Neurobiology and Brain Physiology

Neurobiology and Brain Physiology

  • Molly Boutin added an answer:
    Difference in reagents for dissection/growth of mouse vs rat cortical neurons?

    Hello all, 

    I usually perform dissections and culture postnatal rat cortex (P0-P3) and am about to try out the same dissection in mouse. I use a modified version of the brainbits protocol for cell dissociation (papain, etc). I was wondering if anyone had come across reagents that work well for rat cortical dissections but not mouse? Or additional reagents/issues/anything relevant for mouse, that doesn't come up in rat? Thanks for your help in advance!

    Molly Boutin · Brown University

    Thank you very much Yuan Lee for your help. I have now performed a couple mouse dissections. I have very high cell viability immediately after dissection/dissociation (>95%) but my attached neurons look sad- slowly growing extensions, dead cell accumulation, etc. I was wondering if, because the cells are initially alive, this could be a medium issue? You did not make any modifications to your growth medium formulation or the way that you treated the cells in culture? Thanks so much!

  • Victoria Burmester added an answer:
    Have oxytocin receptors been found in the olfactory bulb of humans?

    I am aware that they have been identified in the olfactory nuclei but have they also been found in the bulb?

    Victoria Burmester · Kingston University London

    Thanks Nilesh.  I know it's established that oxytocin receptors are in the rat olfactory bulb.  What I am trying to find out is whether this is so for humans.

  • Tiina Kauppinen added an answer:
    Can I use BV2 microglial cell instead of rat/mice primary microglial brain cells ?

    In most studies the primary microglial cells of the rat brain are use for experiments and I have the BV2 cells ,, So can I use the BV2 cells instead of primary microglial cells of the rat pups brain?

    Tiina Kauppinen · University of Manitoba

    Like previous answers already stated there are differences between BV2 cells and primary cells. BV2s are cell lines and cell lines are programmed to proliferate and thus expression patterns and active signaling pathways differ from primary cells - microglia like any other cells go through maturation/differentiation process (which is quite nicely captured in Prinz & Priller, Nature Reviews Neuroscience 15, 300–312 (2014) ; Fig 2). Phagocytosis relies on various receptor expressions and while BV2 cells phagocyte effectively they might lack the control or selectivity that primary microglial cells posses.

  • Katherine Poinsatte asked a question:
    How do I reduce vessel staining after BDA injection?

    I am iontophoretically injecting BDA (biotinylated dextran amine) into the motor cortex for anterograde axonal tracing. My BDA is tagged with Alexa594. I'm doing 5 minute injections with a current of 5 uA, alternating 7 seconds on and 7 seconds off with pulled glass pipettes of 20-50 um. I'm having really nice staining of the axons but the BDA also stains the vessels near my injection site. Is there any way to avoid this or is this just an inevitable issue with the method? 

    I've attached a picture. Has anyone encountered this before?

  • Tariq Ahmed added an answer:
    Why is anisomycin not suppressing my late LTP experiments of synaptic plasticity in the hippocampus?

    I am having trouble to replicate one of the main postulates about the expression of late LTP, concerning its dependence on de novo protein synthesis. I performe fEPSP recordings on the Schaffer collateral to CA1 synapse in rat hippocampal slices. Stimulate with 4xHFS (100 Hz, 1 sec), every five minutes. However, I cannot see an effect using anisomycin 30 µM on late LTP, even if I preincubate the slices in anisomycin for 2 hours before the experiment.

    Villers et al. (PLoS One. 2012;7) already reported no effect of anisomycin or cycloheximide on late LTP. In this paper they argue that the protein-synthesis hypothesis for development of late LTP should be reconsidered.

    What do you think? Do you have any idea about why these experiments are not working?

    Thank you in advance for your help!

    Diego Fernández

    Tariq Ahmed · KU Leuven

    How do you prepare your anisomycin? You may be oxidising it.

    If that fails tets emetine and puromycin to confirm the mechanism is protein synthesis dependent.

  • Patro Nisha added an answer:
    Does any one know a good IgG mouse anti nestin to stain neurons in brain sections?

    I am using 40u   free floating mouse brain sections.

    Patro Nisha · Jiwaji University

    Thanks!

