er this er this er taurine release in the (into the probe) so and and th- then you describe that the taurine is is actually going through these volume-sensitive channels has that been shown </S3>
<S2> yes </S2>
<S3> so so and and with (xx) you can compete that </S3>
<S2> yes it's er blocker actually er it's a blocker of er a volume-sensitive chloride channels it means that it occupies the channel (xx) so it wi- it has been shown that taurine can come through these channels </S2>
<S3> can go through the channel </S3>
<S2> yeah </S2>
<S3> both ways , can go in and out </S3>
<S2> yes , no , i don't know at least it can go out @@ </S2>
<S3> okay and then when you block the taurine er er transporter at minu- one minimal concentration then you get more taurine out </S3>
<S2> yes </S2>
<S3> which which er which means that actually the taurine transporter is limiting the taurine [(outside)] </S3>
<S2> [yes that's right] yes it's it seems that in er resting conditions er taurine exits through the chloride channels and returns back to the cell through the taurine transporter so it's [(xx)] </S2>
<S3> [or or or] is this actually in the induction of this exchange transport </S3>
<P:05>
<S2> i think no because in a control conditions the er transporter is mainly directed from outside of the cell to inside of the cell </S2>
<S3> has somebody seen how it is directed . so how can a membrane molecule switch switch er is it somehow circling there or or </S3>
<S2> no it's not actually er <COUGH> <COUGH> </S2>
<S3> <POOR SOUND QUALITY> </S3>
<S2> yeah that <POOR SOUND QUALITY> into that channel however it's not the <POOR SOUND QUALITY> means that it's like a open war er it works like er . it exposes the binding site from to the outer er part of the membrane and when it gets the , when it binds for example taurine and er sodium then it can switch and open er this binding site to be accep- acceptable from the inner part and the molecules then diffuse to the er (outside) the cell so it works like that so er the work of the transporter is directed to the er difference in the concentration in sodium and if in the resting conditions we have er much more high high much higher concentrations of sodium er outside the cell and inside the cell so this difference in the concentration directed the flow of taurine and the sodium </S2>
<S3> okay and then if this resting membrane potential is completely messed up then you then you get <S2> [yeah] </S2> [a (xx)] case or or more even balanced er sodium concentration and then this transporter is supposed to be reversed </S3>
<S2> yes it is i understand it be- come to be non-organised or even it can be reversed however i can s- i can't imagine a situation that the concentration of sodium is much higher inside the cell so maybe er it's more correct to say that the direction is non-organised it's like still (question) </S2>
<S3> alright so what happens if you put er glutamate here into the probe </S3>
<S2> then glutamate will er diffuse from the probe to the surrounding tissue and it may act on glutamate receptor and then er evoked all types of (reactions) connected to the glutamate [receptors] </S2>
<S3> [yeah] but i i was mostly meaning what happens to the taurine release </S3>
<S2> @ah@ <GASP> mhm statics show that the glutamate can evoke the release of taurine , so acting by- acting to NMDA receptor and that's a (xx) receptor it can er evoke the the release of taurine and this release is er nitrical oxide sensitive </S2>
<S3> <S2> [(xx)] </S2> [are these er] are these about the same concentrations as as er as are th- there are in (the external) fluid i mean you do need er glutamate much more than the concentrations that they have in the for example in this this in vivo microdialysis system </S3>
<S2> mhm yes of course there should be higher concentrations , if er in case of the microdialysis study er the concentrations of the effector substances should be at least ten times higher than you (are) expect them to be in the extracellular space of the brain and after that it should be er higher compared to the basal concentrations (xx) </S2>
<S3> okay so so this paper was really interesting and er and er showed us that the the concentration of taurine is actually rather high in the extracellular fluid and i guess that already means that er that there cannot be any high affinity binding sites for the taurine in the brain </S3>
<P:04>
<S2> if [it's] </S2>
<S3> [there's no] way because you have 25 micromole concentrations </S3>
<S2> [if that's] </S2>
<S3> [so you can't] have any high affinity binding sites then , otherwise they would be all the time occupied </S3>
<P:04>
<S2> in this in this case it means that er GABA A and glycine receptors are should be also occupied or <S3> mhm </S3> (what are you) (xx) <S3> mhm </S3> and it doesn't , so it means that maybe there is [(xx)] </S2>
<S3> [so the affinity may be may be] er you you probably need more in order to produce this ac- [activation] </S3>
<S2> [yeah] yes that's that's right </S2>
<S3> okay good so let's go to the next one so it's er it's the paper two mechanism or mechanisms of enhanced taurine release under calcium depletion and the authors are the same and it's published in neurochemistry international and er and er the method is also the same so you only only would need to explain explain er the results </S3>
<P:04>
<S2> so in this manuscript we studied the phenomenon on of evoked taurine release er by emission of calcium ions and we focused on er mhm (buffering) of taurine release under these conditions and some regulatory possible regulatory mechanism the- which can evoke this release of taurine and we were able to show that under er calcium depletion taurine is not released by er , volume-sensitive (chloride) ions , how it happens in the basal conditions in these conditions it doesn't happen but er it's it seems that it's released through the reversal action of taurine transporter and after that we were able to show that er this release may be first more or less needed by in- inhibitors of all the sensitive calcium channels and on the opposite when these inhibitors are applying in the absence of extracellular calcium ions they were able to diminish the release of taurine so from these results we conclude that er under er calcium omission some part of taurine is released due to the non-specific influence of sodium ions from er voltage-sensitive calcium channels and of course this effect is er , not likely to occur in the normal condition becon- because in a normal condition it can't happen that we build up calcium and (xx) in extracellular (xx) however some part of taurine are released in response to mhm calcium omission may be related to er some calcium-dependent ions so er it may be related to the in- decreased influx of calcium for er voltage-sensitive calcium channels and er this decreased influx may somehow (attend) the taurine transportion and maybe e- enhance its er work in a reversed (xx) </S2>
