Science topic

Neurochemistry - Science topic

The study of the composition, chemical structures, and chemical reactions of the NERVOUS SYSTEM or its components.
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Tell me the literature or articles where you can find the neurochemistry of forgiveness (which systems work at the same time)
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Acceptance, gratitude, ocean-like deep and broad mind, ability to rise above colour of skin and religion, a cosmic unconfined spiritualty, an unfurling of a deep-consciousness, a persona beyond ego, egotism, and narcissism, ability to acknowledge error and learn incessantly and much more is required to learn the art of forgiveness. Forgiveness is Divinity on Earth and beyond. Neurochemistry of forgiveness is but one component that makes the wholistic concept of a compleat (no typing error) human. Forgiveness deftly and more commonly grudgingly overcomes the stress-response and imparts an out-of-this-world or Universe experience -- a learned response that carries the frailty of life and breath to another Dimension even while on Earth. 03-May-2024. ORCID iD: 0000-0002-6770-5916
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I now that alot of artificial networks has appeared now. And may be soon we wil not read articles and do our scientific works and AI will help us. May be it is happening now? Wat is your experience working with AI and neural networks in science?
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Artificial intelligence definitely could help in neuroscience due to the fast development of AI nowadays. During the coronavirus period, AI helped in fast genome sequencing, and consequently, very fast vaccines have been developed. Similarly in neuroscience requirement for real-time analysis during the treatment of neuroscience patients to find out new proteins and genes for particular functions and diseases also.
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I analyzed the internal standard "2-methyl-L-cysteine hydrochloride" using HPLC and the chromatogram shows three peaks instead of one which is weird!!
I repeated the preparation process two times to check if there is a contamination problem but the chromatogram still showing three peaks. So what do you suggest?! Is there a possibility that the internal standard is converting to other compounds?!
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Dear Rawaah Y. Al-sharrab,
Thank you so much for your interest. Actually, it is very difficult to solve the problem of internal standard. That is why, most of the researcher are now developing their method using external standard and to use matrix matched standard for avoiding the use of internal standard. Anyway, if you interested to work with internal standard, try to use the solvent as same as mobile phase, it may helps to minimize the problem. I read the answer made by
William Letter, he described many things, you may follow his advice. Thank you.
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I would like to scan calcium-indicator-dye-loaded neurons after fixation, just to know, which cells have been loaded. Fura and Carbodiimide fixation have been reported to retain fluorescence but I prefer paraformaldehyde fixation for several reasons. Is there a single calcium dye, which is not quenched after paraformaldehyde fixation?
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May I ask if you continue your experiment and find a good agent? I am also thinking about fixing my cells on a coverglass right after treating with Fura-2 AM and I am wondering if fixation processes by 4% PFA may effect the membrane permeability and cause Ca2+ fluorescence to be lost.
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The incredible thing about Physarum polycephalum is that whilst being completely devoid of any nervous system whatsoever (not possessing a single neuron) it exhibits intelligent behaviours. Does its ability to intelligently solve problems suggest it must also be conscious? If you think, yes, then please describe if-and-how its consciousness may differ {physically or qualitatively ... rather than quantitatively} from the consciousness of brained organisms (e.g., humans)? Does this intelligent behaviour (sans neurons) suggest that consciousness may be a universal fundamental related more to the physical transfer or flow of information rather than being (as supposed by most psychological researchers) an emergent property of processes in brain matter?
General background information:
"Physarum polycephalum has been shown to exhibit characteristics similar to those seen in single-celled creatures and eusocial insects. For example, a team of Japanese and Hungarian researchers have shown P. polycephalum can solve the Shortest path problem. When grown in a maze with oatmeal at two spots, P. polycephalum retracts from everywhere in the maze, except the shortest route connecting the two food sources.[3] When presented with more than two food sources, P. polycephalum apparently solves a more complicated transportation problem. With more than two sources, the amoeba also produces efficient networks.[4] In a 2010 paper, oatflakes were dispersed to represent Tokyo and 36 surrounding towns.[5][6] P. polycephalum created a network similar to the existing train system, and "with comparable efficiency, fault tolerance, and cost". Similar results have been shown based on road networks in the United Kingdom[7] and the Iberian peninsula (i.e., Spain and Portugal).[8] Some researchers claim that P. polycephalum is even able to solve the NP-hard Steiner minimum treeproblem.[9]
P. polycephalum can not only solve these computational problems, but also exhibits some form of memory. By repeatedly making the test environment of a specimen of P. polycephalum cold and dry for 60-minute intervals, Hokkaido University biophysicists discovered that the slime mould appears to anticipate the pattern by reacting to the conditions when they did not repeat the conditions for the next interval. Upon repeating the conditions, it would react to expect the 60-minute intervals, as well as testing with 30- and 90-minute intervals.[10][11]
P. polycephalum has also been shown to dynamically re-allocate to apparently maintain constant levels of different nutrients simultaneously.[12][13] In particular, specimen placed at the center of a Petri dish spatially re-allocated over combinations of food sources that each had different protein–carbohydrate ratios. After 60 hours, the slime mould area over each food source was measured. For each specimen, the results were consistent with the hypothesis that the amoeba would balance total protein and carbohydrate intake to reach particular levels that were invariant to the actual ratios presented to the slime mould.
As the slime mould does not have any nervous system that could explain these intelligent behaviours, there has been considerable interdisciplinary interest in understanding the rules that govern its behaviour [emphasis added]. Scientists are trying to model the slime mold using a number of simple, distributed rules. For example, P. polycephalum has been modeled as a set of differential equations inspired by electrical networks. This model can be shown to be able to compute shortest paths.[14] A very similar model can be shown to solve the Steiner tree problem.[9]"
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To bardzo trudne pytanie.
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Background:
Professional-school student with extensive ACE (child adverse events) history along with severe depression and anxiety diagnosed over previous year, presented with recent severe ADHD (I-Type) diagnosis at age 26.
Documentation confirmed maximum dose step therapy for various Amphetamine-based stimulants was completed but still not found to be fully affective.
Unexpectedly, they are currently prescribed daily 50mg Mydayis (Mixed salts of single-entity amphetamine product) along with 80mg Prozac, and consumming 300-400mg of caffeine.