  • Michele Bastide asked a question:
    Does anyone have a good enzymatic dissociation protocol for mouse cerebral vascular smooth muscle cells for patch-clamp recordings?

    I try to dissociate middle cerebral arteries and basilar artery by enzymatic procedure. Unfortunately, I couldn't obtain healthy cells. They are very fragile and incompatible to patch-clamp   recodings. I used a two-step protocole with first papine and DTT and second collagenase H, collagenase II or F and DTT.

  • Jeffrey N Joyce added an answer:
    What is the best in-vitro parkinson's disease model?

    Im trying to conduct a neuroprotective assay with SH-SY5Y cells differentiated with RA and TPA to dopaminergic neurons. 

    There seem to be multiple cell lines capable of similar feats, and some that seem to be used more without differentiation.

    Is there any reason to go through the trouble of dual differentiation? or would it be easier and more reliable/reproducible to work with N2A/CATHa/PC12 cell lines, as they seem to be the standard in neuroprotective research. 

    I understand that SH-SY5Y cells are of human origin, but even still, they dont seem to be heavily used when compared to mouse line.

    Jeffrey N Joyce · Kansas City University of Medicine and Biosciences

    Obviously since my laboratory years ago did a lot of characterization of the RA/TPA differentiation of SH-SY5Y cells, I favor using those cells. We always got high yield and felt that there was a reaons to utilize human cells vs non-human cells. Any of the immortalized cell lines are going to be different from slice culture or primary cultures.

  • Federico Iovino added an answer:
    Is there any in-vitro system (commercially available) that mimics blood brain barrier?

    I am trying to design carbonate apatite nanoparticles that might cross the endothelial layer of brain capillary (blood brain barrier-BBB). Thus, an in vitro system would be a better option (easy to understand and faster) to check the permeability of particles before moving to an in vivo model. I am looking for an in vitro BBB system that can be used for this purpose. I have seen the co-culturing systems discussed by many researchers. Are the co-culturing system with the filter available commercially?

    Federico Iovino · Karolinska Institutet

    HBMEC (Human Brain Microvascular Endothelial Cells) are a well-known and developed in vitro model for the brain endothelium (=blood-brain barrier). 

    You could also consider a co-cultivation of HBMEC and astrocytes. The BBB is not only brain endothelial cells but astrocytes represent the supporting component of the blood vasculature. In my opinion, HBMEC+astrocytes represent the most complete in vitro system. The issue is to be able to culture them together, since they are two different types of cells. It might be worth to give it a try...good luck!

  • Yolanda de Pablo added an answer:
    Does anyone have good rat cortex neuronal culture protocol?

    Dear all,

    We are trying to obtain rat cortex neuronal (pyramidal) cell culture with no success so far. We currently use P1-3 rat pups and procedure is as follows:

    Decapitation ->

    Decapitated head immediately placed into 96% EtOH @RT for ~5sec. ->

    Then head is immediately transferred to ice cold dissection media (PBS with glucose and pen/strept) and brain is removed (procedure takes 2-3 min)->

    Brain then placed to fresh ice cold dissection media and cerebellum, olfactory bulbs, meninges and etc. are removed this takes ~8 min. (per brain). ->

    Cortex then is transferred to the fresh ice cold dissection media and is finely (~1x1 mm) chopped with the scalpel blade (takes ~2 min) ->

    Pieces of cortex then collected and transferred to the preheated (37 C) Versene media, gently mixed and incubated 5 min in 37 C gently mixing the solution every 1 min. -> 

    After Incubation with Versene 2 different diameter fire polished Pasteur pipets are used for trituration still in 5 mL of Versene solution (this procedure additionally takes ~5 min.) ->

    After trituration 5 mL of ice cold growth media (Neurobasal-A supplemented with B27, L-Glutamine, FBS and Pen/Strept) is added to the cell suspension and mixed to deactivate Versene. ->

    To wash cells of Versene, suspension is then centrifuged 250 x g @4C and supernatant then removed. ->

    Cells then are resuspended in 5 mL of ice cold growth media and transfered to the new tube through cell strainer. ->

    Cells are counted in the Neubauer chamber, yield of live cells is normally 1-2*106 cells/mL ->

    Finally cells are placed on laminin and poly-L-lysine pretreated 24x24 cover-slips (concentration is 5*105 cells/mL) and grown at 37C 5% CO2 incubator. ->

    Next day after procedure half of growth medium is replaced with preheated fresh growth media. Same procedure is performed later every 3 days.

