- Moshe Turner added an answer:4Releasing agent vs. reuptake inhibitor?
Selective norepinephrine and serotonin reuptake inhibitors (SNRI's) are commonly used in the treatment of cataplexy because cataplexy is the result of down dysregulated norepinephrine, itself the result of missing or greatly diminished orexinergic signaling.
However, cataplexy is also occasionally treated with Phentermine, which is a norepinephrine releasing agent.
What happens when a person takes both a releasing agent and a reuptake inhibitor (not necessarily specific to this example, but generally)? Is the result a greater concentration/availability of the protein you're after than you'd get with either individually?
That's the kind of thing I was hoping for! The abstract is very clearly written and already has given me some insight, so I'm expecting to enjoy the full text article. Thanks!Following
- Jordan Jahrling added an answer:12Does anyone have a protocol for growing bEnd3 cells on Corning Transwell inserts?
I am attempting to do some in vitro blood-brain barrier experiments using Corning Transwell pore inserts. Unfortunately, I have not had any luck getting my bEnd3 (mouse brain endothelial) cells to grow on the pore inserts. I have tried altering my seeding density but to no avail. The inserts are TC treated; nevertheless, I am planning to coat the inserts with collagen or lysine unless anyone is familiar with a working protocol. Any suggestions are appreciated. Thank you.
I've never had to coat my plates, though I always use 10cm plates from Corning and have never used flasks. I can't imagine this would make much difference, but it's something. What density are you plating at? bEnd3 'prefer' to pack fairly tightly and do not grow well if they are plated too sparsely.Following
- Manuel Gesto added an answer:115HIAA/5 HT turnover ratio in depression?Can anyone explain me the meaning/use of 5HIAA/5 HT turnover ratio in depression? Why is this ratio important from the utilization of serotonin in the brain? In simple terms.
Maybe you can try this reference:
Shannon, N.J., Gunnet, J.W., and More, K.E. 1986. A comparison of biochemical indices of 5-hydroxytryptaminergic activity following electrical stimulation of dorsal raphe nucleus. J. Neurochem. 47: 958–965.
- Maria Rosa Peraita-Adrados added an answer:4Do you know any article concerning myelinogenesis and sleep?
The process of myelinogenesis starts at birth and last until 22 years of age.
Sleep may play an important role in this process.
In demyelinising diseases such MS sleep disturbances are the rule.
Perhaps it will be interesting for this studies the myelin mutant taiep rat model
by José R. Eguibar, Instituto de Fisiología, Universidad Autónoma de Puebla. Mexico)
Thanks for your reference that I did not know.
- Vitthalrao Khyade added an answer:8What is the physiological or anatomical difference between place cells and grid cells in the hippocampus?
In the medial temporal lobe,there are specific types of neural cells such as place cells, head-direction cells, grid cells, and boundary vector cells which involved in cognitive map and spatial memory. Hippocampal “place cells” encode the rat’s location within an open environment independently of its orientation and fire in the specific position. The complementary encoding of the orientation, independently of location, is done by “head-direction cells” .I think all of them are pyramidal neurons. So Is there any physiological or anatomical difference between these kinds of cell?
Research Group, A.D.T. And Shardabai Pawar Mahila Mahavidyalaya, Shardanagar, Malegaon(Baramati) Dist. Pune – 413115.
“Dr. APIS” SCIENCE SPECTRUM
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Fenton's reagent is a solution of hydrogen peroxide and an iron catalyst that is used to oxidize contaminants or waste waters. Fenton's reagent can be used to destroy organic compounds such as trichloroethylene (TCE) and polychloroethylene (PCE). It was developed in the 1890s by Henry John Horstman Fenton as an analytical reagent.
Iron(II) is oxidized by hydrogen peroxide to iron(III), forming a hydroxyl radical and a hydroxide ion in the process. Iron(III) is then reduced back to iron(II) by another molecule of hydrogen peroxide, forming a hydroperoxyl radical and a proton. The net effect is a disproportionation of hydrogen peroxide to create two different oxygen-radical species, with water (H+ + OH−) as a byproduct.