<S3> okay so you used again er like er like er anesthetised er young rat what is a young rat and er what does anaesthesia do do to the to the to the brain </S3>
<S2> er we were , they were not @not (exactly young)@ @@ they were at least one month old <S3> yeah </S3> so it's a young adult (xx) </S2>
<S3> yeah <S2> [and] </S2> [but wha-] of course i wanted to know what is [young] </S3>
<S2> [yeah] yes that's right [@@] </S2>
<S3> [okay] so it's one month </S3>
<S2> yeah it's about one month so it's young adults and er here we used the halothane anaesthesia and as i understand er i i should say that i , don't really know enough about the mechanisms of the effect of halothane anaesthesia i know that it's related to er the modulation of glycine (xx) neurotransmitter systems in er er basal er er regions of the brain in the lower regions of the central neurosystem of the brain </S2>
<S3> yeah so so halothane actually er li- a- and this er inhalation of anaesthetics they have many (side effects) </S3>
<S2> yeah [probably i i will (xx) er yeah] </S2>
<S3> [so they they they little bit facilitate the] these inhibitory systems like glycine <S2> [yeah] </S2> [and] GABA and might also interfere with the possible taurine actions actions on those and er or or potentiate them and then they have many actions on calcium channels one on potassium channels so they have basically are very difficult er systems </S3>
<S2> yes that's right </S2>
<S3> so they produce a lot of changes in the in these er in the in the function of the neurons </S3>
<S2> mhm-hm </S2>
<S3> but er er i guess er the other possibility would have been that you you would er you would have used the like er chronic (xx) and and then then er you you would er you would er wake up the animal and er and just er let it heal and er then insert the probes probes while they are <SIC> wake </SIC> so can you do that </S3>
<S2> yes it was possible however it's more time-consuming and as i decided it's more easier to do these types of experiments because anyway er we have the base line and we can study some effects of our er pharmacological agents compared to the base line and er at least er the effects of calcium emission on taurine release were shown also in the (medium) so we can compare that to the results are (xx) </S2>
<S3> yeah i i i i sure accept this because i can't i can't make up any story why why how anaesthetics would have been [messed up in the] </S3>
<S2> [yes that's] </S2>
<S3> whole system in such way that [these results would be possible] </S3>
<S2> [yeah they they can affect] but </S2>
<S3> okay so erm er did you did you test whether any of these ef- effects are like er reversible </S3>
<S2> no i didn't (xx) </S2>
<S3> and also in the in the first study you didn't do any any any any reversibility tests </S3>
<S2> no , actually in er the figure two of er this manuscript er when we studied the (flow) of taurine transporters in the means of er taurine erm we used the er guanidinoethanesulfonate which is <SIC> competititive </SIC> competitive inhibitor of er taurine transporter and we (promoted) it during er one hour and after that we omitted the substance from the er medium and of course the application of er this substance by itself (implies) the extracellular concentration of taurine by inhibiting the transport and if we can if we will achieve er control conditions where we had only er guanidinoethanesulfonate er after interruption of er , application of this substance er the levels of taurine went into the base line so it means that it is the effect of the substance was tempotar- temporary </S2>
<S3> so you mean with this with this er with this GES subs- [substance] </S3>
<S2> [yes] yes that's right </S2>
<S3> okay so how does it work </S3>
<S2> it , as i understand it acts on the same site as taurine or the taurine transporter </S2>
<S3> okay so i- it prevents the taurine reuptake or something like that </S3>
<S2> yes </S2>
<S3> and then the taurine concentrates go up </S3>
<S2> yes that's right </S2>
<S3> and [what then] </S3>
<S2> [and when we] when we (promote) this substance inside the cell it does again the same as taurine if er taurine transporter works in the opposite direction by <S3> [yeah yeah] </S3> [(sub-releases)] it can (compete) to the binding site er now from the inner side of the membrane so it actually does the same but in <S3> [yeah] </S3> [in] this case it er , <S3> [so that's] </S3> [in the (xx)] of the release of taurine </S2>
<S3> that is of course </S3>
<DISC CHANGE>
<S2> (xx) of that however i think that that's not the case because er we applied guanidinoethanesulfonate only for one hour and there er maximum release was above 150 per cents from the base line (mediums) however er some other condition conditions may evoke er much greater release of taurine so it means that there are , <S3> [(xx)] </S3> [the releasable] taurine pool inside the cell is much higher that er the taurine which erm is released during er this one-hour application of G-E-S </S2>
<S3> yeah of course it depends er what is released . okay so er as er for the pharmacologist this this figure would have been better if you if you have the would have shown concentration dependence </S3>
<S2> mhm </S2>
