From Matthew.Kirkcaldie from removeutas.removeedu.au Sat Nov 1 17:42:43 2008 From: Matthew.Kirkcaldie from removeutas.removeedu.au (Matthew Kirkcaldie) Date: Sat Nov 1 19:05:15 2008 Subject: [Neuroscience] Re: Rat (Long Evans) Cerebellar Cortex Size References: <7357694e-1405-4c68-b956-f16d61f58c0b@f37g2000pri.googlegroups.com> Message-ID: In article , Kerry wrote: > Sorry, I should have been more explicit w/ the given purpose. I am not > trying to use this information for any quantitative analysis. For now > I am just trying to get an estimation of width (e.g. Euclidean > distance from the most extreme sagittal points end to end). However, > if I new the volume of the cerebellar cortex I could calculate the > convex hull volume of the section I have to get a more exact > proportion. I don't know if a convex hull is really the right way to approach this in terms of proportion - the cerebellum is highly involuted, and its function is quite clearly arranged in terms of the sheet (parallel fibres running in one direction and the dendrite trees of Purkinje cells orthogonal to those fibres, contacting tens of thousands of them). Really the only meaningful proportion of the cerebellum is the proportion of the cortical sheet area, or the proportion of total Purkinje cell numbers. Your parasagittal sections are good, however, because of course the folding and involution is mostly at right angles to the sagittal plane. A good first-draft approximation of sheet proportion would be to look at an unfolded surface and draw lines parallel to the midline. I don't think the convex hull is going to help because the depth of infolding varies considerably. > I would guess the total volume is more readily available, > but if I could find a picture w/ a scale bar of the cerebellum viewed > from the rostral or caudal direction I could get an idea of how much > section I have. The latter may be quantitatively meaningless but > qualitatively, for those who have knowledge of the cerebellar cortical > size and shape, one could infer what proportion of the cortex I am > studying. A crude approximation is good enough as I am just trying to > provide a cartoon that gives a rough idea (e.g. it is meaningful to > say whether I have closer to 1% or 90% of the entire cerebellar > cortex). If you want a dimensionally accurate view of the cerebellum in three planes, I'd recommend you to the atlas by Paxinos and Watson, The Rat Brain in Stereotaxic Co-ordinates, 6th edition 2007. It's the standard reference work on the subject. If you're not sure how far from the midline your sections were taken, the atlas has a parasagittal series at known distances from the midline which may allow you to match up your sections. It also had a series sectioned in the horizontal plane which should show you the full lateral extent of the cerebellum at most levels. Another major variable to consider is tissue processing and the resultant shrinkage. A fixed brain can shrink by up to 15%, and the degree of shrinkage varies in white matter and grey matter, making things even more complicated! Sorry for such a fastidious answer, but with a complex three-dimensional structure like the cerebellum, back-of-the-envelope approximations are often startlingly wrong. Cheers, Matthew. ** Posted from http://www.teranews.com ** From kbrownk from gmail.com Sun Nov 2 20:33:05 2008 From: kbrownk from gmail.com (Kerry) Date: Sun Nov 2 21:03:45 2008 Subject: [Neuroscience] Re: Rat (Long Evans) Cerebellar Cortex Size References: <7357694e-1405-4c68-b956-f16d61f58c0b@f37g2000pri.googlegroups.com> Message-ID: <55faaf26-27c9-44d6-9161-44ebc798df02@u18g2000pro.googlegroups.com> On Nov 1, 5:42 pm, Matthew Kirkcaldie wrote: > In article > , > > Kerry wrote: > > Sorry, I should have been more explicit w/ the given purpose. I am not > > trying to use this information for any quantitative analysis. For now > > I am just trying to get an estimation of width (e.g. Euclidean > > distance from the most extreme sagittal points end to end). However, > > if I new the volume of the cerebellar cortex I could calculate the > > convex hull volume of the section I have to get a more exact > > proportion. > > I don't know if a convex hull is really the right way to approach this > in terms of proportion - the cerebellum is highly involuted, and its > function is quite clearly arranged in terms of the sheet (parallel > fibres running in one direction and the dendrite trees