(1) Fe2+ + H2O2 → Fe3+ + HO• + OH−
(2) Fe3+ + H2O2 → Fe2+ + HOO• + H+
The free radicals generated by this process then engage in secondary reactions. For example, the hydroxyl is a powerful, non-selective oxidant. Oxidation of an organic compound by Fenton's reagent is rapid and exothermic and results in the oxidation of contaminants to primarily carbon dioxide and water.
Reaction (1) was suggested by Haber and Weiss in the 1930s as part of what would become the Haber–Weiss reaction. Iron(II) sulfate is typically used as the iron catalyst. The exact mechanisms of the redox cycle are uncertain, and non-OH• oxidizing mechanisms of organic compounds have also been suggested. Therefore, it may be appropriate to broadly discuss Fenton chemistry rather than a specific Fenton reaction.
In the electro-Fenton process, hydrogen peroxide is produced in situ from the electrochemical reduction of oxygen.
Fenton's reagent is also used in organic synthesis for the hydroxylation of arenes in a radical substitution reaction such as the classical conversion of benzene into phenol.
(3) C6H6 + FeSO4 + H2O2 → C6H5OH
A recent hydroxylation example involves the oxidation of barbituric acid to alloxane. Another application of the reagent in organic synthesis is in coupling reactions of alkanes. As an example tert-butanol is dimerized with Fenton's reagent and sulfuric acid to 2,5-dimethyl-2,5-hexanediol.
The Fenton reaction has importance in biology because it involves the creation of free radicals by chemicals that are present in vivo. Transition-metal ions such as iron and copper donate or accept free electrons via intracellular reactions and help in creating free radicals. Most intracellular iron is in ferric (+3 ion) form and must be reduced to theferrous (+2) form to take part in Fenton reaction. Since superoxide ions and transition metals act in a synergistic manner in the creation of free radical damage, iron supplementation must not be done in patients with any active infections or in general any diseases.
Henry John Horstman Fenton (18 February 1854 – 13 January 1929) was a British chemist who, in the 1890s invented Fenton's reagent, a solution of hydrogen peroxide and an iron catalyst that is used to oxidize contaminants or waste waters. Fenton's reagent can be used to destroy organic compounds such as trichloroethylene (TCE) andtetrachloroethylene (PCE). Born in London, Henry Fenton was educated at Magdalen College School, King's College London and Christ's College, Cambridge. He became the university demonstrator in Chemistry at Cambridge in 1878, and was University Lecturer in Chemistry from 1904 to 1924.
1. Fenton H.J.H. (1894). "Oxidation of tartaric acid in presence of iron". J. Chem. Soc., Trans. 65 (65): 899–911. doi:10.1039/ct8946500899.
3. Haber, F. and Weiss, J. (1932). "Über die Katalyse des Hydroperoxydes". Naturwissenschaften 20 (51): 948–950. doi:10.1007/BF0150471.
4. Juan Casado,Jordi Fornaguera,Maria I. Galan (January 2005). "Mineralization of Aromatics in Water by Sunlight-Assisted Electro-Fenton Technology in a Pilot Reactor". Environ. Sci. Technol. 39 (6): 1843–47. doi:10.1021/es0498787. PMID 15819245.
5. Brömme HJ, Mörke W, Peschke E (November 2002). "Transformation of barbituric acid into alloxan by hydroxyl radicals: interaction with melatonin and with other hydroxyl radical scavengers". J. Pineal Res. 33 (4): 239–47. doi:10.1034/j.1600-079X.2002.02936.x. PMID 12390507.
6. E. L. Jenner (1973). "α,α,α',α'-Tetramethyltetramethylene glycol". Org. Synth.; Coll. Vol. 5, p. 1026.
7. Robbins and Cotran (2008). Pathologic Basis of Disease - 7th edition. Elsevier. p. 16. ISBN 9780808923022.
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Acknowledgement: Girija Girish Tambe of Vaishnavi Xerox helped for Collection of images in the Science Spectrum of 5 September, 2015. All the mistakes in the collection of information from website, it’s compilation and communication belongs exclusively to :
Vitthalrao B. Khyade (And not to his pace making Shardanagar). Please do excuse for the mistakes.