<S3> if you would have shown that er when you increase more er the concentration of GES when you are loading it you would then got er get like more inhibition of the of the taurine release afterwards . but of course er that does not er does not alter the the other possibilities , okay good , and then you have measured these erm these calcium channel effects and er and they will also affect this er and you come with the with the scheme er figure six can you explain what you mean with this with this er with the scheme </S3>
<S2> mhm-hm er in the first case (on the) calcium omission we had the release of taurine through the reverse action of taurine transporter and it it's random (both) by decrease in er intracellular concentration of calcium which is the result of the decreased influ- influx of calcium er from outside and er th- the release of taurine is also (driven by) the non-specific influx of sodium through er voltage-sensitive calcium channels and when we apply (xx) of er voltage-sensitive calcium channels we prevent the this non-specific influx of sodium however we still have the decrease in intracellular concentration of calcium so we have we still have some release of taurine but it's reduced because we were able to inhibit one complement of er this mechanism of evoked taurine release and when we apply , mhm , calcium omission with the er (borders) of er voltage-sensitive calcium channels er we also have the same situation actually so we have this rele- release of taurine but it's reduced compared to just calcium omission er because we blocked the channels and er the idea is that the second picture mimics the conditions which we have in the first picture and the third picture shows that we can reduce the release of taurine by closing these channels voltage-sensitive calcium channels </S2>
<P:04>
<S3> okay it's not easy easy to figure out <S2> yes [but] </S2> [on] this basis but er but might be might be okay so what happens to these volume-sensitive chloride channels here </S3>
<S2> they don't act it seems or at least when we inhibit the volume-sensitive chloride channels we didn't have any decrease in taurine release so it seems that they don't (xx) of that </S2>
<S3> and the and the calcium enhanced release </S3>
<S2> yes , yes </S2>
<S3> okay good let's go to the third one , and this is still in vivo work and this is now a little bit more complicated <READING ALOUD>  interstitial concentrations of amino acids in the rat erm er striatum during global forebrain ischemia and potassium-evoked er s-spreading depression </READING ALOUD> so all kinds of models could you please go through these erm </S3>
<S2> [so] </S2>
<S3> [systems] </S3>
<S2> the main idea of this manuscript er differs a little bit fro- from er the main topic of er PhD dissertation actually and the background for this manuscript was that er there are some studies er published or which were done on humans or isolate human tissues where they er people attempt to mhm describe the possible er damage to the brain tissue by analysing the changes in extracellular concentrations of amino acids however in these studies they focus only upon er some neurotransmittory amino acids like glutamate and GABA and we wanted to show here that actually glutamate and GABA are released quite easily from the nervous tissue and for example if in the case of a stroke the er level of er tissue damage er will not contribute much to the release or non-release of this amino acid and in this paper we compare er the release of quite wide range of amino acids in er case of ischemia like a quite serious (impact) and in case of spreading depression and this work was done o- er were done or was done using in vivo microdialysis however erm in case of ischemia we combined er model of er animal ischemia to the microdialysis approach and in case of spreading depression we evoked the the membrane depolarisation by application of high er concentration of potassium to the perfusion medium so in what case it's a combined model er global forebrain ischemia (plus) microdialysis and in another case it's just microdialysis and spreading ischemia was induced (xx) , and we were able to show a difference in the release of er certain amino acids namely er threonine er , serine and asparagine and er , we conclude that in general it will be beneficial for these clinical studies er to measure the wide range of amino acids like we did here because er we've got the same release in for aspartate , glutamate er the GABA in both cases in ischemia and in spreading depression however er these two conditions differ profoundly differ on the on the release on non-neurotransmittory amino acids so that was the main idea of this manuscript but the interesting part er of the manuscript important for this PhD study was er properties (xx) release of taurine under ischemia and spreading depression and we were able to show mhm two interesting features of taurine-evoked taurine release er taurine was released at er on a response to (xx) ischemia and spreading depression and the interesting thing is that the maximum release of taurine actually happens quite late compared to the release of glutamate the maximum release of glutamate was just after (with the) first sample during er ischemia for example or spreading depression and er the maximum release of taurine was little bit delayed and er the concentrations of taurine remain elevated for quite long time so they are two characteristic features of the evoked taurine release </S2>
<S3> okay thank you so the quality of the figures er is not so so good here in this paper so so they were ei- either printed very poorly by this journal or or the originals had something that you know some <S2> [(how so)] </S2> [some some] er something to be improved but er again we have to remember that these are now anesthetised animals again and anaesthesia is [is er is] </S3>
<S2> [(the same) er] </S2>
<S3> may may protect the brain and produce some other changes there so but still i don't know whether it it should now directly affect the the release </S3>
<S2> yes it shouldn't at the (time) i i know that er there are some experimental approaches where er people managed to er evoke ischemia in the absence of anaesthesia however i think that it's quite difficult studies and i think that it's enough to use (xx) anaesthesia for these types of research </S2>
<S3> so how do you know that er that that was ischemia </S3>