Due to initial medication-only use producing very minimal stabilizing effects, but found to increase at re-introduction of SSRI and further increase with Caffeine reintroduction.
No adverse effects (cardiac, neuromuscular, neurocognitive) have been reported/measured in 4 months of aforementioned therapeutic combination.
NOTE: Adverse reaction to methylphenidate-based medications were identified early on.
Assessment of (remaining) presenting symptoms seems to overlap with tentatively defined SCT Criteria.
NOTE: Student has never been prescribed Strattera (only presently confirmed SCT-symptom relief medication)
Specific question:
Recent research has shown SCT + ADHD to correlate with much greater impairment in adults, do you think a combination of severe ADHD + SCT may result in required use of excess pharmacotherapy dosages that surpass established safe therapeutic/combination parameters?
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Remember both PTSD and major depression can produce significant cognitive impairment including issues with attention equal to ADHD.
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Hi everyone ^_^
I was wondring if there is a specific technique to study the chemical change responsible for DID and I want to know if I could follow a specific procedure to the neurochemicals behinde this disorder and how the alteration vary between a normal person and a DID patient? also, the most important thing is finding a specific drug to cure this disorder. I know that there are drugs that help the patients endure anixty and depression come with this disorder but there is no direct and specific cure for DID.
I read that contextual treatment helped the patient somehow to know themselves and some of them return the control and can change from one personlaity to the other when they want but it didn't work for most of them although it benfits them somehow but not cure them. So; I read a lot of artiles and reviews from the litreture but most of them focused in the contextual treatment not chemical based treatment!!
I don't know if this is right or not but I think this return to neuropsychopharmacology and I'm a chemistry master student so I want to know if there is a possibility to study this in my thesis project and if I need a high tech equipments to do that?
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If you want to specifically measure chemicals in the brain, then you cannot do it when the subject was alive. Psychiatry is still clouded in mystery, as we cannot objectively measure many things we theorize. It was likely easier to measure brain wave or structural abnormalities from X-ray or MRI. However, even those do not provide reliable outcome, as the changes vary depending on the individuals and specific cut off to determine which is normal and which is not is still debatable. I'd like to brief you about the difficulty faced when measuring chemicals, hoping that one day any one can overcome it.
When we measure chemicals in the brain, we cannot use chemicals in the blood. The problem is because there is a tight clutter of cells preventing large chemicals to pass through, which is termed blood brain barrier. As neurotransmitter (neurochemicals in the brain) cannot pass through this junction, even if we measure it's concentration in the blood, it's hard to make connection with it's level in the brain. It might even be possible that the level in the blood increases while the level in the brain decreases. There are already methods to measure neurotransmitter in urine as representative of it's concentration in the blood, but it's very much debatable as there is no reliable argument of how it reflects anything in the brain.
The second problem is that the neurotransmitter in the brain mainly exists inside neurons (neural cells). When a neuron is active, it would then release neurotransmitter to the next neuron to pass electrical signals. It was a very quick release through junction between neurons, which is called synapse. Once the neuron goes inactive, the excess neurotransmitter in the synapse get recollected (reuptake). Even if we measure the neurotransmitter inside the neuron, it might not be a good clue about how much the chemical is used during neuron activation. And measuring it when it was in synapse for a very brief time is quite an obstacle. Instead, we may be able to measure the amount of neurotransmitter receptors the neuron has. As receptor is necessary during release of neurotransmitter, the increasing amount of receptors reflect the increasing activitty of neurotransmitter. However, receptor is not soluble and remain attached to the neuron. One of the way to visualize it is by sending "dummy neurotransmitter" attached with radioluscent materials visible in X-ray or MRI (so that they attach to the receptors and we can see how much receptor is there). but even then, the result is variable as we mostly use animal study, which may or may not have psychiatric disorder (animal model is yet another obstacle as we cannot diagnose animal with interview).
The third problem is that many neurons use the same neurotransmitter, while they may convey completely unrelated information. Let's assume that we see three neuron and name it neuron A, neuron B, and neuron C. Increase of total amount of neurotransmitter in a single place does not necessarily mean all individual pathways got more active. Even if certain neurotransmitter increases by two-fold the concentration, it doesn't mean the activity of neuron A is increased as well. And each of these three neurons may govern completely different pathways and completely different inputs, even when the target area is close by.
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The Elevated Plus Maze Model (EPM) is basically tested for the anti-anxiety activity. Currently, I am working on learning and memory activity in rats by the Barnes Maze and EPM test. Is the EPM reliable for nootropic activity? whether the electrophysiology of hippocampus part of rats brain suitable or not to know the mechanism of action for tested drugs?
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Our linear maze is a good instrument for memory tasks. I attach a picture in my paper (page 125). All measures are added. Ope4en-field is also pictured and it is is a good apparatus for fear and anxiety:
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Does anyone know how/if its possible to measure erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) in living humans?
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آسف السؤال ليس من اختصاصي
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Currently doing a research project for my Honor's Chemistry class, lately I've been looking into Brain Machine Interfaces on the side for research later on this summer, however, I was hoping to find a relationship between BMI's and chemistry so that I can use this project as an addendum for my internship.
The scope of the project is essentially on current research in chemistry, or historical importance of chemistry.
that being said I'm aware there is some chemistry involved in BMI's however, I can't find anything to specifically research on.
Any ideas or advice?
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Bump
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GABA can be released via GABA Transporter (GAT) through reversion of this transporter. But how this 'reversion' works? In which situation this transporter starts to release (and not uptake) GABA? Is the mechanism described?
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Hi SIlva,
Please see the following link. I hope it will be useful to answer your question.
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Can systems dynamics (e.g. Braun, 2001) and Fisher’s account of temperament (Fisher et al., 2015; Brown et al., 2013), be used to explore the "emergence" of values? For example, a child developing in a setting which rewards and reinforces curiosity and creativity might become increasingly sensitised to feelings associated with their dopamine system (Fisher, below), and subsequently seek experiences which offer that particular feeling of reward. This might establish a reinforcing cycle (Braun, 2001), through which the child’s temperament becomes predominantly expressive of the dopamine system. From Schwartz’ human values perspective would this child’s goal choices tend to consistently express openness to change, independence and self-direction values? Would the child’s choices be values-based insofar as they are infused with feelings, refer to desired goals, and exhibit consistency that transcends specific situations (Schwartz, 2012)?   