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Compiled for: Science Association, Shardabai Pawar Mahila Mahavidyalaya, Shardanagar (Baramati) – 413115 India.
With the Best Compliments From: Shardanagar (The Agro – academic Heritage of Grandsire Padmashri Dr. D. G. Alias Appasaheb Pawar).
All the mistakes in the collection of information from website, it’s compilation and communication ( through email ) belongs exclusively to : Vitthalrao B. Khyade (And not to his pace making Shardanagar).Following
- Luiz Ataíde added an answer:17Do brain frequencies like "delta" and "theta" change independently while approaching sleep?
I am researching about drowsiness and sleep detection. We know in general, frequencies from 1Hz to 16Hz (Delta, Theta ,Alpha, low Beta) increase while approaching sleep, while frequencies with 17Hz and higher (high Beta, Gamma) decrease. So do these signals change independently? For example, can we consider that the increase in Delta frequency is independent of the decrese in Gamma? Can we just see the Delta and say: "OK, you increased then the person is sleepy now" ?
I have an special interest on your research.Following
- Serkan Cakir added an answer:3How can I measure brain opioids and how can I store the chicken head samples?
I will take blood samples during the slaughter of chicken, and also collect to head (as a result, brain ) samples.
Even ıt is not exact procedure,
I want to measure some opioids in blood and brain samples.
Endorphine, encephaline, substantia P, noradrenaline, serotonine, cortisol etc ib blood and brain samples
1-How must ı store the brain samples as soon as ı got after slaughter. May ı store the head samples (washed quickly) in -80 in 10 seconds for unchanged opioids level in brain.
2- Which opioids are most important to define possible pain during nonstunning-slaughter?
I hope one expert can help me?
Many thanks for your detailed help.Following
- Staffan Holmqvist added an answer:2How many astrocyte cells are in in the rat brain?Do we know how many neurons are in the brain (human, rodent)?
It seems that the more interesting feature of human vs rodent/primate astrocytes is not the relation in cell counts, but rather the shift in complexity of glia. It has been estimated a normal rodent astrocyte could support/modulate around 90,000 synapses within its domain. Human atrocytes with larger size, processes in combination with higher synaptic density is estimated to support 2 million synapses.
For further reading I recommend Oberheim, Goldman and Nedergaards review articles on the subject.
2006 - doi:10.1016/j.tins.2006.08.004
2012 - doi:10.1007/978-1-61779-452-0_3Following
- Serguei N Skatchkov added an answer:2Any suggestions on chromatin during brain development?
this is another of my mysterious questions. I am working with brain chromatin during development, in particular with P4 and P60 mouse brains, and I am always getting a weird result. whether I am preparing chromatin for ChIP or just isolating it after Mnase digestion, I always get a huge amount of DNA from P4 brains but very small amount from P30. Before you ask I treat every sample with almost 200ug of PK for 2 hours. My first thought was that DNA could get stuck to proteins and so I could lose it during my phenol chloroform extraction. I always start from same amount of nuclei so DNA amount should be same. Anybody can help me solve this?
The key players are polyamines (PAs), spermidine and spermine. Why? PAs work as casks to hold DNA. This stabilizes DNA structure and an extraction of DNA from brain is a process which should count (i) PA unbinding from DNA procedures and (ii) cell types you are isolating: (1) neurons versus (2) glial cells. Since in adult brain Glia outnumber neurons about 13 times in brainstem and ~3.5 time in cortex, so the nuclei of astrocytes, NG2 cells, oligodendrocytes, ependyma (all glial cells) contain most of DNA sampling from brain. Are you sure that the samples of collected nuclei are containing both cell types or you are loosing one of the types due to just isolation procedure?
Another key is the age. While young brain is not much differentiated (glia versus neurons), after establishing adulthood (for rats and mice it is from 21 day to 60 days) the glial cella are maturated and glia outnumber neurons. Therefore, how do you collect nuclei and separate them, glial versus neuronal?