<S2> mhm , unfortunately we didn't (share) it we didn't measure the local blood flow in the brain in the nervous tissue er however the model of ischemia by itself is quite hard so we , er collect both er , (xx) and decreased the main blood pressure by half so it's it's not very hard to control by itself because there are still two vertebrate arteries which supply tauri- er supply blo- blood to the brain so it's not very hard erm model of ischemia compared to for example four-vessel occlusion er however it's quite obvious that i- if er you'll do everything correctly and then it will be ischemia @@ </S2>
<S3> so <S2> [but] </S2> [did] did you do this surgery for the for the ischemia </S3>
<S2> yes </S2>
<S3> okay so so many changes were happening here and er and actually really great changes for example in GABA and glutamate , and also taurine flows changed a lot and again this change was was prolonged quite long </S3>
<S2> yes </S2>
<S3> why was glutamate reduced so much and also also came back very slow </S3>
<S2> so it seems that in this case the glutamine er the extracellular glutamine concentrations were to reduced due to er its usage for the synthesis of glutamate so er glutamine was used by (xx) to (fulfil) the releasable flows of glutamate </S2>
<S3> yeah that's what i (xx) , okay <ORGANISING PAPERS, P:09> so it seems that i have very little questions here <P:09> yeah actually coming back to this er prolong- er glutamate er er prolonged taurine release you are most likely aware of the helmut haas group studies by by by <NAME> and others that they have studied this er this er er corticostriatal glutamate pathway and its effects er i- it's it's like a synaptic efficacy in the brain slices using (xx) physiology and they found that when they applied taurine and and er then the- they applied er stimulation to the cortex and then then measured responses from the from the from the striatal cells and they kept like er er very long-lasting enhancement of the responses after they applied this this taurine how is that possible because taurine is supposed to be under under under inhibitory system </S3>
<S2> they there's that recent publication of the same authors there they suggest the er mhm mechanism for this enhance- <S3> [okay] </S3> [enhancement] of the responses however i i don't have any explanation in mind they proposed quite er er sophisticated modulation by , dopamine (xx) impulse and er by GABA (xx) connections so they sub- suggested that mainly neurotransmittory systems are evoked in this phenomenon er but the the interesting thing is that er during the application of taurine they actually had the reduced response and they have the (xx) only after mhm a medium taurine buffer buffer so i know i- it's hard [for me to say that that's er there's a] </S2>
<S3> [so yeah so was] was that then an effect of intracellular taurine so that had an excitatory effect </S3>
<S2> they don't agreed on that they studied that and </S2>
<S3> so what do you think </S3>
<S2> it's hard for me to say it th- that's (release) (xx) studied to answer (that) the question i don't have any ideas and the interesting thing is that this phenomenon was studied on er corticostriatal pathway and in hypocampus and if i remember right er they show that er taurine transporters may take part in this <POOR SOUND QUALITY> process and another thing is that while you study the properties of release of taurine er the er guanidinoethanosulfonate is a quite good tool for studying that however it may h- again have a side effects so for example it was shown that it may act o- as a antagonist at the glycine receptors so very probably when they these groups studied the role of taurine transporters in this enhancement of taurine and they applied guanidinoethanesulfonate it has (some) side effects so , again w- we can't say about the (xx) of a taurine action just by using er inhibitors of taurine uptake er wh- when we study actually the effects of taurine when we we study the release of taurine it's little bit easier </S2>
<S3> but er didn't they also study this er this er knock-out mouse line </S3>
<S2> no they no [no] </S2>
<S3> [tau-] taurine-transported knock-out mouse line </S3>
<S2> no they didn't i er i- at least i didn't remember if they combine the knock-out mice and this approach </S2>
<S3> so we have to checked this out <S2> @@ </S2> so may- maybe they (xx) with that system </S3>
<S2> maybe </S2>
<S3> okay so so er their study together with this prolonged action which it might might might somehow somehow be that er this er at least part of the effects are certainly from the intracellular site , okay so we will go to the to the fourth paper which is now an in vitro study using these corticostriatal slices <READING ALOUD> taurine attenuates D aspartate release evoked by depolarisation in ischemic corticostriatal slices </READING ALOUD> <COUGH> so can you explain wha- what er what did you do here and er and why did you study this kind of thing </S3>
<S2> <COUGH> er it was proposed actually it has been proposed for erm some period of time that taurine may be neuroprotective in ischemia and the evoked taurine release under ischemia we have neuroprotective (xx) and taurine may act on some- something er promoting the cell survival after ischemia and exce- excitatory impacts and actually there are quite little studies aimed on er studying the effect of taurine on the glutamate release as a most one of the most important component of the erm , cell death pathway under ischemia so er under ischemia glutamate is released i- released in the response of the reduced blood supply namely oxygen and glucose supply and these high concentrations of glutamate affect the er glutamate receptors and that (xx) actually starts up all pathological reactions wh- which we (xx) during ischemia in er nervous tissue so we decide to study if taurine can erm inhibit er this ischemia-evoked taurine release as in first step in the response er of cells to the ischemia conditions and it it can be shown that indeed taurine was able to suppress the release of glutamate evoked evoked by oxygen-glucose deprivation and er chemical ischemia and after that er , here we tried to study little bit the mechanisms of er this effect and it happened so that er , er GABA