of Purkinje cells > orthogonal to those fibres, contacting tens of thousands of them). > Really the only meaningful proportion of the cerebellum is the > proportion of the cortical sheet area, or the proportion of total > Purkinje cell numbers. > > Your parasagittal sections are good, however, because of course the > folding and involution is mostly at right angles to the sagittal plane. > A good first-draft approximation of sheet proportion would be to look at > an unfolded surface and draw lines parallel to the midline. I don't > think the convex hull is going to help because the depth of infolding > varies considerably. > > > I would guess the total volume is more readily available, > > but if I could find a picture w/ a scale bar of the cerebellum viewed > > from the rostral or caudal direction I could get an idea of how much > > section I have. The latter may be quantitatively meaningless but > > qualitatively, for those who have knowledge of the cerebellar cortical > > size and shape, one could infer what proportion of the cortex I am > > studying. A crude approximation is good enough as I am just trying to > > provide a cartoon that gives a rough idea (e.g. it is meaningful to > > say whether I have closer to 1% or 90% of the entire cerebellar > > cortex). > > If you want a dimensionally accurate view of the cerebellum in three > planes, I'd recommend you to the atlas by Paxinos and Watson, The Rat > Brain in Stereotaxic Co-ordinates, 6th edition 2007. It's the standard > reference work on the subject. > > If you're not sure how far from the midline your sections were taken, > the atlas has a parasagittal series at known distances from the midline > which may allow you to match up your sections. It also had a series > sectioned in the horizontal plane which should show you the full lateral > extent of the cerebellum at most levels. > > Another major variable to consider is tissue processing and the > resultant shrinkage. A fixed brain can shrink by up to 15%, and the > degree of shrinkage varies in white matter and grey matter, making > things even more complicated! > > Sorry for such a fastidious answer, but with a complex three-dimensional > structure like the cerebellum, back-of-the-envelope approximations are > often startlingly wrong. > > Cheers, > > Matthew. > ** Posted fromhttp://www.teranews.com** Thanks for the advice. I'll have to take a look around for an appropriate atlas or pick up the one you mentioned. Thanks again, K From simak.alexandr from gmail.com Mon Nov 17 03:11:23 2008 From: simak.alexandr from gmail.com (BadCript) Date: Mon Nov 17 11:52:57 2008 Subject: [Neuroscience] PhD in Brain Science, Institute of Statistical Science, Academia Sinica (Taipei) Message-ID: <2f1862a4-cf1d-4580-8966-ae2e42b2ffe1@f37g2000pri.googlegroups.com> Research team at the Institute of Statistical Science, Academia Sinica seeks applicants for graduate research assistantship. Research opportunities are in several different areas of brain science. Research will involve EEG/MEG data processing and source localization, structure images, functional MRI and 4D PET image analyses. Ideal candidates will need a strong background in computer science, statistics or numerical methods and be really interested in working with and studying in brain science. Applicants must have a MS degree in computer sciences, applied mathematics or related areas. Research assistantships include a monthly stipend, health benefits, scientific supervisor and tuition remission. Please contact Dr. Michelle Liou at michelleliou@gmail.com. From rgiuly.group from yahoo.com Tue Nov 18 09:47:22 2008 From: rgiuly.group from yahoo.com (Rick Giuly) Date: Tue Nov 18 11:40:59 2008 Subject: [Neuroscience] Ion channel distributions Message-ID: <44e366f0-a97b-492f-a9fc-1f29a0636d1b@x16g2000prn.googlegroups.com> Hello All, I'm interested in modeling neurons and I have a few questions: (1) In adult animals, are ion channel distributions constant? (2) Do particular cell types have well defined ion channel distributions that are similar among all cells of the type? (For example does a Purkinje cell have a predictable ion channel distribution?) (3) Are the answers to questions (1) and (2) different for vertebrates and invertebrates? Any comments or paper recommendations are appreciated. I've done some literature searching and found some fragments of information on these topics but nothing definitive yet. -Rick From gmsizemore2 