Finally, In the cells ~80% of spermine and spermidine are bound on RNA and DNA. During development (and specifically during aging) PA amount is dropped down (the well known fact). As so, the amount of chromatin isolated will be also dropped down. For example your problem may be related to the concentration of PAs in your samples and therefore to amount of DNA isolated. Check your methods, how does it related to PAs cleavage from DNA, etc. Does my answer helps conceptually?
- Yuan Li added an answer:4Any suggestions on the roles of hexokinase II ?
Hexokinase I is predominant form of hexokinase family in mammalian brain. But under hypoxic conditions, there is no obvious change in its expression. In contrast, hexokinase II, which is mainly expressed in insulin-sensitive tissues and malignant tumors, experiences a robust upregulation. I think it is a norm that only when a protein's function has been compromised, then its subsitute may undergo a change to adjust to this situation.So my question is why hypoxia exposure can induce a significant uptegulation of hexokinase II whereas it has no effect on hexokinase I in brain? Is it possible that it may play some other roles in hypoxic situations? Thanks ~~
Thaanks for your suggestion, Olivier Diaz. I have seen this paper before, but the source of lactate is not from neurons themselves. Howeve, under hypoxic conditions, hexokinase II is upregulated in neurons, even they can use lactate as fuel, they may also cause acidification within cells since it is acid.Following
- Alexandre Hiroaki Kihara added an answer:3Do you think hanging drop 3D cell culture can be used for synapse study?
This question is with regards to 3D culture. Do you think hanging drop 3D cell culture can be used for synapse study?
Yes, but I also recommend hidrogel based culturing.
- Bert Bosche added an answer:1Has anyone worked with Mannitol in a pig or other large animal model for blood brain barrier disruption?
I'm trying to use Mannitol as a positive control for blood brain barrier disruption in a piglet model. I've seen a lot of work done in rat but that involved cervical artery catheterisation and large volumes of drug. I would prefer to do IV or subcut but am not sure on the dose required.
Any experiences working with mannitol would be greatly appreciated.
I.V. Mannitol was and is still used to temporary "prevent" BBB disruption in large (or so called malignant) hemisphereic stroke or in other neurocritical diseases with BBB break-down and brain swelling (long-term outcome changes were disappointing). It works only temporary to decrease clinical makers like ICP most likely by its onkotic pressure (so in Neuro-ICUs it is sometimes life-saving). Therefore, I.V. injections may be critical for your purposes to disrupt the BBB. However, Paracelsus is calling: It all about the dose.
I.P. can not work at all, because mannitol as a large molecule should not enter the circulation that good. It is rather made for not crossing endothelial barriers.
To disrupt BBB with mannitol you may need rapid changes of mannitol and thereby the onkotic pressure in the brain cirulation, then mannitol have a paradox effect leading to an "opening" of BBB. That means I.A. mannitol injection or as a compromise I.V. injection is the only way to bring it to the brain circulation in a rapid and higher dose.
I hope these information will help a bit for planing your experiments.
Bert Bosche, MD/PhD
Specialist in Neurology, Neurocritical Care,
Neurosurgical Research Fellow - Post-doc Scientist
in the Laboratory of Prof. Dr. R. Loch Macdonald
Division of Neurosurgery
St. Michael's Hospital and Li Ka Shing Knowledge Institute
209 Victoria St., Room 517
Toronto, Ontario, Canada M5C 1N8
Phone: +1 416-864-6060 ext. 77641
Mobil: +1 647-204-4757 or +49 173-7076734Following
- Nicolai E. Savaskan added an answer:7What is the difference between BV2 and N9 microglial cell lines?.
BV2 cells are more common although this says nothing about the suitability for assays. We can provide you with both such cell lines.
- Radim Brixi added an answer:11How consciousness actually worksAs the basis for further discussion, I would like to suggest several of my articles proposing a new paradigm of the physics underlying cyclic cosmogenesis... With potential consciousness (awareness, will, qualia) being the fundamental subjective nature of unconditioned pre-cosmic absolute (0°K) space.