and glycine receptors are d- don't play any role in that pro- neuroprotectory effects of taurine and again we we applied here (the) guanidinoethanesul- sulfonate to avoid if er the er site of taurine action is extracellular or intracellular so and in this case we can't say anything for sure because guanidi- dinoethanesulfonate actually er <COUGH> suppresses the release of glutamate by itself so it means that in a presence of guanidinoethanesulfonate er taurine still has the protectory effect but guanidinoethanesulfonate by itself has the same effect so we don't know for sure is that a side effect of guanidinoethanesulfonate or it's related (back) on a inhibition of er taurine uptake so it (xx) er extracellular concentrations of taurine in these (annulated) concentrations of taurine may affect somehow in a some extracellular site so this case we can conclude only that er this effect of taurine is not related to GABA and glycine receptors and after that we modelled pharmacologically different ways of glutamate release under ischemic conditions so first during the early first minutes of the ischemia taurine is released by synaptic exocytosis and after that after the full depletion of er a ATP pool inside the cell er it start to be released due due to the (reverse) section of glutamate transporters and er due to the release through volume-sensitive chloride channel because er under ischemia cells also swell and s- try to regulate the its volume by extorting (xx) subs- substances and it seems so they we modelled this (xx) pharmacologically and it seems so that taurine were was able to inhibit er glutamate release evoked by activation of sodium channels so it's more or less synaptic release but it doesn't have any effect on the particular er hypo-osmotic er (buffer) -mediated release or transporter-mediated release , so that my conclusion of this manuscript was that taurine is indeed able to reduce release of glutamate and it's more likely connected to its effect on the depolarisation <SU-3> (xx) </SU-3> the state of depolarisation of the membrane </S2>
<S3> okay and , so <COUGH> so this is now in vitro and why di- why did you er er select these corticostriatal slices </S3>
<S2> er first of all it's er like a continuation of the present of the former work so at fir- first we studied the properties of the release of taurine from the striatum and now we moved to some effects of taurine so it would be would keep the er target system the same </S2>
<S3> so is <S2> [and] </S2> [is] er is is er glutamate now released from the striatum or from the cortex </S3>
<S2> no it's from the cortex actually there there are no glutamatergic (xx) in terms of glutamatergic connections in the striatum so er </S2>
<S3> so but er but the- there are terminals </S3>
<S2> yeah but it's also good to have er neurons to have something to stimulate so we s- chose to work with the corticostriatal preparations to have this glutamatergic cor- corticostriatal pathway erm more or less intact </S2>
<S3> yeah it just makes this system more complex </S3>
<S2> yes that's right but , of course it's possible to evoke the release of glutamate from the striatum but i thought that m- maybe then we will lose some regulation mechanisms which happen in the (xx) </S2>
<S3> so would it have been technically possible to continue this study longer so you have like er this now 50 minute </S3>
<S2> yes [it's possible] </S2>
<S3> [influx study] it is possible </S3>
<S2> yes </S2>
<S3> and er and er after you have applied er er taurine you could have again taken it off and you can you could have followed what happens to the aspartate release </S3>
<S2> yes [that's right] </S2>
<S3> [and maybe you ha-] y- you would have been able to figure out what is going on in in these in these haas haas electrophysiological studies </S3>
<S2> yes maybe @@ </S2>
<S3> basically you should you shou- you should then see that er this application after this application of taurine when you remove it [er aspartate release is is going going up] </S3>
<S2> [maybe i didn't (xx) yes (xx) possible] yeah </S2>
<S3> er you didn't have any more tubes or or so so you you stopped it at 50 minutes or or how was it </S3>
<S2> er actually at that time er it was er interesting for me to get the it was quite curious and surprising and after that i tried to describe this effect and for these purposes 50 minutes is more than enough so i was not so much interested in the long-term effects </S2>
<S3> yeah so it seems that you have been loading loading this (xx) a lot so many many big experiments </S3>
<S2> yeah </S2>
<S3> what a work maybe somebody has helped you </S3>
<S2> yes of course er i i did a part of the experiments by myself but er er our laboratory technician <NAME> did the major part of the experiments </S2>
<S3> okay , so you already es- e- explained this er this er guanidinoethanesulfonate effect here and erm and then we can look a little bit at table two you have here many rate constants which you explained how you you measured these or calculated the ra- rate constants they have been on on page 17 figure six there is a good explanation how to how to calculate those rate constants so so in er in table two you have these rate constants for the basal release and evoked release and er and you have er when you have this er this glutamate er er transporter (xx) there er , you you get er you get like er increased rate constants and er in the during the evoked release and er so this is not this is not very easy for me to understand because then you have like in the in this in this er base line condition you you just had it with the with th- in- nothing added there this evoked rate constant is er 1.57 and er in the presence of this THBA you have like 1.44 and er and you say that it is it is er significantly different but still if you used for example if you would use for example analysis of variance most likely you would not see any any difference there </S3>
<S2> <S3> [(or)] </S3> [er] it's significantly different compared to er the constant of basal release so of course it doesn't differ much from other (groups) i (xx) that is different from these er (zero point fifty three) </S2>
<S3> excuse me maybe i'm i'm looking wrong at this table , so there is B if i see [it] </S3>