    So the question is: We get lots of glia (that we once were suppresing by AraC, but do not do it anymore) but only few if at all live neurons, so does anyone have any suggestions what is wrong with our protocol or what can be improved, or maybe by any chance you do have another protocol that would work for you and that you could share with us? Thank you in advance.

  • Logan Beach added an answer:
    What is the best Golgi staining method for human tissue that has been fixed in formalin for many years?

    There is less information available for human cortex about optimal golgi staining than for mouse or rat. But the tissue I am working with is human and has been in formalin for many years.

    I am specifically looking for dendritic spines for classification purposes.

    Any protocol ideas or publications for the optimal human cortex golgi staining protocols?

    Logan Beach · Brigham Young University - Idaho

    These guys say that past 6 months it gets too inconsistent to try: http://www.neurostructural.org/default.asp?page=80788

    But seeing as that is not very helpful these guys posted some papers on it in another thread on research gate.  Look at the responses.  They may help  https://www.researchgate.net/post/Can_anyone_help_with_Golgi_staining_and_spine_analysis

  • S. K Sharma added an answer:
    How can I store MPTP?

    Does anyone know how to store MPTP? I just buy 10mg powder MPTP from SIGMA. And the product information says that the aqueous solutions may be stored for 24 hours at 4 ° C.

    However, I need 0.1mg of it dissolved in 1 μl saline in each experiment. If I dissolve all the MPTP in saline at once, I am afraid after 24 hours the left MPTP can not be used any more.

    So I wonder very much how to store MPTP, should I have to divide the powder MPTP into 100 pieces and dissolve it in 1 μl saline each time before each experiment?

    Does anyone can help me, thank you very much!

    S. K Sharma · Saint James School Of Medicine

    Dear Dr. Hong, 

    I fully agree with the above researchers.  The Parkinsonian Neurotoxin, 1-Methyl, 4-Phenyl, 1,2,3,6 Tetrahydropyridine (MPTP) is a Mepridine analog and belong to the category of Tetrahydro-isoquinolines. 

    In addition, I would like to keep MPTP in an amber container as it is photo-labile. So it will be good idea to make the serial or log dilutions in amber tubes. 

    It is hygroscopic also, So it will be better to store it in a dessicator with dehydrating silicon balls.

    You will be using a face mask. a hood, and reconstitute the entire powder in saline directly by using 1 ml microsyringe with a needle and a vent. Please do not open the vial just mix the entire solution couple of times and then aliquot in micrioials.

    I would also suggest you to please use cryo-vials if you need to store the reconstituted dilutions of MPTP at -30C or at -80C.  

    Avoid keeping the solution at room temp and under direct sunlight.  As instructed above, use it as fresh as possible. Repeated freezing and thawing can cause chemical degradation of the product. 

    I hope it is helpful and universal for any other toxic substance as well. 

    Good luck with your research. 

    Respectfully, 

    Sushil Sharma, Ph.D; D.M.R.I.T

    Professor of Pharmacology & 

    Course Director

    Saint James School of Medicine

    Plaza Juliana 4, Kralendijk

    Bonaire, Dutch Caribbean

  • Huong Ha added an answer:
    How to increase the frequency/amplitude of AMPAR mediated mini EPSCs in culture neurons?

    Hi all,

    I am interested in AMPAR mediated mini EPSCs in hippocampal neurons. My current recording configuration (hibernate E as bath solution and Cs gluconate as internal) seem to allow me to record from them up to day in vitro (DIV) 25 for about 30 min with fairly stable access. The sad story is that I do not see a lot of mini events (1 - 2 events every 3 - 5 seconds --> much less than 1 Hz). I can see quite a lot spontaneous events starting at DIV 11 already (1 - 2 event every second or so). Does it sound like something you experience before? How would you recommend troubleshooting it? Maybe, like, changing the recording condition or culture condition to have more mini AMPAR EPSCs? Thanks a lot!!!