from yahoo.com Tue Nov 18 18:14:17 2008 From: gmsizemore2 from yahoo.com (Glen M. Sizemore) Date: Tue Nov 18 23:09:24 2008 Subject: [Neuroscience] SSRIs bind to post-synaptic 5-HT receptors and function as competitive antagonists? Message-ID: <49234c1f$0$26361$ed362ca5@nr5.newsreader.com> Someone I know claims that SSRIs bind at post-synaptic serotonin receptors, and function as competitive antagonists. Anyone know anything about this? Thanks ahead of time... G. From gmsizemore2 from yahoo.com Wed Nov 19 05:26:55 2008 From: gmsizemore2 from yahoo.com (Glen M. Sizemore) Date: Wed Nov 19 12:59:28 2008 Subject: [Neuroscience] Re: SSRIs bind to post-synaptic 5-HT receptors and function as competitive antagonists? In-Reply-To: <49234c1f$0$26361$ed362ca5@nr5.newsreader.com> References: <49234c1f$0$26361$ed362ca5@nr5.newsreader.com> Message-ID: <4923e9c8$0$26332$ed362ca5@nr5.newsreader.com> "Glen M. Sizemore" wrote in message news:49234c1f$0$26361$ed362ca5@nr5.newsreader.com... > Someone I know claims that SSRIs bind at post-synaptic serotonin > receptors, and function as competitive antagonists. Anyone know anything > about this? Thanks ahead of time... > > G. Correction: SOME SSRIs From r_s_norman from _comcast.net Wed Nov 19 08:01:14 2008 From: r_s_norman from _comcast.net (r norman) Date: Wed Nov 19 12:59:35 2008 Subject: [Neuroscience] Re: SSRIs bind to post-synaptic 5-HT receptors and function as competitive antagonists? References: <49234c1f$0$26361$ed362ca5@nr5.newsreader.com> <4923e9c8$0$26332$ed362ca5@nr5.newsreader.com> Message-ID: <1f38i4ptqiifib4vmg6d6elrrm30t0t6s0@4ax.com> On Wed, 19 Nov 2008 05:26:55 -0500, "Glen M. Sizemore" wrote: > >"Glen M. Sizemore" wrote in message >news:49234c1f$0$26361$ed362ca5@nr5.newsreader.com... >> Someone I know claims that SSRIs bind at post-synaptic serotonin >> receptors, and function as competitive antagonists. Anyone know anything >> about this? Thanks ahead of time... >> >> G. > >Correction: SOME SSRIs Could (s)he be thinking of the action of pindolol which is sometimes used in conjunction with SSRIs? From gmsizemore2 from yahoo.com Wed Nov 19 19:21:49 2008 From: gmsizemore2 from yahoo.com (Glen M. Sizemore) Date: Thu Nov 20 13:07:54 2008 Subject: [Neuroscience] Re: SSRIs bind to post-synaptic 5-HT receptors and function as competitive antagonists? In-Reply-To: <1f38i4ptqiifib4vmg6d6elrrm30t0t6s0@4ax.com> References: <49234c1f$0$26361$ed362ca5@nr5.newsreader.com> <4923e9c8$0$26332$ed362ca5@nr5.newsreader.com> <1f38i4ptqiifib4vmg6d6elrrm30t0t6s0@4ax.com> Message-ID: <4924ad7c$0$28062$ed362ca5@nr5.newsreader.com> "r norman" wrote in message news:1f38i4ptqiifib4vmg6d6elrrm30t0t6s0@4ax.com... > On Wed, 19 Nov 2008 05:26:55 -0500, "Glen M. Sizemore" > wrote: > >> >>"Glen M. Sizemore" wrote in message >>news:49234c1f$0$26361$ed362ca5@nr5.newsreader.com... >>> Someone I know claims that SSRIs bind at post-synaptic serotonin >>> receptors, and function as competitive antagonists. Anyone know anything >>> about this? Thanks ahead of time... >>> >>> G. >> >>Correction: SOME SSRIs > > Could (s)he be thinking of the action of pindolol which is sometimes > used in conjunction with SSRIs? Hi Dr. Norman, I can now answer my own question, but I'll post the provisional answer for those that might be interested, and I'll provide a reference for those that are interested. Some SSRIs do, indeed, bind at the 5-HT2c receptor, and function as competitive antagonists. Cordially, Glen From nin from neurohost.org Thu Nov 27 11:11:22 2008 From: nin from neurohost.org (Jose Guzman) Date: Thu Nov 27 13:51:34 2008 Subject: [Neuroscience] Cuttting solutions for electrophysiology Message-ID: Hi everybody, I am currently working in hippocampal brain slices to do patch-clamp electrophysiology. Due to some experimental issues, I will have to record to neurons in old animals (P21-30). The problem is that the neurons I need are very susceptible to hypoxia , so the preparation requires some special cutting solutions. I collected the 3 major types of cutting solutions, and I would really like to know if any of you tried these in his/her experiments . These solutions have some minor variations in other publications (may contain additionally APV or kyneuric acid to prevent glutamate excitotoxicity), but basically are described as follows: 1.- Sucrose based solution: total or partial replacement of NaCl by Sucrose. (Aghajanian and Rasmussen, 1989, Moyer and Brown,1998). 2.