<S2> [yeah] it's B [and , yeah] </S2>
<S3> [okay so] </S3>
<S2> compared [to the] </S2>
<S3> [so it means] that er it is different from the er from the basal release in the [(xx)] </S3>
<S2> [yeah] yeah [from the basal release in the absence of (xx) stimulation] </S2>
<S3> [yeah yeah yeah okay okay] </S3>
<S2> er the reason for this actually they were additional experiment <S3> [yeah] </S3> [experiments] required by the reviewer so first of all er we use er the THBA which is a inhibitor of er taurine transporter to confirm that er we indeed are studying the release of glutamate er evo- or mediated by the reverse action of glutamate transporter and we have however when we apply the inhibitor of transporter we will have er an (an elevated) extracellular concentrations of the substance and in this case we have two choices that's more or less that's more methodological actually but not er scientific er first of all er you can apply er you can preload the inhibitor during the basal level and during this basal level er the release not the release but actually er the extracellular concentration of the substance in question will be quite high however after that you can remove er this inhibitor from the medium and you will still have this inhibitor inside the cell and after that you can apply the stimulation and see if this preloaded inhibitor affect the stimulation (volumes) and in this case like i describe if we will have this inhibitor only during the basal period we will be able to statistically compare er two experimental groups er during the period of stimulated release not the basal release but stimulated release , in and there is another opportunity if we will have this inhibitor during all the experimental time er then we will be able to statistically compare the level of basal release and level of stimulated release er within the same experimental group however we can't compare er values obtained from this experimental group to other experimental groups because er </S2>
<S3> and why not </S3>
<S2> because the inhibitor it's is still present and in this case it will affect the release , evo- er mediated by the (xx) of the transporter and it still will er maintain the high extracellular levels of a compound (xx) here and D-aspartate so it will interfere it will affect both if we would say that for example under depolarisation er or in some other things the direction of the transporter is not no- not agonised so it can work in both directions and if we will have er inhibitor from each side it will affect er both directions er both fluxes thus i- inside out flux and er i i understand this little bit different </S2>
<S3> @@ yeah this this this becomes very complicated and then when you @@ you calculate anyway these constant er these ratios , you know the K-2 per cent from er er per cent of K-1 and er and you calculate the thi- this for these two conditions and you come up with different percentages but but er you know anyway the rate constant is is more or less the same </S3>
<P:10>
<S2> so during the basal conditions without any the rate constant is zero point three when we had this inhibitor this constant is anyway (75) (xx) the inhibitor affects the uptake of D-aspartate so it's [like it's] </S2>
<S3> [so so] does this mean that that the superfusion is not fast enough to remove this er released er compound </S3>
<S2> it shouldn't be so but </S2>
<S3> so it is so slow that some of the released er er glutamate is taken up , you know i'm i'm only confused with this erm er heteroexchange and homoexchange and er how how you can really study it , it becomes very complicated and er and er probably you would need to to to draw some figures on the on the on the board and er and explain us how these er how these transporters are now supposed to be doing at that different phases , okay but er , but er i think that this is very difficult to interpret this er this this data in terms of er what kind of transporter systems glutamate is doing </S3>
<S2> yes unfortunately it's quite difficult however erm to my knowledge if you don't have knock-out mice it's only one way to isolate this (inverse) section of transporter so yes that's that's quite difficult that's why i <S3> [yeah] </S3> [prefer] to have the this transporter only during the basal conditions and after that <S3> [yeah] </S3> [to remove it] to get rid of this , (elevated) concentrations </S2>
<S3> so i have one more question on this paper , er it is er it is on the concentration of taurine <S2> yes </S2> what i- what is what is a relevant concentration of taurine that can be that can er you know relevant in terms of er of er neuroprote- protection in the brain </S3>
<S2> [yes] </S2>
<S3> [in] comparison with the with the concentrations that you used here </S3>
<P:06>
<S2> okay , we used here erm ten millimolar concentration of taurine </S2>
<S3> you actually also used five millimolar </S3>
<S2> yeah but it didn't have any , so ten millimolar (concentration) and we have shown that basal concentration of taurine is 25 micromoles and under ischemia this concentration may be elevated to about er like . 3,000 per cent so if we multiplied 25 (with) five three thousands we will have <CALCULATING, P:04> 75,000 micromoles so it means that it's s- <CALCULATING> 70 , five millimoles so it's very high e- or i am i wrong in the calculations </S2>
<S3> yeah maybe , maybe a bit , yeah anyway these concentrations are high but also ischemia is inducing er [like like er] </S3>
<S2> [yes yes] the the <S3> [right yeah] </S3> [idea is that basal] concentration of taurine are very high and ischemia-induced (xx) release of taurine so many times so it may reach quite high levels so maybe yes maybe ten millimolar concentration of taurine is quite high but it's also determined by the experimental (set-up) if we will apply taurine quite locally we would maybe require less concentrations of [taurine] </S2>
<S3> [yeah] so they may ma- match in terms of of er of these kind of studies they may match <S2> yeah </S2> so be above the reasonable levels , okay er then we go to the last paper which is here as a manuscript . <READING ALOUD> inhibitory effect of taurine on veratridine-evoked er D-aspartate release from mur- mu- murine er corticostriatal slices involvement of chloride channels and mitochondria </READING ALOUD> , so could you go through it again </S3>