    Best,

    Huong

    Below are some more information if you would like to know....

    When the neurons are younger (Div 12 - 15), there are a lot of action potential driven EPSCs [huge events, > 100 pA]. And when they get to Div 25, there are mostly very small events (20 pA, more or less). The small events decay time is approximately from 4 - 30 ms. 

    Regarding the culture, Coverslips are coated with 1 mg/ml Poly D lysine. I plate the neurons from E17 - E 18 hippocampi at 1.4 millions neurons per 100 mm dish containing 6 coverslips. The coverslips are submerged in serum containing media. The coverslips have wax feet so I can flip them up side down into 60 mm dishes with neurobasal + B27 + glutamax + 20 % media conditioned by astrocytes [which facilitates the growth of a lot of astrocytes underneath the neurons].

    Sometimes I also co-culture the neuron coverslips in dishes with astrocyte feeder layers [in which the cells are not touching each other  i.e. Banker culture]. The viability and development look fairly good. I can see a fairly dense network of dendrites already at DIv 11 and it just gets denser over time. Cells are evenly distributing across the coverslip, not much fasciculation or any sign of substrate problem. I feed them twice a week after the first week.  

    Huong Ha · Stanford University

    @Thomas: As always, many ideas! Thank you! :) It is very helpful indeed!

    - Regarding your concern for LTD, I did not see any reduction of spon events within once cells. About 2 weeks ago, I recored from ~ 4 week old neurons (who were not co-cultured with astrocytes) and they did not have much of the big EPSCs. This week we started to have Banker cultured neurons at ~ 3 week old and these all seem to have very robust spontaneous EPSCs and look very nice (3D-looking, smooth membrane, nice processes).

    - I have not analyzed the data but looks like these new neurons have higher mini frequency already (still lower than 1 Hz though). I might still need to heat them up or try your suggestion with the KCl to get more response and reduce recording time. Thank you for this! 

    - The density is about as high as we can do considering other factors. I tried higher density before but the media just got acidic so quickly and the neurons were not healthy...

    -For your question about cortical and hippocampal neurons, no, they did not look like they had the same final density. The cortical neurons usually looked slightly denser than the hippocampal neurons even if we plated them at same density. I don't have any quantification on this yet though. This was mostly from my quick glance at the traces

    @Saak: That is super insightful! I do use Cs gluconate as internal with all of the voltage clamp experiment but did not really think about the geography of culture processes that hard. :) From my staining experiments, culture neurons do have synapses everywhere, and their dendrites stretch quite far. I have not measured and compared these numbers to in vivo neurons. Would be interesting to learn more.

    I did not know that culture neurons have mainly GABAergic synaptic activity... I saw quite a lot of mini-like activity (events <~ 20 pA, frequency is way higher than 1 Hz) when recording w/o drugs.  And when I added APV, picrotoxin and TTX, the frequency of tthese is like... below 1 Hz. I was thinking the activity was mostly driven by AMPAR and NMDAR since I am holding the cells at -70 mV (close to Cl- reverse potential). Did not at all think of GABA... Did you do some measure with this? 

  • Jochen F Staiger added an answer:
    How can we verify that our brain slice manages to preserve the thalamocortical pathway?

    I just get very puzzled when reading some paper results about brain slice work, when the author stimulates one intracortical region and records the response of another intracortical region, how can I know whether it is direct connection between these two regions or may involve other subcortical regions, eg the thalamus? I didn't find any evidence in the paper about preserving the thalamocortical pathway or not. How can I understand the results in these kinds of paper?

    Hope anybody expert in brain slice work can help me with this. Thanks

    Jochen F Staiger · Universitätsmedizin Göttingen

    Tracing is necessary. An alternative to DiI is biocytin. See attached PDF.

  • Saak V. Ovsepian added an answer:
    How do I program P97 puller to make a glass recording electrode for brain slice hippocampus LTP?

    How do I program P97 puller to make a glass recording electrode for LTP?