Might Fisher’s Explorers express Schwartz’ Openness To Change values, Builders express Conservation values, Directors express Self-enhancement values, and Negotiators express Self-transcendence values?
Might accounts connecting neurochemical systems with values and feelings (e.g. Lövheim, 2012) improve the utility of the worthwhile, satisfied, happy, anxious and social trust dimensions in the subjective wellbeing literature (e.g. Michaelson et al., 2012) and enrich the goal choice (e.g. Knafo and Sagiv, 2004) and productivity literatures (e.g. Parks and Guay, 2009)?
Fisher Four broad temperament dimensions:
·         Explorers expressive of the dopamine system linked with traits including: being curious, creative, spontaneous, energetic, risk-taking, novelty-seeking, mentally flexible.
·         Builders expressive of the serotonin system linked with traits including being: traditional, conventional, following the rules, respecting authority.
·         Directors expressive of the testosterone system linked with traits including being: analytical, logical, direct, decisive.
·         Negotiators expressive of the oestrogen system linked with traits including: being empathetic, emotionally expressive, good with people.
Schwartz (2012) values super groups:
·         Openness to change values: emphasise independence of thought, action, and feelings and readiness for change (self-direction, stimulation)
·         Conservation values: emphasise order, self-restriction, preservation of the past, and resistance to change (security, conformity, tradition)
·         Self-enhancement: emphasise pursuit of one's own interests and relative success and dominance over others (power, achievement).
·         Self-transcendence: emphasise concern for the welfare and interests of others (universalism, benevolence)
References
Braun, W., 2001. The Systems Modelling Workbook. Berlin: Springer, 2001. [Online]. [Accessed 19 January 2017]. Available at http://www.albany.edu/faculty/gpr/PAD724/724WebArticles/sys_archetypes.pdf
Brown, L.L., Acevedo, B. and Fisher, H.E., 2013. Neural correlates of four broad temperament dimensions: testing predictions for a novel construct of personality. PloS one, 8(11), p.e78734.
Fisher, H.E., Island, H.D., Rich, J., Marchalik, D. and Brown, L.L., 2015. Four broad temperament dimensions: description, convergent validation correlations, and comparison with the Big Five. Frontiers in psychology, 6.
Lövheim, H., 2012. A new three-dimensional model for emotions and monoamine neurotransmitters. Medical hypotheses, 78(2), pp.341-348.
Knafo, A. and Sagiv, L. 2004. Values and work environment: Mapping 32 occupations, European Journal Of Psychology Of Education, 19 (3).
Michaelson J., Mahony, S. and Schifferes, J. 2012 Measuring Well-being: A guide for practitioners, London: New Economics Foundation.
Parks, L.,and  Guay R. 2009. Personality, values, and motivation, Personality and Individual Differences, 47, pp. 675–684
Schwartz, S. H. (2012). An Overview of the Schwartz Theory of Basic Values. Online Readings in Psychology and Culture, 2(1). http://dx.doi.org/10.9707/2307-0919.1116
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Hi Glen, sorry that I missed your question.
Thank you for this thoughtful and thought provoking proposal.
Yes, i think that temperament should provide a basis to some of the variation in values. I will update as we progress with this part of the project.
Ariel
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Hi everyone,
I have an Amyloid beta 1-42 fragman, but i dont know how can I prepare this. In water or another liquid. There are not enough paper in web about that. I asked to many people, but they did not give an answer clearly.
I want to inject AB 1- 42 into the brain parenchyma. 
Is there any advice?
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Hi Canan, 
You need to solubulize the peptide in hexafluoroisopropanol and dry it as films. These films can be reconstituted in DMSO followed by PBS or water. For more details see this paper. 
W. L. Klein, A[beta] toxicity in Alzheimer's disease: globular oligomers (ADDLs) as new vaccine and drug targets. Neurochemistry International 41, 345 (2002)
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What is the normal range NT-proBNP using ELISA kit in the healthy adolescents?
Reference. please
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2.00-4.00 or 200-400 ?
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Dear all,
I am currently working with ghrelin (a gut peptide) and I would like to check if the peptide crosses the blood brain barrier and if it is possible to find out in which areas it reaches. I know that the question is a bit general, but I've been puzzled, since there are no previous studies investigating that question and there are only some hypotheses out there which don't seem to help me. Ideally, I would like to somehow trace "the flow" of the peptide through the BBB and its distribution across the brain areas. 
Any ideas or suggestions would be highly appreciated!
Thank you all in advance.
Regards,
Lydia
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You could fluorescently label the peptide with a small fluorophore, such as FITC. There are a number of commercially available kits which you could buy to do this. Then administer the peptide or perform in situ brain perfusion and fix the brain with paraformaldehyde. You can then look at its distribution using fluorescent microscopy. You can check whether the peptide reaches in an in tact form or not by running a brain homogenate from an unfixed brain on a gel. If you see the labeled peptide at the correct molecular weight this would indicate it reached the brain in tact.
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For research purposes
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You could try contacting the researchers who introduced the compound:
Hedlund PB, Leopoldo M, Caccia S, Sarkisyan G, Fracasso C, et al. (2010) LP-211 is a brain penetrant selective agonist for the serotonin 5-HT(7) receptor. Neurosci Lett 481: 12–16 10.1016/j.neulet.2010.06.036 [doi]. doi: 10.1016/j.neulet.2010.06.036
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It has been known for a long time now that cotinine, a metabolite of nicotine, is a nootropic and more importantly, is somewhat neuroprotective for PD and AD.  It is also known that cotinine is well tolerated by human subjects and has none of the negative side effects of nicotine itself.  Given today's great concern regarding both PD and AD, how can it be that research on this compound, very closely approximating a "silver bullet" if you will, is not being conducted with great intensity by NIH and/or the pharma industry?  Why does this seemingly magical solution to so many problems sit on the side while less practicable molecules get to dance?  
I don't get it. There is tons of research supporting this but not much is happening.  Why?
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Yes, Paul, I agree.  I was being a bit hyperbolic and put "silver bullet" in quotes for that very reason.  Thanks for the links; I read one of them previously.  I have read much on this and chatted with someone very active in research in this area.  The question still stands:  Why is this going nowhere?