<S2> <SIGH> so here we isolate er this effect of taurine on er depolarisation-induced glutamate release and our main aim was to study the mechanism how taurine may affect the release of glutamate again we applied , er first of all we star- we tried to characterise the release of glutamate w- what we are working with and er we applied different criteria the only calcium-free solution and <P:07> and er , er the inhibitor of taurine transporter and we were able to show that it seems that under the general depolarisation er so here in this case we induced the release of glutamate by activation of sodium channels and we were able to show that the activation of sodium channels may actually evoke the release of glutamate through both er synaptic er vesicular rel- release and by the (reverse) section of taurine transpor- of er glutamate transporter again so it means that er the veratridine-induced is not like purely synaptic release but it seems that er taurine may affect on the both types of this release and it's quite er interesting things thing and it from my point of view it may be explained only by the thing that taurine affect on some er general factor which will induce after that er the the release of glutamate by these two types and then we focused on the mechanisms and we were able to show that er when we (omit) the (chloride) ions from the medium er we have the effect of taurine to be reduced so it means that it's possible and some chloride channels er are involved in the effect of taurine we were able to show that GABA A and glycine receptors don't have anything to do with this taurine effect however when we applied the less specific inhibitor of both GABA and glycine receptors picrotoxin we were able to er er significantly decrease the effect of taurine so it seems that picrotoxin may prevent the effect of taurine and the second question er was if er the recollection of calcium er concentration by mitochondria has something with this er suppressive effect of taurine on the glutamate release and we actually inhibit the uptake on calcium to the mitochondria by application by inhibiting the electron transport chains in mitochondria in wi- in this case of course we get the (profound) release of er glutamate because we in these conditions we have the mito- the (xx) mitochondria however in this case we didn't have the effect of taurine at all in (case) for example of application of CCCP (xx) and the effect at least the effect was that (mitochondria) diminished so we conclude that the taurine may suppress the release of glutamate by affecting some unknown chloride er channel which may be (xx) some receptor and by affecting of er er regulation of calcium concentrations by mitochondria so both mechanisms may be involved in this phenomenon </S2>
<S3> so so you think that picrotoxin is affecting of blocking some other anion channels and er and that's the </S3>
<S2> er [yes] </S2>
<S3> [that's] the target of taurine </S3>
<S2> yes </S2>
<S3> so we come back to that again that er that there is a receptor for taurine that this anio- un- an- ion anion channel and er can be blocked by picrotoxin but it is not GABA A receptor and not not er strychnine-sensitive er </S3>
<S2> yes that's right </S2>
<S3> receptor yeah yeah erm that of course may be may be er may be right but er in pharmacology we would have liked to have like like more concentrations in terms of er of these effects er to see clearly that there is no effect with with some or some other positive control that er these concentrations of bicuculline and and strychnine would have er blocked blocked these receptors </S3>
<S2> yes that's right </S2>
<S3> so picrotoxin in the is is acting on on different site that bicuculline for example </S3>
<S2> yes <S3> [so] </S3> [bicuculline] is er competitive er antagonist so it means that it binds to the s- same or about the same site er as GABA for example and picrotoxin binds to the pore walls of the receptor </S2>
<S3> so is is taurine now binding to the to the to the [pore or i-] </S3>
<S2> [yeah yeah it it's su-] it's suggested that in case if taurine will affect the GABA receptor it will bind on the same site as GABA </S2>
<S3> okay good so this was accepted also </S3>
<S2> yes </S2>
<S3> so i didn't find any any major major mistakes here so so it will be probably printed printed rather soon okay so er er people have used also some taurine antagonists i remember some compounds i don't eve- even now remember the names of these compounds but there has been some some compounds er that have been [(analysed)] </S3>
<S2> [yes] yes [that's right] </S2>
<S3> [taurine antagonists] </S3>
<S2> yes [that's right] </S2>
<S3> [did you did you] think about using those </S3>
<S2> yes , unfortunately , yeah the- there is at least one substance which is quite er widely characterised the substance with the very low (label) but the abbreviation is T-H-E and , er however it's not produced er it's not in the market so we are not able to find it and er that's why it's quite difficult to plan any experiments with this substance </S2>
<S3> yes that's that's how it often is so this remains a little bit er like a mystery how the- how these effects are produced , or or is it is it is it here </S3>
<S2> it's not here unfortunately i , maybe i speculate but probably if picrotoxin was able to prevent the effect of taurine maybe there is some er receptor which er is very similar to that of GABA and glycine receptors so maybe the structure is more or less the same so maybe it's it belongs to the same sub-family of er proteins and it's <S3> [so] </S3> [quite] possible that er it's more or less specific to taurine [however] </S2>
<S3> [so so] do y- do you really think that there is er there is er some gene or still to be found that would be so homologous to GABA A and and glycine receptors that people would ha- not have seen it </S3>
<S2> maybe no-one has tried to do that , there are not so many research on taurine that [nowadays] </S2>