The underlying "holonomic" (ref: Bohm, Pribram) "Astro-biological coenergetic" (ABC) fractal field theory offers logical proof why experiential or phenomenal consciousness cannot be an epiphenomena of the brain or its neural processes... And concludes that the working mind and long term memory are independent, radiant higher frequency phase order sub quantum harmonic hyperspace standing wave fields (that are resonantly interconnected with each other and the brain's overall EM field) — which carry the 3D information of conscious as wave interference patterned holograms on their 2D surfaces.
This information is detected, reconstructed holographically, and perceived by zero-point consciousness (located at the ZPE center of the harmonic hyperspace fields) by means of projection and reflection of coherent radiation... e.g., visual consciousness is located at a zero-point in the pineal gland source of the brain's EM field (as well as its sleep and mood inducing hormones). All other sensory consciousness is non locally perceived at zero-points in the center of particular cells located throughout the body, and linked perceptively to the global center of individual physical self consciousness (located in the naval plexus) by BEC entanglement of ZP space. The focal points of higher states of consciousness are located at ZP centers of the six other chakra fields along the central axis of the body.
The articles are published (with illustrations) at: http://knol.google.com/k/how-it-all-began# (ABC theory) and at: http://www.jcer.com/index.php/jcj/article/view/85 (PDF)
http://leonmaurer.tripod.com/ (web reprint of JCER article)
There are many states of consciousness. Some states seem not to need brain or body.
- Michael Hunter added an answer:12Why does gray matter volume increase and decrease?
Does anyone have an idea why sometimes gray/white matter increase over time due to skill learning, and sometimes they decrease?
I've heard people say that maybe it decreases because the structures are becoming more efficient, but do we know what distinguishes or predicts increase versus decrease due to learning?
Cognitive and neuroscientific research over the past 60 years has shown that cognitive abilities, and their underlying neural architectures, change depending upon the experience of learning itself (Raisman, 1969; Cicchetti and Blender, 2007).
Here is another paper/citation I hope you find useful:
Driemeyer, J., Boyke, J., Gaser, C., Büchel, C., & May, A. (2008). Changes in Gray Matter Induced by Learning—Revisited. PLoS ONE, 3(7), e2669. http://doi.org/10.1371/journal.pone.0002669
Recently, activation-dependant structural brain plasticity in humans has been demonstrated in adults after three months of training a visio-motor skill. Learning three-ball cascade juggling was associated with a transient and highly selective increase in brain gray matter in the occipito-temporal cortex comprising the motion sensitive area hMT/V5 bilaterally. However, the exact time-scale of usage-dependant structural changes occur is still unknown. A better understanding of the temporal parameters may help to elucidate to what extent this type of cortical plasticity contributes to fast adapting cortical processes that may be relevant to learning.
Using a 3 Tesla scanner and monitoring whole brain structure we repeated and extended our original study in 20 healthy adult volunteers, focussing on the temporal aspects of the structural changes and investigated whether these changes are performance or exercise dependant. The data confirmed our earlier observation using a mean effects analysis and in addition showed that learning to juggle can alter gray matter in the occipito-temporal cortex as early as after 7 days of training. Neither performance nor exercise alone could explain these changes.
We suggest that the qualitative change (i.e. learning of a new task) is more critical for the brain to change its structure than continued training of an already-learned task.Following
- Sean Patterson added an answer:4What is the best way to do a synaptosomes preparation from Postmortem Human Cortex?
Dear all, I am trying to do a synaptosomes preparation from Postmortem Human Prefrontal Cortex (frontal). I read few articles, they used 1 to 5 gram at the beginning. I started with low amount of frozen brain, around 300 mg (PMI < 4 hours). From 300 mg weight of prefrontal cortex, I generally obtain ~25 micrograms of total protein from isolated synaptosomes.
Is anyone have any idea to increase the yield at the end (beside increasing the weight of the brain)? I do not have a high amount (weight) of human brain.