    Saak V. Ovsepian · Deutsches Zentrum für Neurodegenerative Erkrankungen

    Dear Kaichuan, please follow simple steps below to get things going :  (1) check the filament and make sure that it is installed properly; (2) run a ramp test - this is quite easy (just google for the details or check Sutter Instruments website); (3) use the readouts of ramp as your starting parameters for the filament and glass you have (4) adjust the temperature, delay and velocity until you will get electrodes with parameters you would like to use. Keep in mind that with higher temperature the tip of electrode becomes smaller, which increases the resistance. Typically, for field potentials electrodes should not exceed 3 mega Om, while for whole cell you need pipettes with higher resistance (up to 6-7 mega Om). Good luck -    

  • Sumana Chakravarty added an answer:
    Does anyone have experience with in utero injections of mouse embryos?

    I did in utero injection of 0.5 microliter of fast green with 26 g hamilton injector into cerebroventricular of 16 days mouse embryo, but it was born dead. I don't know why? What is falls? May it be due to infection or because injection into the brain? How could I do that?

    Sumana Chakravarty · Indian Institute of Chemical Technology

    Thanks Bruna. I will also try glass capillary next time. In fact, I do not sterilize the needle as we don't have multiple needles and once you do one animal again you have to do for next animal (I think it is impractical for my case)...so I take care of needle very carefully for not to touch anywhere during the process and in between animals I whip it by alcohol swab...wait to dry...then use it again. FYI, I use another clean needle/ syringe set for empty vector.

    I will be happy if it works for you.

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  • Fatiha Chigr added an answer:
    How can I isolate choroid plexus from mouse brain?

    As it is obvious, choroid plexus in mouse brain is very small and I am not sure that I can have enough quantity of it for RNA extraction.

    Fatiha Chigr · Université Sultan Moulay Slimane

    Personnally I prefer to get CP from all ventricles. This gives more chance to perform your RNA extraction.

  • David J Hodson added an answer:
    Can someone recommend a neuronal tracer that diffuses in short time in fixed tissue?

    I used DiI and I traced very well some fibers, but a disadvantage is that when it's exposed to UV light in a microscope, loses fluorescence very quickly. I need a tracer in fixed tissue because it's easier to administrate the tracer in the zone that I'm studying than in stereotaxic. Thanks.

    David J Hodson · Imperial College London

    Justin's answer pretty much covers everything. The only thing I would add is to maybe think about using multiphoton excitation if you continue to have issues. Dil can be excited at ~ 700 nm and this would significantly reduce photobleaching, as well as increase axial/lateral resolution due to the two-photon effect.  

  • Saivishal Daripelli added an answer:
    Which region is best for implanting reference electrode for measuring theta oscillations in CA1? How can I get peak frequency of 7-8Hz in CA1 region?

    I want to measure modulation of theta oscillation in CA1 (Hippocampus) region by stimulating NPO (Nucleus Pontis Oralis) in rats. I have gone through the literature where researchers used Frontal bone or ear bar or cerebellum as reference. Can anyone suggest me, which region is best for reference electrode to get synchronized theta waves?


    In literature it was mentioned 0.5 to 3.0V current is required to get synchronized theta. But we are getting synchronized theta in CA1 (cerebellum as reference), only when we stimulate NPO by 3.0V and peak frequency was 4-5Hz. Is there any possibility to get peak frequency of 7-8Hz with low voltage stimulation?


    Thank you

    Saivishal Daripelli · Suven Life Sciences Limited

    Thanks Bali and Molden for your replies. My apologies for the delay in acknowledging the same.

    Your suggestions are really helpful for us.

    By using constant current  (60 uA) for stimulation now we are able to get theta oscillations of 5-6Hz with cerebellar reference, but after Donepezil treatment we are observing decrease in amplitude of theta (which was reported to increase). What could be the reason for this?

    thank you.

  • Benjamin A Suter added an answer:
    Has anyone had problems with a channel rhodopsin AVV killing neurons?

    I purchased the CamKIIa-ChR2-eYFP from the UNC vector core.  This is the one from the Deisseroth lab.

    My problem is that when we infected some rats with this virus and took fresh slices for electrophysiology (~5weeks post injection), it looked like almost all of the neurons at the injection site were dead under the infrared camera. There were still plenty eYFP positive projections, but almost no soma.