<S3> [no no] i mean that the people who are screening thr- through the through the er DNA libraries and er and er sequences </S3>
<S2> i don't [know] </S2>
<S3> [so] so they they they they have er gone through and they they claim that there are no more GABA A receptor supplements for example <S2> mhm-hm </S2> no everything is found <S2> mhm-hm </S2> and er so so it's not so clear that whether that would be anything it must be something different , and of course that's that's a possibility okay going back to to er the page 27 , er i'm just er ma- ma- (you know) i think that we we went through these er effects on er on these er ischemia stuff and and er you have this table here table two on page 27 you have this taurine uptake inhibitor effects and and then these er these er er chloride channel inhibitors are these are these arrows in the r- i- in the in the right direction </S3>
<S2> i hope so @@ </S2>
<S3> so so did you get er reduce the taurine release by this er by this uptake blocker or increased <P:08> and i- did these er these er er chloride channel blockers now increase the release or decrease the release </S3>
<S2> yeah it seems er yes er i'm very sorry it seems that they mis- they , did a misprint here </S2>
<S3> yeah </S3>
<S2> [because obviously] </S2>
<S3> [you know] when i was reading it i i got here very mixed up so i i thought that i understood something and and then then they were all all upside down because these tables are really great because they summarise all the all the data and er and er and that usually makes these er these thesis er summaries very good , okay so er we have not we have not discussed yet er er another possibility to change er neurotransmittory release and it is er presynaptic receptors <S2> [yeah] </S2> [so] would would taurine has have any effects of the presynaptic er receptors that er that regulate er calcium release </S3>
<P:05>
<S2> i don't know </S2>
<S3> so how does this er this er er compound that is in the clinics in the treatment of alcoholism acamprosate work , so it's calcium acetyl homotau- taurine this compound </S3>
<S2> mhm </S2>
<S3> it is not pure taurine </S3>
<S2> oh i i personally was not so much interested in that [so i didn't (xx)] </S2>
<S3> [yeah so that] that compound was first supposed to be GABA receptor agonist then then er er glutamate receptor antagonist and now it seems to be that it is it is an antagonist of er of one type of these metabotropic glutamate receptors </S3>
<S2> [a-ha] </S2>
<S3> [then] then <S2> [yeah] </S2> [you know] nobody really knows because it has low (xx) so the alcoholics they have to take a lot of that compound and then they have diarrhoea and all kinds of side effects and er some of then actually benefit of that compound so i'm not sure whether it has anything to do with the effects of taurine but er but it might er er maybe maybe you could have inclu- included these presynaptic receptors here s- here as as one of the target sites to modulate the glutamate release , okay i think we have er we have gone through now the the detailed examination and er and if i'm right if we stop now here i may i may , i may say some words what i'm going to write in the in the statement <ARISES FROM THE CHAIR, P:08> <COUGH> so i'm not going to er to read all this and this is not final yet so i got i got revise this a little bit so to the faculty of medicine er opponent's statement on the PhD dissertation of er <NAME S2> on the topic release and neuromodulatory effects of taurine in the rodent er striatum er <NAME S2> has continued the tradition and series of studies on taurine in tampere in the supervision of er professor <NAME> and and er emeritus professor <NAME> er this er thesis er has potential scientific and practical significance er because taurine modulation is important somehow it must be important for the brain function but we don't really know how to how to get it working maybe those energy drinks er maybe they are not the final word yet so the basic research area areas are neurochemistry neuropharmacology and neurobiology and er especially when we go to neuro-degeneration and and ischemia and er and er these kind of studies so we are we are then among the highest possible competition internationally so these are very very much studied studied areas so in that sense er this area is is er is er up-to-date and and very important so then i describe here will describe er here little bit er about the aims and er and er and also how the thesis was put together of five publications and and erm and also i mention the contribution of er of the disputant er that was clear- clearly a decisive role in the in the production of the data and and er planning and er and also also er er also like er like er summarising the results , so the experimental methods have been er quite demanding of course one could have made them even more demanding but er but somewhere is the limit and er and er er the disputant has mastered these methods very well so then i will describe here some of the of the main findings er but i will not repeat those now here so during this public examination the disputant has er has described the work in such a detail that it's it's very clear you understand all the theories and and interpretations behind these er these er these studies the usage of english language is very good and also also spoken and written so i'm not sure how has how has finally written this this thesis but er anyway during this er this discussion everything is everything was more or less clear and er the disputant er defended her r- er her results and interpretations and conclus- conclusions er with actually very great enthusiasm on the work which is always th- l- like the driving force in getting these kind of studies done without that it's almost impossible , er and based on on the assessment of the written thesis and and this dissertation er er er public er dissertation er i'm happy to recommend er this er thesis er the acceptance of this thesis er for the doctoral degree </S3>
<P:06>
<S2> i thank you professor <NAME S3> for the comments of high (value) which i (xx) concerning my dissertation er i now respectfully ask those who wish to comment my dissertation to request the <FOREIGN> kustos </FOREIGN> to for permission to speak </S2>