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Brain tissue was homogenised in 25 mM Tris - 4 M urea buffer. After a freeze thaw cycle noticed that the tissue formed clumps. Which is not getting dissolved. Why it is so?
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Thank you for your valuable suggestion Markus.
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Just recently, features of psychiatric disorders such as schizophrenia were suggested to be due to a decrease of brain memory center’s inhibitory neurons. HSV-infection is primarily limited to mucosal epithelial cells and neurons, and autophagy is critical in antiviral defense in neurons. Given that type I IFN treatment failed to completely block HSV-1 replication and induce cell death in neurons, some xenobiotical factors might be considered triggering replication of neurotropic HSV-1, and lead to a loss of inhibitory neurons.
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So selective neural damage, mediated by activation of HSV1, would be triggered by an environmental toxin? Interesting idea, but in terms of translational research, you may have a long road ahead... HSV1 has been suggested to be the culprit in many neurological and psychiatric diseases, but so far the evidence is anything but convincing. On the other hand, there is no denying that HSV1 works in mysterious ways http://www.neurology.org/content/83/21/1888.short
J
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As we know, microdialysis is an acceptable method for detecting the concentration of many metabolites, such as lactate, glucose, glutamate etc. in human brains. However, I wonder weather this technique shrank the true value for which the taken interstitial fluid was diluted in this process. Thus, could you provide some evidences to clarify the true value of C. lactate in human brain? 
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Why would you want to use an invasive method such as microdialysis to measure lactate levels in human brain when it can be measured non-invasively using magnetic resonance spectroscopy?
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Orch-OR theory for consciousness asserts that the microtubules are the neural structures that support the quantum effects. Let's assume that it is true. Therefore, if they have to play a role in the brain, they need to effect the signal transmission in the brain. Is there any indication for such an effect?
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Dear Abolfazi,
Indeed microtubules are important in function of ion channels and neurotransmission. I think most importantly the microtubules are involved in transport of proteins (including ion channels), neurotransmitters/modulators, organelles (including mitochondria) from soma to the periphery. This microtubular role is very critical for axonal transport of neurotransmitters to the pre-synaptic terminal, without it the terminal will not have enough neurotransmitter and energy required for synaptic transmission. Anything that disturbs or reduces microtubular function results in significant neuronal dysfunction.
best wishes,
Refik
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I used neutrophil elastase antibody to stain brain sections and got a lot of background staining. I used 3% H2O2 and 2% serum blocking.
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If you are using FFPE, you may also want to play around with your slide prep. Many commercially available elastase antibodies don't do well with heat-retrieval as the epitope is degraded. Citrate-retrieval has also been reported to work better with many NE antibodies in FFPE. Good luck!
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Hello!
I am looking to utilize ifenprodil in slices of rat cortex.  I have read that it can also act on α-adrenergic receptors which I wish to avoid.  Most work seems to utilize 0.1-10 micromolar, and I see EC50s of approx. 0.3 micromolar.  Does anyone have experience with using ifenprodil in slices and have a recommendation of a selective concentration? 
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I had used 2.5 μΜ of ifenprodil to block NR2B-containing NMDA receptors in adult mouse slices. I wouldn't recommend going too high with the concentration, as it might act on other receptors as well. Which receptors do you wish to block exactly?    
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I have had this ICC working for several years and have used it for multiple experiments, but recently it has stopped working. I am now having nearly every cell in the brain labeled in every trial I do. I initially tried ordering a new lot of the primary antibody, but that made no difference. I have modified the concentration of the secondary and the time in the DAB and neither of those helped. I tried doing a no primary control to rule out nonspecific binding of the secondary and a no primary or secondary control to rule out ABC binding to endogenous biotin in the tissue. Both of those trials came out clean, with no staining, suggesting that the problem is not with the secondary or the ABC. We have tried replacing the paraformaldehyde (our brains are not perfused, so fixation is the first step of our IHC), the methanol, the H2O2, the normal goat serum, and the DAB tablets and none of these things have solved the problem. We also thought that contamination in the water may have been the problem, but I got the same result when using all solutions made with HPLC grade bottled water. Finally we thought there may have been a problem with the tissue since it had been in the -80 freezer for 2+ years and may have experienced temperature variation, but I recently included sections from a brain that was collected and sliced within the previous week and that did not solve the problem.  I have run out of ideas for what could be causing the excess staining.  Does anyone have suggestions on what I could try?
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The steps you are following to identify the culprit are all right. Write down a flow chart of the problems. Some suggestions for it: Are you getting the same high background when using other atbs? If NO, follow recommendation from Srinivasan, above. If YES, go to a colleague who is now running (successful) IHC and repeat your experiment in his/her setting. If GOOD results there, start replacing reagents, one by one, etc.Good luck
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I need to know where the cholinergic afferents that reach the temporal side of the perirhinal cortex are coming from. Thanks.
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In what species?
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Dear expert,
I need some works about diffusion mechanism (How molecules are transferred from blood into the brain tissue) in the brain .or an up-to-date brain anatomy.
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Do not omit to read the studies of MW Brightman.
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Since, the primordial Na-pump on the plasma membrane is the ultimate source of energy of animal cells; it appears that the remaining Ca-signaling networks will join the leading Na-pump, and the potential energy is used by remaining networks for homeostasis. Hence, the cellular bioenergetics initiated and managed by the NaAF regulated Na-pump, by turn regulated by the provisional Ca-pump and Ca-signaling, is the leading-center of a cell’s holistic-networks.
We visualized operation of the ion-pump under Ca-signaling as an all-time allosteric dancing of the Na-pump.  During operation, the ubiquitous NaAF acts as the dancing-partner cum gate-keeper of the double-gated (two α-subunits) Na-pump allowing simultaneous transport of Na (in) and K (out), while the process is controlled by Ca-signaling. Intensity of the dancing will be affected by the isoform-nature of the Na, K-ATPase which varies in various bodily cells in a tissue-specific manner except brain where the Na-pump is area-specific. The extremely specialized functions of the area-specific brain (of human) are of prime interests now-a-days.
The Ca-signaling is conducted periodically by the provisional Ca-pump which is an altered form of the cell-specific Na-pump. The provisional Ca-pump is entrusted to pump out the excess (inhibitory) local Ca for continuation of Na-pump function for homeostasis, thus making it a stop-and-go type dance.