    However, I also ran a second batch of adult animals at a separate time point. When I perfused rats 8 weeks post-AAV injection and slice/mount the fixed tissue, I can see very dense eYFP projections at the injection site (similar to what was seen with the fresh e-phys tissue).  Oddly, the soma are completely devoid of eYFP (looks like dark holes in the tissue, figure attached), but that pattern looks similar to representative images I see in other publications. There is also still DAPI positive staining in the nuclei. I've been told that this is just the result of the ChR2-eYFP fusion protein moving into the terminals. This makes me think there wasn't much of a problem, at least in this batch.  I've had mixed results running behavior with optogenetic stimulation.

    Has anyone had a problem with cell death using AAV5-CamKII-ChR2-eYFP?

    Benjamin A Suter · Northwestern University

    Did the injection volume vary between your animals, or between the two batches of surgeries? I've heard of people seeing vastly increased cell health at the injection site and increased axonal transfection at projection sites after reducing the volume of virus injected. While it likely depends on titer, a volume of ~30-50 nl per site seems appropriate in cortex for typical (e.g. UPenn) AAVs; not sure for your injection locations.

    Regarding appearance of soma: with ChR2-fluorophore fusion proteins, I believe it's common to have a dim somatic signal since it's just in the membrane and not cytosolic, while the dendrites and axons may appear to have a more discernible signal.

  • Benjamin A Suter added an answer:
    Does anyone know the distance a certain optical light can diffuse in the brain issue?

    Hi, I am trying to use optogenetics to study the connection of two nucleus within rat brain. However, the distance between this two nucleus is only 1mm. Does anyone know the distance a certain optical light can diffuse in the brain issue? Thanks!

    Benjamin A Suter · Northwestern University

    Bottom line: if you're using a fiber, and place the tip ~200 um from the stimulation target (a common distance), your target will see only ~10% of the light intensity measured at the tip of the fiber.

    Another useful source, that synthesizes from a number of publications:

    http://www.openoptogenetics.org/index.php?title=Transmission_Through_Brain_Tissue

    This includes measurements by Svoboda's group that look at the lateral spread for an LED on the cortical surface.

  • Michael Alexander added an answer:
    Is chemotherapy-induced cognitive dysfunction in cancer survivors due to effects on microglia or endothelial cells?

    If you are aware of the fact that cancer patients treated with standard chemotherapy over the past 20 or so years have cognitive problems with reasoning and motor skills, I'd like your input on why, and why ten to twenty years later.  Microglia (brain macrophages, which is a misnomer) can initiate the process of neurodegeneration, which leads to ex-cancer patients, who've survived through chemotherapy, down the path of significant brain damage.  There is an initial insult due to systemic inflammation, but I'm thinking that endothealial damage and their ability to secrete inflammatory lymphokines perpetuate or exhasterbate chemotherapy-induced cognitive problems in cancer patients.

    What do you think?

    Michael

    Michael Alexander · Widener University

    Thank you Peter that was very helpful.  This section of the book I'm writing has to do with surviving cancer and it's treatment.  The book itself is an overview of how cancer changed our society.  The book, like my others, is an effort to translate the information from the large variety disciplines involved with this societal change.  It looks at both the 'Big Picture' and 'Detailed Picture' of cancer and society.  I'm very thankful for the input from you as well as a myriad of other professionals in seemingly unrelated disciplines that have helped me put together this work.  

  • Jennifer H Steel added an answer:
    Does anyone have an idea what can be the problem during our RNA ISH on free-floating mouse brain sections?

    We do RNA ISH on formalin fixed mouse brain slices (not paraffin embedded or frozen)(40 µm) with DIG-labelled RNA riboprobes as described in the attached file.
    The problem is that the success of the protocol is about 40%. Once we got sign, then there is no signal, even if the circumstances are almost the same. May be the duration of perfusion with DEPC treated PB(± 2 min) and PFA-fixed brain slicing differ time to time, could it cause significant mRNA degradation in the tissue?
    The other possibility we thought is the degradation of the probes. We store them at -20°C and dilute during ISH as described in the protocol (in the attached file). What is the best way to check the integrity and functionality of the probes? Or could be something else the problem?
    We started using this technique recently, so we have not so much experience in RNA ISH. We appreciate every help!