- Glycerol based solution: the same as the previous but with Glycerol replacement. (Ye et al., 2006). 3.- KGluconate based solution: without Ca2+ or Na+: specially indicated for patching dendrites (Dugu? et al., 2005). A major problem I found is that these papers are not very much cited in other publications (for example, Ye et al., 2006 has only 6 citations). Personally, I found that Sucrose-based solutions made an improvement (although not very big) but I would really like to hear from your experiences. Thank you very much for your feedback! From connelly.bill from gmail.com Sun Nov 30 02:44:20 2008 From: connelly.bill from gmail.com (Bill) Date: Sun Nov 30 13:35:36 2008 Subject: [Neuroscience] Re: Cuttting solutions for electrophysiology References: Message-ID: <5a7ed4e7-77ab-4e29-bea9-b6c7d4d19152@k36g2000pri.googlegroups.com> Hi Jose, Just off the bat, lets remember that 21-30 days in not 'old'. Indeed, it is barely mature. Old is 18 month old rats. Secondly, for 21-30 do not 'need' specialized cutting solution. The biggest change you see between slices from neonatal and ~30 day old brain slices is nothing to do there being more dead cells, it is simply that it is harder to see the neurons (though there are some areas of exception, e.g. highly mylinated areas). The fact that the sucrose based solution didn't make a big difference informs you that depolarization toxicity is not your problem. It would have been nice to know where your slices are from, so I could be certain, but assuming your working from some kind of cortical region: Sucrose is good. You might as well do it all the time >P14. AP5 is a waste of time, acute depolarization induced neurotoxicity is NMDA receptor independent. Incubate your solution at 35 degrees for half an hour after slicing, esp good for small cells. If your neurons are looking a bit desicated/crinkly/hard try 1mM Pyruvate and 3mM ascorbate in your incubating/holding aCSF What brand of slicer are you using? As your brain gets older, this gets far more important. You can cut nice slices from 12 day old brain with your hand and a razor blade, those neuros don't care. Make sure you're vibrotome has been serviced some time in the not too distant past. I've seen old Camden slicers that bounced around on the table because they needed salt crust chipped out of the motor and some lubrication. But I stress this. If you're making slices from 21 day old animals, and your neurons look unhealthy (vs just harder to see) you are doing something wrong, indepedent of cutting solution. I worked on 8 week old mice for 2 years, and you can't get the neurons looking as round and full as 2 week old slices, and you can't get the neurons as visible (talk to someone who works on young cat thalamus if you want to hear about invisible neurons). But from a 21 day old animal, the neurons should still be like lovely round grapes, just harder to see than in a 14 day old. And if you're working with pyramidal cells, remember to keep your slice plane in the plane of the dendrites, those guys really hate having their dendrites cut. Also, if you're working from an ultra mylinated structure, like the brain stem, it is vaguely impossible to get reasonable slices from 99% of that area in animals over ~21 days old, give or take a couple days. I hope you haven't found this too didactic, but this is my experience (I'll probably get told this is completely wrong by someone else). Oh finally, you can try intracardiac perfussion of your neuroprotective cutting solution. Its a pain, and its fiddley, I don't think it helps with the neuroprotection, but I think it helps makes the neurons in the slices more visible. On Nov 28, 5:11?am, Jose Guzman wrote: > Hi everybody, > > I am currently working in hippocampal brain slices to do patch-clamp > electrophysiology. Due to some experimental issues, I will have to > record to neurons in old animals (P21-30). The problem is that the > neurons I need are very susceptible to hypoxia , so the preparation > requires some special cutting solutions. > > I collected the 3 major types of cutting solutions, and I would really > like to know if any of you tried these in his/her experiments . These > solutions have some minor variations in other publications (may contain > additionally APV or kyneuric acid to prevent glutamate excitotoxicity), > but basically are described as follows: > > ?1.