This brings us to the question we are asking. Are the molecular natures of the provisional Ca-pumps the same as the isoforms of the PMCA well-known in the vast Ca-ATPase literature?
It will be very interesting issue to investigate. 
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I think there is a misunderstanding, Bridget Bengtson. The Ca-pump (PMCA) is wellknown to be present in normal animal cells. however, we found that the PMCA reported in the literature is an altered form of the Na, K-ATPase (Na-pump) which acts as a provisional Ca-pump in high-Ca situation to remove excess Ca from the cell. 
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Can I know if I can detect calcineurin levels in brain of hamsters after prion infection in fresh sacrificed animal brain only or is it possible to detect levels after the freezing of brain sample for future analysis? I would like to use calcineurin activity assay kit.
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As long as you can preserve protein with out denaturation or proteolysis. You can use for the assay later. If you can do it immediately, always better!
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Hello, I am trying to find a source of Deoxynivalenol (vomitoxin) HRP conjugate and having no luck. I could make it, but am hoping to find a commercial supplier. Anyone ever come across this?
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Please Join Solu Link to get the method of preparation.
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The concentration of alpha synuclein is 25-100 micromolar and i use 800 rpm, 37 degree celsius. I am concerned about the sample volume (30 microlitres in 1.5 ml tubes) and whether agitation helps at small volumes.
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I've always wondered about the relevance of this. I mean, 800 rpm would have to be a pretty fast & continuous hula hooper. When we were working on synuclein, some 5-7 years ago, we used to use 250 rpm at 37C and had huge problems forming fibrils. Without agitation it would take a month to form fibrils and if you follow the reaction by 15N-NMR you can see synuclein usually starts to have all kinds of degradation products over that timescale (of course this would depend on purification scheme). It's pretty much why we worked on non-fibrillar states of synuclein and switched to other amyloids. We found a key issue in synuclein fibrillization is a boiling step in the purification where you heat the cell lysate to get rid of contaminating proteins. We did countless mass spec experiments (unpublished) to prove to ourselves nothing was happening to the covalent structure (like deamidation) but it made a huge difference in the speed of fibrillization whether the samples were boiled or not. Presumably heat was non-covalently disrupting some fibrillatization nucleus, and since these are nucleation reactions, it doesn't take much. I think others may have published on this more recently.
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I'm successful in separating the presynaptic and PSD fraction using a method that published in a Neuron article. I have some native gel running complication with the pre-synaptic membrane fraction as the gel goes bizarre. I found that the triton X-100 in the presynaptic fraction causing this while running native page gel. I even found the same while running in SDS/PAGE but I was successful after subjecting the presynaptic fraction to acetone precipitation.  But the acetone precipitated presynaptic fractions is not compatible to resolve you native proteome as acetone disturbs the lipid bilayer thus no longer the native condition of your membrane proteins maintained. At the separation step of presynaptic and PSD fraction we use 1% triton at pH 8. During this step the presynaptic fraction is resolved in the supernatant along with 1% triton. I used to filter (used 100 K esp for running native page also used 10 K to 30 K filters) and exchange the presynaptic fraction buffer composition (ph 8 to 7.4) to get rid of triton but instead it gets concentrated to form a micelle I persume so. How do I get void this triton from this fraction without affecting the nativity of a proteome? Does anyone have any idea how do I over this situation?  
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Actually I'm working on blue native but the problem I face is that the presynaptic fraction contains triton x-100. The presynaptic fraction is the supernant part which I get after separation. Therefore when I compare the presynaptic and the PSD fraction (is the pellet)  I have to maintain the equal amount of protein (Does it make sense to compare both the presynaptic membrane fraction and the PSD fraction?). But always I find that after staining the gel the presynaptic fraction is  less protein concentration than the PSD fraction despite that I get more more concentration OD value for the presynaptic fraction. How far dialysis would help me that I don't want to remove completely triton x-100 from the presynaptic fraction as I need to run blue native and also I need to compare it with the PSD fraction in which case we need to extract (extraction buffer will contain the detergent) the proteins from the pellet. But the Amicon filtration doesn't help me. 
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I’m working with a new Haas type interface chamber, called BSC-HT Brain Slice Chamber System Haas Top (Harvard Apparatus). The problem I encountered is as follows: after calibrating the perfusion, the slice doesn’t look dry, yet the fEPSP amplitudes facilitate during the recording (increases by about 400 uV in an hour).
The ACSF is saturated with carbogen and is at room temperature, carbogen is also directly flown into the chamber to produce carbogen-steam. I tried different perfusion speeds (1-4 ml/min), but it didn’t make significant difference. I previously worked with a similar Haas type chamber, and managed to overcome similar difficulties.
I wonder what may couse the facilitation in the fEPSP amplitude - any suggestion is welcome.
Thanks in advance.
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Gabor,
Like you and the others, I also observed as steady increase in extracellularly-recorded fEPSP amplitude, and PS amplitude, when recording for long duration from a single slice (several hours) in an interface-style recording chamber. Over this same period of time I observed that the slice changed from opaque to semi-translucent. This was not a matter of the slice 'drying out', which can occur when the electrode opening is too large leading to a tough 'skin' on the surface of the slice.
I suspected that there were structural changes in the slice to cause these effects, so together with Dr. Lew Haberly, a former colleague who is an excellent anatomist, we examined several such slices using an electron microscope. We found that the slice had indeed become much reduced in volume, primarily due to a contraction of the extracellular space. We therefore inferred that the size of the field potentials increased due to an increase in the extracellular resistivity. We did not measure the extracellular resistivity to confirm, so this remains speculation. When I modified the chamber to a submersion-style perfusion these changes did not occur.
Good luck to you!
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I've been using trizol to homogenize frozen nerves. I've pooled 2, 4, and 6 nerves and ran qPCR for GAPDH. The Ct values for all combinations were about the same (~23). So I used 2 nerves to run a data set, but this time my Ct values for GAPDH ranged from 26 to 31. I think I may just not have any RNA, and the readout of the RNA concentrations is something else. What protocols have worked for others?