Each minced prep is immediately homogenized by applying 20 slow stokes using a teflon-glas tissue grinder (grinding chamber clearance is 0.15 mm). Then I use layering of discontinuous sucrose gradient.
I am thinking to use a glas-glas tissue grinder (grinding chamber clearance is 0.025 - 0.076 mm).
I welcome any idea.
I've never heard of anyone preparing synaptosomes from chemically-fixed tissue and, frankly, I doubt it would work. Certainly, the material you are purifying would need characterization by electron microscopy to ascertain its composition. After formalin fixation, even the enrichment of synaptic markers by Western blot would be problematic. I really don't think that your problem here is the homogenization step.Following
- Graham Peter Michael Burnett added an answer:32Why does music evoke emotions or feelings?Music has many bodily effects. This is not trivial.Following
- Khalid Eldahan added an answer:8How can I label brain hemispheres for free-floating histology?
I am running immunohistochemistry on rat brain tissue, and the sections will be stained while free-floating. However, we need to reliably know the left from right hemisphere. We cannot take a notch out of the cortex (which we have done previously), because we are taking a full survey of the brain.
Could someone recommend a method to label the hemispheres? Is there some sort of marker or pen that is visible to the eye but would not affect staining?
- Thank you -
I would use a little dab of lipophilic dye, like DiI:
Are you planning to use fluorescent IHC? If you do, just make sure that the dye doesn't overlap with the emission profile of the fluorophore you will use!Following
- Yoshiyuki Numasawa added an answer:14Do you know any direct efferent projections to cerebral cortex from pontine nuclei other than cerebellar cortex?
The neurons in the pontine nuclei have the axons which project to the purkinje cells. But I don't know whether the pontine nuclei have the projections direct to CEREBRAL cortex or not. Does anyone have data or suggestion?
Thank you, Nikolaos
Yes, I'm also interested in those broadly projection neurons.Following
- Jeff S Davies added an answer:6Could stress increase proliferation in the hippocampus ?
All studies, to my knowledge, show that stress (whatever duration or kind of stressor) decrease proliferation, survival and neurogenesis. I was wondering if a stress paradigm applied to rodent could result in a transient increased proliferation in an attempt to "counteract" the effect of stress? Thanks in advance for your answers. :-)
Check this paper out. Good luck.
- Aleksandra Somogyi added an answer:4Why is Neuraminidase 1 not localizing to lysosomes in cerebellar granule cells?
I used an anti-Neu1 antibody in order to examine the localization of Neu1 in cerebellar granule precursor cells and wondered why it is not localizing to lysosomes as expected. So I used HeLa cells for positive control and there the localization is correct. Does someone know if there is a functional reason why it is different in my cell line? Or did someone else check this and has different results?
No, I haven't... but the other two enzymes that are usually in complex with Neu1, beta-galactosidase and PPCA, localize to lysosomes with this approach.Following
- Jill Miotke added an answer:6Can 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.
I also agree with Justin's comments. However, you don't mention how the DiI is being exposed to UV light--are you also using another stain, like DAPI/Hoechst for nuclei, or Fluorogold? I did find out the hard way that one should image the UV channel last when viewing DiI labeled axons in a tissue that has also had Fluorogold, but then I label a very small number of fibers in the tissue (on purpose). If you are also labeling with a second dye that is excited in the UV, be sure to acquire that channel last.
In addition to the lipophylic dyes available from Molecular Probes, there are also the NeuroVue dyes (available from Polysciences here in the US). A publication from Bernd Fritzsch is posted here on ResearchGate:
I also have a question for David--have you had good success with MP of DiI? We have been underwhelmed, to say the least, with the signal (it could be the thickness of our tissue, though). If you have any hints, I would greatly appreciate hearing them.
Best of Luck,
- William Nickell added an answer:16What is the result of collision of action potentials: annihilation or penetration?
In axons, action potentials can move both in ortho-dromic (normal) direction as well as in anti-dromic direction, if stimulated in the right way. But what happens if two action potentials are generated simultaneously, one in the distal axon end and one at the soma, that are moving towards each other to collide? Will they penetrate (move past each other) or annihilate?