    Jennifer H Steel · Imperial College London

    Do you have a positive control?  All our ISH was controlled (on frozen or paraffin sections) using beta actin as a positive control probe, done on a separate section.  This should tell you whether the RNA is well preserved. Then your specific target can be detected using your own probe on other sections. In my protocol, the anti-dig antibody is incubated at room temperature for 1 hour but the NBT-BCIP substrate detection is on overnight. Many probes, optimal dilution, require overnight NBT-BCIP.  If the signal appears quickly it is often non-specific in my experience.    Another consideration is the probe size and hybridisation temperature.  I have always used 50°C overnight, for any probe longer than about 100 bp.  Oligos can be hybridised at 37°C.

  • Alexander von Holst added an answer:
    What is the best way to enzymatically dissociate mouse brain tissue that gives a high yield for all cell types (neurons, astrocytes, microglia)?

    We currently use mechanical dissociation, but our astrocyte yield is low. We've tried papain (I'm not sure which concentration we used), but it did not give a good yield for neurons. We want to minimize cell death during dissociation as well as maximize preservation of cell surface markers (e.g. CD11b). Any suggestions? Thanks!

  • Michael Alan Bozarth added an answer:
    Why is I.c.v. injection of vehicle solution affecting the performance of mice undergoing a fixed ratio 1 schedule of reinforcement?

    I have a batch of C57BL/6J male mice (3 month/old) undergoing a fixed ratio 1 schedule of reinforcement (1h/day, 2 hrs after dark onset, 10% sucrose) with an implanted cannula into the later ventricle. They press the active lever 80-100 times, and the inactive lever 10-20 times. They are now really accustomed to the injection procedure since I did 7-10 days of sham injections, but every time I inject the vehicle solution (1ul in 3mins; I tried PBS, then saline and lastly aCSF) their performance drops at 30-40 concerning the active lever, and 0-5 concerning the inactive lever. I am running the sessions 2h following i.c.v. injection but before the session the mice already look "unwilling" and not really active. I also tried to inject them every day, but their performance stabilized at a low level (40-50 lever presses).

    Any advise/suggestion is very welcome.

    Many thanks in advance.

    Michael Alan Bozarth · University at Buffalo, The State University of New York

    I haven't worked with mice, so please take any comments or suggestions I make here with the proverbial "grain of salt" (pun not intended but somewhat amusing anyway).

    I would be suspicious of two things, both related to possible trauma caused by the injection procedure. First, make sure that the injection cannulae are placed properly within the ventricle. Remember that drug solutions flow up (not down) following a microinjection-- they follow the path of least resistance which is up the cannula shaft. It's possible that the injection is actually causing pressure/trauma to tissue directly above your cannula placement. Have your tired other A-P injections sites? (I'm presuming that you've already histologically confirmed your cannulae placements and checked for gross tissue damage in the surrounding area.)

    Second, consider the injection speed. I understand that your intended infusion rate is acceptably slow, but is it in actuality? Movement of the PE or other flexible tubing typically used to connect the injection pump or other device to the injection cannua often leads to uncontrolled infusions. Have you checked for flow after removing your injection cannula? Simply making a subsequent injection into air should confirm that the system is working properly--if you don't see a nice, round, well-formed fluid 'bubble' on the end of your injection cannula after making a single injection 'into air' following the intended microinjection into the subject, you have inadvertently injected an uncontrollably large volume into your subject. I always made at least two subsequent 'air injections' that should appear to be about the same size (Although one obviously can't accurately visualize a one microliter volume, with experience it is fairly easy to tell if the volume appears to be correct; I typically used a 100 nl volume and learned to readily visual its approximate size in air. My microinjections tended to be very good or associated with an occasional problem, making this simple post-injection fluid-visualization test basically a 'go or no-go' evaluation of the probable microinjection accuracy.).

    Finally, I published the design for a low-pressure injection device in the Journal of Neuroscience Methods around 1980 (sorry, but I don't have an electronic copy of this article). This device uses gas pressure which will compress when meeting significant tissue resistance thus infusing solution more at a rate readily acceptably by brain tissue. Histological examination revealed minimal tissue damage using this method when compared with traditional microinjection procedures.