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You can try to put RNA on agarose gel and perform clasical electrophoresis. You should see something like this https://www.promega.com/~/media/images/resources/figures/10900-10999/10950ta_334px.jpg?la=en
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I have been staining brain sections of humanized mice with a number of human microglial and macrophage markers but nothing is showing up. The same antibodies are positive for peripheral tissues. Has anyone looked at human macrophage markers in the brains of these mice. These mice are supposed to have human CD34+ CD45+ and CD68+ cells in the brain. But I have failed to see any of these yet.
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I doubt you'll see any human cells in the brain, especially macrophages (myeloid lineage development is not very robust in NSG mice). 
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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.  
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Huong, my gut reaction is that something is wrong with your primary neurons.  Cultured neurons are typically ridiculously active, pumping out minis at 10-15 hz, which just increases over time.  In fact most neurobiologists studying activity-dependent processes in cultures have to "quiet" neurons down by inhibiting activity with APV or TTX for hours to days prior to stimulation.  That you should be looking for ways to increase mini frequency and amplitude suggests to me that your cultures are not as healthy as they should be.
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In the past we have used Diasorin's ACTH IRMA for assays in human and rhesus plasma.  This kit has been discontinued, and I am looking for a replacement.  I have tried MP Biomedicals RIA and MD Biosciences ELISA.  The ELISA has given more comparable results, but many of our samples are near the limit of detection which results in high variability in duplicate measurements.  Does anyone have any other suggestions for a replacement plasma ACTH assay?
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Dear Pat,
we used recently in our investigations in human pituitary glands the antibody provided by Abcam (Abcam AB8615) - worked fine at least in WB!
Best regards
Joerg
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I am need to estimate the level of serotonin in my sample, for the that I have to compare my results with standard curve. I had tried lot of methodology but failed. So please suggest the working methodology (for serotonin standard by using serotonin creatinine sulfate monohydrate) to come up with good results.
Thanking you in advance for your kind courtesy.
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It would be helpful to know what methods you have tried so that we could help troubleshoot what might be going wrong. There are pretty standard methods of detecting serotonin in samples. HPLC for example is a useful tool for detecting a wide range of neurotransmitters and their metabolites. Also, there are standard ELISA kits that are effective in detecting serotonin.
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I am performing patch-clamp in whole brains in vivo and including a fixable intracellular dye
In order to locate the cell I have recorded from more easily, I figured if I add some Hoechst to the intracellular solution, then; as it is highly fluorescent, membrane-permeable and does not interfere with living cellular functions, it would serve as a general locational marker.
Has anyone else tried this? I am expecting to see a nice UV patch in the cortex I have recorded from that should guide me to where the cell I have patched is :D
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I agree with Alexander. Use need to use a cell non-permiable dye. If you do not want to do a post-stain to see your cell, AlexaFlour dyes are nicely biocompatable. Very little Hoescht will stain any cell your pipette got close to and make your pipette solution into a potent mutagen/carcinogen/teratogen. 
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I'm patch-clamping horizontal cells from goldfish retinas, and the membrane potential is constantly reading 10-30 mV (from "I = 0" setting on my AxoPatch 200B amplifier). I don't know if it's physiological, pathological, or a problem with the equipment. When I put on glutamate, the cells repolarize towards the glutamate reversal potential (0 mV), so I think it's not an equipment problem - but I'm all ears if you have advice/ideas.
I've tried less papain during dissociation; this didn't change anything. I've tried whole cell and perforated patch over and over - no difference. I've replaced solutions and tried different ones. I've checked my osmolarity and tried different ones. I've tried new batches of fish. Any ideas?
Please help! You'll be my hero!
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Try to record from GCs in whole cell V- clamp mode, run I-V protocol and see if cells are normally spiking if so then nothing is wrong with equipment or solutions ruther something is wrong with dissociated HCs, 
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I'm interested in recording fiber volleys in CA1 elicited via stimulation of Schaffer collaterals in Organotypic slices.  I have been looking around for an explanation of what type of stimulation electrode to use and how to place the stimulator/recording electrodes but have not been able to find a clear explanation.  
Should I pull large glass pipettes for the recording electrode so that I have a lower resistance?  Or does this not matter?  Should I place the concentric bipolar electrode directly into the slice or should I place it in a glass pipette?  
Thanks for your help.
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Chris, bipolar stimulating electrode goes into the slice a little bit (touch the slice, and drive maybe 50 um). Place stim in stratum radiatum as Amber mentions.
Place recording electrode (resistance not very important) about 1 mm from the stimulating electrode in stratum radiatum (dendritic field) or pyramidale (somatic field). Lower resistance recoding electrodes are a bit less noisy.
Use a bipolar pulse 100 us about 2-10 V and you should get a response.
The thing with the organotypic slice is that a lot of the connections are long gone, and you are left with cells, and few axons. Since the fiber volley is the response of the axons depolarizing, you may get very very small ones in the organotypic slice. The response will be a down deflection immediately after the stimulus artifact and before the field response.
Good luck
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I'd like to perform an experiment on mice inducing EAE with whole myelin as the immunogen. The most important reference seems to be "Oct;21(4):749-57.
Myelination in rat brain: method of myelin isolation. Norton WT, Poduslo SE.J. of Neurochemistry 1973" that obviously is about rats.. Thank you in advance for your help.
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Just a few simple pratical suggestions.
For an optimal immunization animals must receive an intradermal injection of the antigen in a depot form that will release it slowly. For this purpose any lyophilized preparation must be reconstituted in water or in saline solution up to the appropriate concentration and the suspension must be emusioned with Complete or Incomplete Freund's Adjuvant until a thick compound similar to a cream will be obtained. This is usually carried out connecting two opposed syringes with a two/three way connector one containing  the Freund's adjuvant and one the water/saline suspension (a connector can also be built in house, connecting a  large caliber needle with the cap of a secon identical needle). Pumping grdually the water solution in the oil a nice thick emulsion will be easily obtained. The thickness of the emulsion is critical for a efficacy of the depot.
Animals can be injected in the foot paths or in the skin around shoulder or hind limb joints, with  insulin syringe and needle. There isn't much room for large volumes of emulsion in these sites, therefore the concentration of the antigen in the emulsion must be carefully planned in advance.
Purification of Myelin from brain or spinal cord is simple, all the methods are based on gradient centriugations. In addition I believe that at least bovine and mouse purified  myelin prparations can be purchased from the market.