According to classical Hodgkin-Huxley model and theory of neurophysiology they will annihilate due to the in-activation of the sodium conductance. This effect has also given rise to the experimental method called "the collision test", which is used to confirm axon projection from one brain region to another by means of antidromic stimulation.
Nevertheless, a recent paper claims that two colliding action potentials will penetrate just as two colliding waves on a sea of water:
My question: Does anyone know the original literature about collision of action potentials? This must be back in the 1950'ties or 1940'ties. Who did the investigation and what are the publication references? I have been trying to find the original papers, because I am sure that scientist investigated this back in those days. The only one I could find was this:
I. Tasaki, Collision of Two Nerve Impulses in the Nerve Fiber, Biochim. Biophys. Acta 3, 494 (1949).
I'm going to jump in here with a quick comment:
One of the authors (Heimburg) has been pushing a crank theory that the HH model is wrong and that action potentials are actually 'solitions' (sound waves). I have collected objections here: http://www.quora.com/Are-nerve-impulses-solitons
Collision annihilation is, of course, a routinely observed phenomenon. There are few papers after 1949 simply because there is no question to be investigated.Following
- James Park added an answer:15Has anybody struggled with a sudden difficulty picking cells from tissue using LCM, even though protocols and equipment have not changed?
As of late, I am having trouble picking any cells from brain tissue slices using immuno-LCM techniques. We have successfully picked single and pools of single cells from brain tissue up until recently. Now it seems that we are consistently unable to pick up any cells. Nothing significant has changed with respect to the equipment, protocol, or reagents (to my knowledge). I have been troubleshooting for a while now and am nowhere closer to any sort of solution for this mystery. We use the following protocol for tissue sectioning and immuno-staining:
slice OCT embedded brain tissue using cryostat at -20C (10um thick slices)
store slices on charged slides (for better adhesion)
store sections at -80C until staining and picking
we fix slides using ice-cold acetone (1min)
air-dry slides post fixation (~ 2-3 min)
block with 3% BSA solution
rinse with 1X PBS
add primary Ab solution (incubate for 3 min at room T)
rinse with 1X PBS
add secondary Ab solution (incubate for 3 min at room T)
rinse with 1X PBS
dehydrate in a series of EtOH solutions of increasing conc. (70%, 75%, 95% 100%, 200%)
final dehydration in xylene (100% 1 min, 100% 4 min)
air dry and store in 50mL tube with dessicant for 10-20 min
I have tested a variety of factors targeting various aspects our protocol:
longer acetone fixation times (5-10min), room T acetone (2min), longer EtOH baths (from 30s to 2 min), longer xylene baths (5min-10min), used new reagents (unopened bottles), new caps, uncharged slides, longer dessicant drying times (as long as 45 min), and have adjusted environmental conditions as best as I can in the LCM room (relative humidity ~mid-30%, which we have been able to pick cells successfully in the past). The IR-laser is able to wet the caps fine as well. We are able to pick up cells from older tissues that have been used in the past, so we do not suspect an issue with the LCM itself.
At this point, my colleagues and I believe it has something to do with the dehydration of the tissue (we note that in tissues that yield successful microdissection, they are typically opaque and white upon fixation and/or xylene dehydration). However tissue that prove difficult to pick from are not nearly as opaque (more a translucent white - faded). I would appreciate any insight from the community as to how we can improve consistency in tissue dehydration and/or techniques that will improve success of tissue microdissection. Any insights or thoughts would be helpful, thank you!
Roberto - thanks for taking the time to post your protocol. I'll take a look and hopefully it'll offer clues as to what may be affecting capture efficiency here on my end. Thanks again!Following
- Fengling Li added an answer:10Any suggestions on the abnormal activities of V1 neurons in PV-Cre mice expressing ArchT ?
Recently, I discovered that there were some abnormal activities of V1 neurons in mice when archaerhodopsin(ArchT) was seletively expressed in parvalbumin(PV)-expressing interneurons.