- Sucrose based solution: total or partial replacement of NaCl > by Sucrose. (Aghajanian and Rasmussen, 1989, Moyer and Brown,1998). > > ?2.- Glycerol based solution: the same as the previous but with > ?Glycerol replacement. (Ye et al., 2006). > > ?3.- KGluconate based solution: without Ca2+ or Na+: > ?specially indicated for patching dendrites (Dugu? et al., 2005). > > ? ? ? ? ? ? A major problem I found is that these papers are not very > ? ? ? ? ? ? much cited in other publications (for example, Ye et al., > ? ? ? ? ? ? 2006 has only 6 citations). Personally, I found that > ? ? ? ? ? ? Sucrose-based solutions made an improvement (although not > ? ? ? ? ? ? very big) but I would really like to hear from your > ? ? ? ? ? ? experiences. > > ? ? ? ? ? ? Thank you very much for your feedback! From connelly.bill from gmail.com Sun Nov 30 03:01:06 2008 From: connelly.bill from gmail.com (Bill) Date: Sun Nov 30 13:35:43 2008 Subject: [Neuroscience] Re: Ion channel distributions References: <44e366f0-a97b-492f-a9fc-1f29a0636d1b@x16g2000prn.googlegroups.com> Message-ID: <6cfa4a20-bdd0-440a-add6-01309defa334@r15g2000prh.googlegroups.com> Hi Rick The short version is 1) No 2) Generally 3) Not especially More complete version 1) Classical examples of varying ion channel distribution included the high punctate density of voltage gated sodium channels at nodes of ranvier along the axon, and high density calcium channels at presynaptic terminals. Other exmaples are in the 1998/1999 papers by Jeff Magee in J neurosci and Nature Neurosci showing the varying density of Ih channels along the dendrites of CA1 cells to achieve synaptic scaling of of EPSPs along the dendrite. There are all kinds of varying concentration of ion channels along dendrites, and dendrites vs axon. And of course a lot of the dendritic ion channels are plastic. Again see papers my Jeff Magee, looking at the modulation of A-type K channels 2) Generally speaking, absolutely. That's why Purkinje cells usually tonically fire. Why Basket cells have a fast spiking phenotype and why layer V dendrites have funny looking action potentials. Just like all humans generally look like humans, but don't look identically. Neurons that fall into one call generally behave very similarly. (Though don't forget the effect passive membrane properties lend to neurons, i.e. big cells are capacitive and have a slow response to membrane currents) 3) Not especially Have a wee look at Curr Opin Neurobiol. 1999 Jun;9(3):288-92 and Nat Rev Neurosci. 2000 Dec;1(3):181-90. On Nov 19, 3:47?am, Rick Giuly wrote: > Hello All, > > I'm interested in modeling neurons and I have a few questions: > > (1) In adult animals, are ion channel distributions constant? > > (2) Do particular cell types have well defined ion channel > distributions that are similar among all cells of the type? (For > example does a Purkinje cell have a predictable ion channel > distribution?) > > (3) Are the answers to questions (1) and (2) different for vertebrates > and invertebrates? > > Any comments or paper recommendations are appreciated. I've done some > literature searching and found some fragments of information on these > topics but nothing definitive yet. > > -Rick From connelly.bill from gmail.com Sun Nov 30 03:03:10 2008 From: connelly.bill from gmail.com (Bill) Date: Sun Nov 30 13:35:48 2008 Subject: [Neuroscience] Who first thought action potentials cause neurotransmitter release? Message-ID: <6110ffd4-746a-4372-af9e-c782cd781d95@e1g2000pra.googlegroups.com> Was it Eccles who first showed that an action potential invades a presynaptic terminal, causes Ca2+ influx and neurotransmitter release? Anyone know the paper? Thanks. From nin from neurohost.org Sun Nov 30 06:26:09 2008 From: nin from neurohost.org (Jose Guzman) Date: Sun Nov 30 13:35:57 2008 Subject: [Neuroscience] Re: Who first thought action potentials cause neurotransmitter release? References: <6110ffd4-746a-4372-af9e-c782cd781d95@e1g2000pra.googlegroups.com> Message-ID: If I recall it propertly, Eccles et al., were more concerned to the study of synaptic integration (temporal and sumation of EPSPs), whereas Katz and collegues were more interested in the basic mechanisms of synaptic transmission. Indeed the calcium dependent neurotransmitter release is atributed to Katz, Miledi and del Castillo. Eccles and colleagues tended to think in terms of electrical synapses, while Katz and his colleagues believed in a more chemical-type synapse (as they later demonstrated). The key paper