However, as already suggested by others, fresh brain white matter or spinal cord, whatever the source, will work nicely as well. In this case fresh tissue can be lyophilized and stored or can be used as a fresh preparation. If used fresh it may not be necessary to dissoleve it in saline solution
Best regards, and good luck with your experiments.
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I am planing some experiments to study difference in basal synaptic transmission accross different animal conditions. I am thinking about producing a fiber volley vs fEPSP slope curve, but I haven't done this before.
Can anyone advise me for this exercise?
Regarding the x-axis of this putative curve, should I give fixed increasing steps for the stimulation intensity (for me this option seems easier) or should I try to find representative FV values (e.g. 0.05, 0.1, 0.15, 0.2, etc...; for me this option seems harder, since the FV size changes among preparations).
It would be great if you can help me.
Best regards,
Diego Fernández
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Dear Diego,
The proper way to do it is to increase progressively the stimulus intensity, and to plot the fiber volley amplitude versus slope of fEPSP
Since you never get the same fiber volley across slices/animals/conditions you just bin the fiber volley say from 0 to 0.5; 0.51 to 1.0; 1.01 to 1.51 (artificial units); etc.
You calculate the average and SEM in each bin for the volley amplitude and slope of fEPSP. This makes a point with double error bars (one horizontal for fiber volley amplitude and vertical for slope of fEPSP.
The difficult part is to get a nice fiber volley, not contaminated by the stimulation artefact. These are key experimental tips:
1. always ensure that your recording electrode and the stimulus electrodes are aligned along the path of the axons (on beam).
2. use twisted wire (i.e. 50 microns NiCr twisted wire) for stimulation, or better glass theta tube with both barrels filled with ACSF (gives the smallest artefact) - my preferred procedure.
3. you have to carefully adjust the distance between the stim and the recording electrode with trial and errors. The closer you are, the more contamination you get from the artefact. The further you are, the more difficult it is to be on beam.
Hope that helps!
cheers
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Hi, nowadays i am trying to apply channel antibody into cerebellar Purkinje cell. However, every time I tried, whole cell condition went bad in 10 minutes (even initial condition was quite good).
Cerebellar Purkinje cell, especially its dendrites, is rather larger than other cells, so I should wait at least 20 minutes until the internal solution spread out enough.
I can see the common problems in Rs configuration; decreasing holding current, increasing leak current by time.
If you know any know-how, please let me know
thank you 
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There is probably something in the antibody solution.  Often commercial antibodies include Azide in the solution and might have high calcium concentrations.  Simply diluting 200-fold may not be enough.  You should remove these things by dialysis.
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The values of the ionic conductances for excitatory neurotransmitters can be found from the paper published by A. L. Hodgkin and A.F. Huxley. Are the values same for inhibitory neurotransmitters? Is yes, then what is the difference?
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The ionic conductance given in Hodgin & Huxley paper are measured during resting membrane potential or spike. If your are intersting in the conductance of GABA LGIC, you need to know the channel subunit composition in order to find the channel conductance. In a more general point of view GABA receptor are permeable to Cl- and HCO3-, therefore you need to control them in both your pipet and extracellular solution.
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Since neuropeptides are small proteins and the samples we collect are very small. I tried some commercial kits that can separately get RNA, DNA and protein from the same sample. But I polled a lot of these samples together and then measured the concentration of whole protein. Even though the concentration of protein can hit the minimum amount used for western blot, no neuropeptides can be detected. I guess most of the neuropeptides either had already filtered through during extracting them by using the kit or the concentration were too low to detect. Is there any good way to handle this? The punch that I use is 0.75 mm in diameter and 0.3 mm in depth. Thanks.
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Dear Jiaqing Y,
First you should need to know the nuropeptide is fully separated or with some complex or mixture of different nuropeptides, if its with mixture of some identical specs means it posses to functional modification so that you got this problem because of this parameter "no nuropeptide detected"
Method to Find Concentration. Just take 0.01mg of sample and try simple loading measurement by esterification method, so that you will find concentration of your neuropeptide sample from that you calculate as for your need, Hope you understood.,
Check this link contain more information, may helpful for you., Any artificial nuropeptide or any bioactive peptide need feel free to ask me...
Good Luck & Regards
R.Selva..........
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I am planning to determine acetylcholine and glutamate level in brain tissue homogenate. Firstly I want to know whether it is possible to detect nerotransmittres in brain homogenate? Secondly if it's possible then how can I get rid of the fats and other proteins in brain homogenate to prepare a sample, that contains only nerotransmitter, to be run in HPLC. As all the papers, I found, to detect neurotransmitters in brain use brain dialysate. 
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Adding up some information about basics of neurotransmitters detection: Mostly HPLC-ECD system is used. Monoamines are simple as they can be detected just after protein purification using 0.1M perchloric acid. Glutamate and GABA will need o-phtalaldehyde derivatization. Ach is little tricky as you have to get brain fairly quick and only using microwave can achieve that. There is some special enzyme-columns which can be used to detect ACh using HPLC-ECD.
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I have no experience whatsoever transfecting cells with cDNA, and I am looking for help/guidance/knowledge from the community.
What electroporation unit do you recommend? I will be using mainly PC12 cells.
Are there particular settings or solutions you like?
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All good over here, don't mind if I answer my own question.
We bought a Biorad electroporator and have gotten good results thus far.
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Does anyone have any clue why the frequency of mEPSCs is so low (<0.1Hz) in rat cultured cortical pyramidal neurons at 14 DIV, even though sEPSCs activity is robust?
We usually generate cortical neuronal culture from E18 embryos and plate the neurons with a density of 10^6 on PDL/Laminin coated coverslip. The neurons were mainteined in BME supplemented with Pen/Strep, Glutamax, FBS, N2 and 2-Me. The medium was replaced every 2-3 days. We transfected the neurons with lipofectamine 2000 at 8DIV and started to record from 14DIV.
For the transfected pyramidal neurons (GFP positive), they have normal morphology including beautiful spines, and robust spontaneous EPSCs (~1Hz). However, mEPSCs frequency is always low. In some neurons, we can't see even one event in 100 second recording. We also tried to culture the neurons up to 21DIV, but the mEPSCs frequency didn't increase over the additional week.