    (FYI: I developed this microinjection method [which I called EMIT -- electrolytic microinjection transducer] for my own work in intracranial self-administration [ICSA]. This experimental method places additional requirements on the contingency as well as the reliability of the microinfusions. I've also used this method successfully for microinjections delivered to subjects habituated to the test apparatus and for not disturbing them by picking them up and the necessary handling restraint while performing the injection procedure 15 minutes after connecting the device (e.g., Hamilton & Bozarth). I don't necessarily suggest that you change to this microinjection procedure because the EMIT system's proper construction and use usually requires some on-site training or other special care not documented adequately in the published literature [A commercial firm tried manufacturing EMIT units, but sloppy construction practices produced gas leaks which caused the system to malfunction; additionally, some special training or care is necessary when using this approach to delivering microinfusions.]. My laboratory has been closed for some time now, or I would invite you for a visit to Buffalo to learn more about the construction and use of this low-pressure microinjection system.]

    Good luck with your continued research. Do take your time and care in setting-up your basic procedure so that you can be confident in your experimental outcomes. I've found that most people simply ignore the problems with conventional approaches to delivering reliable microinjections, relying on calculated estimates or watching a bubble move in the infusion line. Neither of these approaches are sufficient to ensure reliable microinjections. (FYI: I conducted a series of studies in the 1980s using radiolabelled drug and determining the actual amount delivered with conventional and with EMIT microinjection procedures. I found the EMIT system to reliably deliver even delayed infusions into freely moving subjects, while conventional microinjection approaches using a microinjection syringe and PE tubing to be totally unreliable. Unfortunately the full journal report was never submitted for publication and the work appeared only in a Society for Neuroscience Abstract (Mitchell and Bozarth) and presented at the corresponding annual meeting.)

    I'm not sure I can (or will) find my way back to this posting, so if you have any questions you can contact me directly at: bozarth@buffalo.edu

    P.S. I generally used unbuffered Ringer's solution without any problems, avoiding the debate over the proper composition of artificial CSF that was prevalent during my active research years. Also, although I think your one-microliter volume is fine, it might still present a problem with injections delivered inadvertently into tissue or delivered to rapidly. Nonetheless, I would probably stay with this volume to increase the likelihood of reliably delivering your intended drug dose into the ventricles (Of course, the volume would have to be MUCH smaller for tissue injections into a mouse brain.)

  • Adriana Gisler added an answer:
    Does anyone know of a brain structure checklist (focus on frontal lobes) for a chicken embryo model?

    I want to quantify gross brain morphology observations/deviations  in a chicken embryo model, so that I can run an ANOVA, instead of doing it qualitatively.

    Thank you in advance for your help.

    Adriana Gisler · Walden University

    That is very helpful, Matthew!! I will definitely look into the article. Also, I just ordered the atlas you just mentioned. Thanks again for your help.

    Adriana

  • Paul Legrand added an answer:
    Is the extrapyramidal syndrome associated with Midazolam Fentanyl sedation?

    We recently had a extrapyramidal syndrome after a procedure under councious sedation. A Fentanyl Midazolam comination was used with 0.25mg of scopolamine. Has anyone some experience with the management of this problem? 

    Paul Legrand · Mariaziekenhuis

    Ondansetron was not given as there were no nausea problems. The EPS symptoms dissapered after 6 h. teh rest of the follow up was uneventful. 

  • Pardis Td added an answer:
    What is the relationship between hyperprolactemia and mental illness?

    i know that some antipsychotic medications can cause hyperprolactemia as a side effect, but I am wondering whether the dopamine and GABA imbalance produced by hyperprolactemia resulting say from a pituitary adenoma can cause serious mental impairment.

  • Tatyana Pivneva added an answer:
    Can we use TTC stained brain slices for any other purposes?

    I am doing research on stroke animal models and generally use middle cerebral artery occlusion induced focal cerebral model. As you know the infarction degree is quantified by TTC staining and the brain slices are thrown after the photographing. I am suspecting that can we use the TTC stained brain section for other purpose such as protein extraction and western blot or RNA extraction etc. If you have any idea about it please let me know. Thanks in advance.

    Tatyana Pivneva · National Academy of Sciences of Ukraine

    I only used TTC staining for a MCAO model of the  insult .

    Good luck

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