is Katz B, 1969 (where the quantal nature of the transmitter release is described). This papers also describes "the calcium hipothesis" of synaptic release. I would recommend you to take a look to the small article written by Stuart Cull-Candy and Donald Jenkinson in Nature neuroscience on Kartz death. The article is called "Bernard-Katz 1911-2003" and was published on July 2003 in Nat neurosci (vol 6 number 7). I think the article is free, but just send me an e-mail if you do not have access to the magazine, and I will send you a copy. On 2008-11-30, Bill wrote: > Was it Eccles who first showed that an action potential invades a > presynaptic terminal, causes Ca2+ influx and neurotransmitter release? > Anyone know the paper? > > Thanks. From nin from neurohost.org Sun Nov 30 06:45:18 2008 From: nin from neurohost.org (Jose Guzman) Date: Sun Nov 30 13:36:02 2008 Subject: [Neuroscience] Re: Who first thought action potentials cause neurotransmitter release? References: <6110ffd4-746a-4372-af9e-c782cd781d95@e1g2000pra.googlegroups.com> Message-ID: I have just found one paper which you may find interesting: 1.- Jeng JM (2002) Richard Miledi and the calcium hypothesis of neurotransmitter release. Nat neurosci, 3, 71-76 This is a very interesting paper which describes historically the advances in the chemical synapse and the calcium requierement to the neurotransmitter release. I hope you find it useful, regards! Jose. On 2008-11-30, Jose Guzman wrote: > If I recall it propertly, Eccles et al., were more concerned to the > study of synaptic integration (temporal and sumation of EPSPs), whereas > Katz and collegues were more interested in the basic mechanisms of synaptic > transmission. Indeed the calcium dependent neurotransmitter release is > atributed to Katz, Miledi and del Castillo. Eccles and colleagues tended > to think in terms of electrical synapses, while Katz and his colleagues > believed in a more chemical-type synapse (as they later demonstrated). > > The key paper is Katz B, 1969 (where the quantal nature of the > transmitter release is described). This papers also describes "the calcium > hipothesis" of synaptic release. > > I would recommend you to take a look to the small article written by Stuart > Cull-Candy and Donald Jenkinson in Nature neuroscience on Kartz death. > The article is called "Bernard-Katz 1911-2003" and was published on > July 2003 in Nat neurosci (vol 6 number 7). > > I think the article is free, but just send me an e-mail if you do not > have access to the magazine, and I will send you a copy. > > > On 2008-11-30, Bill wrote: >> Was it Eccles who first showed that an action potential invades a >> presynaptic terminal, causes Ca2+ influx and neurotransmitter release? >> Anyone know the paper? >> >> Thanks. From nin from neurohost.org Sun Nov 30 10:32:50 2008 From: nin from neurohost.org (Jose Guzman) Date: Sun Nov 30 13:36:06 2008 Subject: [Neuroscience] Re: Cuttting solutions for electrophysiology References: <5a7ed4e7-77ab-4e29-bea9-b6c7d4d19152@k36g2000pri.googlegroups.com> Message-ID: Well, I agree with you, P21-30 are juvenile animals. I want to get recordings of CA3 pyramidal neurons of the hippocampus. So far, I have not much problems untill P18. However, due to the nature of my experiments, I need to study older animals, and to have relative big number of cells alive (only one or two cells per slice is not enough). Due to the extremely high interconnectivity of those neurons, and the high NMDAR expression I was thinking to use APV to avoid neurotoxicity due to Glu, but if you say it's a waste of time I will think first about other solutions. I am carefully following Bischofberger et al, 2006. My cutting angle seems to be OK, because after filling the cells with biocytine I can clearly see both axon and dendrites (in animals < P18). My vibratome is a Leica VT1200, so the state-of-the art. And YES, my cells look very crinkly (I was looking for that word!) I use a cutting solution in which NaCl is only partly replaced by sucrosse (both for storage and cutting). No Glu-antagonists or anti-oxidant agents like pyruvate or ascorbic acid. I will try soon the Glycerol-based solution, and additionally the solution containing pyruvate and ascorbic acid (with my normal sucrosse solution), and I will let you know how it was. Thank you very much in advance for your wisdom advices! Jose. On 2008-11-30, Bill wrote: > Hi Jose, > > Just off the bat, lets