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I would keep the Ca at "physiological" level, for example at 2 mM. The 20 min of trypsinization sounds OK, but I would use papain instead. Also, the cultures could benefit from using the trypsin inhibitor. Also, the glial proliferation inhibitor (AraC or FuDr) could help, when added a 5-7 days after the plating. It is difficult to say what is wrong with the cultures since there are many parameters that could be critical. In general, the neurons plated on glial layer are much healthier than the high density mixed cultures and display good sEPSCs and mEPSCs.
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I am trying to dilute DPCPX (A1 receptor antagonist) to inject in rats. According to some publications it can be done in a 10% DMSO, 90% saline solution. I tried this and was able to dilute the drug in DMSO, but whenever I add the saline the drug precipitates.
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Firstly, I recomend you to dilute the DPCPX in DMSO to make a stock solution. Secondly, depending on the dose you want to use, take a snall volume and charge it into the syringe and finally, add some PBS into your syringe in order to prepare the injection (depending on the dose and weight of the animal).
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It has been said that some neurons have the capability to spontaneously generate an action potential without receiving any kind of synaptic input, in other words the AP is intrinsically generated.
How does the cell do this? Is this a slow depolarization resulting in tonic low frequency firing? Can phasic firing also be intrinsically generated?
Which brain areas and cell types are particularly known for exhibiting this type of behavior?
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Some (only some!) hints to answer your question can be found here:
1: Tuckwell HC. Biophysical properties and computational modeling of calcium
spikes in serotonergic neurons of the dorsal raphe nucleus. Biosystems. 2013
Jun;112(3):204-13. doi: 10.1016/j.biosystems.2013.01.007. Epub 2013 Feb 4.
Review.
2: Barbuti A, DiFrancesco D. Control of cardiac rate by "funny" channels in
health and disease. Ann N Y Acad Sci. 2008 Mar;1123:213-23. doi:
10.1196/annals.1420.024. Review.
3: Akhavan A. Contribution of pacemaker channels to autonomous electrical
activity of differentiated embryonic stem cells. J Physiol. 2008 May
15;586(10):2425-6. doi: 10.1113/jphysiol.2008.153338. Epub 2008 Mar 27. Review.
4: Ikeda M. Calcium dynamics and circadian rhythms in suprachiasmatic nucleus
neurons. Neuroscientist. 2004 Aug;10(4):315-24. Review.
5: Freeman WJ. Characterization of state transitions in spatially distributed,
chaotic, nonlinear, dynamical systems in cerebral cortex. Integr Physiol Behav
Sci. 1994 Jul-Sep;29(3):294-306. Review.
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What is the best/optimal way to freeze the rat brain - liquid nitrogen, CO2, isoamyl alcohol? When you take out brain from 30% sucrose, what is the next step?
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I also use dry ice for freezing the tissue after 30% sucrose.
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Can anyone tell me about what are the ethical issues in Human Neuroscience studies. Which are Drugs we cannot use? Which are parameters we can use to study Human Brain?
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I would advise you to review some of the BRAIN Initiative, as well as Antonio Damasio's literature. I am currently studying the subject will be glad to Exchange information and sources.
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We tried Santa Cruz but there's a lot of non/specific staining. How about for PSD-95 Western blots?
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Hi,
Santa cruz-546 is a good antibody for western blots and even for immunohistochemistry. When looking in a PSD, just use it as the 1st antibody on the blot and it works. Also, use a 4-12% or 4-15% gel. You will surely see bdnf.
Kind regards,
AM.
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I want to do a western blot using proteins from rat cerebral cortex but I am asking if there will be enough cells that express this?
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I am working on a methylating protein and would like see the expression of gene in mature oligodendrocytes. What is the best method for transient but long lasting expression?
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Hi guys, what sort of transfection efficiency have you got with Fugene and lipofectamine? Thanks!
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I am using a HCN-2 cell line, the growth rate of this cell line is greater than 120 hours. According to ATCC the growth rate of HCN-2 cells is stimulated by treatment with phorbol esters, but I am not getting a good growth rate after treatment with phorbol esters.
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I dont know HCN-2 very well, but more cells slow down there division after differentiation (phorbol). There are a number of ways to increase growth rate all with pretty much the same caveat- phenotype!
1) As Barbara mentioned, increasing serum is one way- did you just change your serum batch? Do you heat it properly?
2) Change medium every day instead of several days.
3) Try a new type of flask or coating- characteristics make a big difference (did you just change your batch of coating?)
4) Check your CO2 and temp settings
5) grow them in a bigger flask to ensure they are not touching, then when ready to use, replate into smaller dishes. If necessary use a multilevel flask.
6) (Barb again) make sure you have not passaged them too long. Cells get old and slow too you know...
*** Any and all of these changes can change the phenotype, so after you have the right growth rate, check for characteristics.****
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I'm working on the regeneration of the auditory cortex in adult rats. In my material, a restricted lesion induces axonal growth from the contralateral cortex, shown using injection of neuroanatomical tracers. Can any suggest antibodies for labeling axonal growth cones in histological sections?
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Growth-associated protein 43 (GAP43) is well known marker of neurite outgrowth. In our recently published paper we have shown that 10 days after cortical injury in the neurons of injured cortex high density of GAP43 staining was concentrated in a particular part of the neuronal cell body forming growth-cone-like structures, which were directed to the lesion site.
Brkic et al. Hyperbaric oxygenation improves locomotor ability by enhancing neuroplastic responses after cortical ablation in rats
Brain Injury (2012) 26 (10):1273-1284,
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What are the important points to be noted while looking at the uncoupled anion currents? The assay will require transfection and over-expression of a membrane protein. A ligand induced current will be looked at. Any inputs regarding these kind of assays?
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Briefly: 1) to block Ca2+ channels, you will need to use ACSF with 0.1 mM Ca2+ and 5 mM Mg2+.
2) to block K+ channels, you will need to use Cs+-based intracellular solution. Additionally, you might add 1-10 mM TEA to ASCF, if necessary.
3) Stimulation protocol: Vhold, -80 mV. Conditioning pre-hyperpolarization -120 mV for 50 ms. Then testing voltages starting from -60 with increments of 5-10 mV.
You can start with all this and then you will see.