Spontaneous local field potential of V1 in PV-Cre mice expressing ArchT were different from both in widetype C57BL/6J mice and SOM-Cre mice expressing ArchT(see the picture attached). This kind of abnormal activity seems not effect on basic response of V1 neurons to visual stimuli, that's to say, receptive field and orientation tuning could be getted.
But, after the V1 cortex was exposed in green laser for sometime, spontaneous local field potential of V1 without laser will exhibit another kind of abnormal activity(also see attached picture). That seems that laser effect V1 neurons irreversibly, inconsistent with the reversible effect of optogenetics. And visual stimuli can aggravate this kind of abnormal activity, which seriously disturbing response of V1 neuron to visual stimuli, even that you can not get the receptive of V1 neurons. It's less happened in SOM-Cre mice expressing ArchT
Is it happened in your experiments? Do you know why this happen? And how to improve the situation?
Look forward to your reply
We didn't inject virus into C57 mice. LFP of widetype C57 was just a blank control.
And now we haven't detailly distinguish the type of the cells we recorded. We assume that LFP mostly results from excitatory neurons, because excitatory neurons are much more than inhibitory neutons. Apparently, two-photon guided recording is the best way to distinguish cell types. But our lab lacks relevant instruments now.
Whole cell recording in slices is a good advice, Thanks a lot .Following
- John N J Reynolds added an answer:3Has STDP been implemented directly in rate neurons?
STDP (spike timing dependent plasticity) is a learning rule for changing the synaptic weights between neurons. It is Hebbian-like in that the weight changes depend on the coincidence of pre- and post-synaptic spikes, but it includes a notion of causation in that the synapse is strengthened if a presynaptic spike precedes a postsynaptic spike, but the synapse is weakened if a postsynaptic spike precedes a presynaptic spike.
Rate neurons are often used in simulations because they are computationally cheap compared to simulating spiking neurons. In order to implement STDP, spikes are generated using an inhomogeneous Poisson process with the rate parameter defined by the current rate of the rate neuron.
What I would like to know is whether a shortcut has been developed to calculate the weight changes due to STDP directly from the temporal dynamics of the pre- and post-synaptic weights without having to explicitly generate spike times through a poisson process.
Have you looked at the Izhikevich model, which uses a spiking network of 1000 cortical neurons with dopamine-modulated STDP to adjust synaptic weights? (Cerebral Cortex doi:10.1093/cercor/bhl152). Apologies of you are already aware of this and it does not help with your question (I'm not a modeller) but it certainly makes good inferences for the need for causality of pre and post synaptic activity in a sea of slower timescale reward events.Following
- Diego Fernández added an answer:13How can I get stable and perdurable LTD in hippocampal slices from adult mice?
I have seen through publications that some labs have been routinaly able to induce very long-lasting in vitro LTD in young and adult animals. I would like to ask you if you can give me some details of the methods to achieve it.
I am trying to get LTD in my hippocampal slices from ~10 weeks old mice, but so far it has been almost impossible. I have been using both electrical stimulation (single 900 pulses at 1 Hz) or the application of mGluR2/3 agonist LY354740. I am stimulating the medial perforant path and recording fEPSPs in the dentate gyrus, at room temperature.
Does anybody have some advice to get stable LTD?
Thank you very much in advance for your contributions.
Thank you everybody for all your comments.
It seems clear that slice health is a critical factor for the induction of LTD, so we will focus on improving our slice preparation and also we will modify our setup to be able to work at more physiological temperatures, following your advice.
These contributions and the papers you've suggested have been very helpful.
Thank you all so much again.
- Erick Arguello added an answer:1Does anyone know how long can squid giant axon survive in sea water for Na/K pump study?
we are trying to study Na/K pump on squid giant axon. But can not fish one in florida during summer time. So we are trying to get one from Woods Hole Marine lab. Just wonder how long it can survive in the sea water via shipping?
Please, see the attachment. For further information, refer to: Gilbert, D.L; Adelman, W.J; Arnold, J.M. "Squids as experimental animals". The attached file was extracted from this book.
I hope it helps! (Please uovote if you liked)