remember that 21-30 days in not 'old'. Indeed, > it is barely mature. Old is 18 month old rats. > > Secondly, for 21-30 do not 'need' specialized cutting solution. The > biggest change you see between slices from neonatal and ~30 day old > brain slices is nothing to do there being more dead cells, it is > simply that it is harder to see the neurons (though there are some > areas of exception, e.g. highly mylinated areas). > > The fact that the sucrose based solution didn't make a big difference > informs you that depolarization toxicity is not your problem. > > It would have been nice to know where your slices are from, so I could > be certain, but assuming your working from some kind of cortical > region: > Sucrose is good. You might as well do it all the time >P14. AP5 is a > waste of time, acute depolarization induced neurotoxicity is NMDA > receptor independent. > Incubate your solution at 35 degrees for half an hour after slicing, > esp good for small cells. > If your neurons are looking a bit desicated/crinkly/hard try 1mM > Pyruvate and 3mM ascorbate in your incubating/holding aCSF > What brand of slicer are you using? As your brain gets older, this > gets far more important. You can cut nice slices from 12 day old brain > with your hand and a razor blade, those neuros don't care. Make sure > you're vibrotome has been serviced some time in the not too distant > past. I've seen old Camden slicers that bounced around on the table > because they needed salt crust chipped out of the motor and some > lubrication. > > But I stress this. If you're making slices from 21 day old animals, > and your neurons look unhealthy (vs just harder to see) you are doing > something wrong, indepedent of cutting solution. I worked on 8 week > old mice for 2 years, and you can't get the neurons looking as round > and full as 2 week old slices, and you can't get the neurons as > visible (talk to someone who works on young cat thalamus if you want > to hear about invisible neurons). But from a 21 day old animal, the > neurons should still be like lovely round grapes, just harder to see > than in a 14 day old. And if you're working with pyramidal cells, > remember to keep your slice plane in the plane of the dendrites, those > guys really hate having their dendrites cut. > > Also, if you're working from an ultra mylinated structure, like the > brain stem, it is vaguely impossible to get reasonable slices from 99% > of that area in animals over ~21 days old, give or take a couple days. > > I hope you haven't found this too didactic, but this is my experience > (I'll probably get told this is completely wrong by someone else). > > Oh finally, you can try intracardiac perfussion of your > neuroprotective cutting solution. Its a pain, and its fiddley, I don't > think it helps with the neuroprotection, but I think it helps makes > the neurons in the slices more visible. > > On Nov 28, 5:11?am, Jose Guzman wrote: >> Hi everybody, >> >> I am currently working in hippocampal brain slices to do patch-clamp >> electrophysiology. Due to some experimental issues, I will have to >> record to neurons in old animals (P21-30). The problem is that the >> neurons I need are very susceptible to hypoxia , so the preparation >> requires some special cutting solutions. >> >> I collected the 3 major types of cutting solutions, and I would really >> like to know if any of you tried these in his/her experiments . These >> solutions have some minor variations in other publications (may contain >> additionally APV or kyneuric acid to prevent glutamate excitotoxicity), >> but basically are described as follows: >> >> ?1.- Sucrose based solution: total or partial replacement of NaCl >> by Sucrose. (Aghajanian and Rasmussen, 1989, Moyer and Brown,1998). >> >> ?2.- Glycerol based solution: the same as the previous but with >> ?Glycerol replacement. (Ye et al., 2006). >> >> ?3.- KGluconate based solution: without Ca2+ or Na+: >> ?specially indicated for patching dendrites (Dugu? et al., 2005). >> >> ? ? ? ? ? ? A major problem I found is that these papers are not very >> ? ? ? ? ? ? much cited in other publications (for example, Ye et al., >> ? ? ? ? ? ? 2006 has only 6 citations). Personally, I found that >> ? ? ? ? ? ? Sucrose-based solutions made an improvement (although not >> ? ? ? ? ? ? very big) but I would really like to hear from your >> ? ? ? ? ? ? experiences. >> >> ? ? ? ? ? ? Thank you very much for your feedback! >