Science topic

Synaptic Transmission - Science topic

The communication from a NEURON to a target (neuron, muscle, or secretory cell) across a SYNAPSE. In chemical synaptic transmission, the presynaptic neuron releases a NEUROTRANSMITTER that diffuses across the synaptic cleft and binds to specific synaptic receptors, activating them. The activated receptors modulate specific ion channels and/or second-messenger systems in the postsynaptic cell. In electrical synaptic transmission, electrical signals are communicated as an ionic current flow across ELECTRICAL SYNAPSES.
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I think both are used mainly for blocking GABAA receptors. Since gabazine is more selelctive for GABAA receptors, is it better to use for studying EPSC? Is it enough to block IPSC by only blocking GABAA receptors? Thanks.
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Hello Lingdi!
Of these two drugs, gabazine is more convenient to use, as it is water soluble and requires a smaller concentration to block the evoked IPSC (in rat cortical slices we use 5-10 uM of gabazine or 50-100 uM of PTX). However, gabazine is more expensive than PTX.
Bicuculline methiodide also blocks the GABAaR-mediated IPSC (it is water soluble and is cheaper than gabazine). Note that it affects the SK-channels.
If the IPSC in your preparation is mediated by both GABAaRs and GABAbRs you should also apply CGP-55845 or use the pipette solution with cesium salts.
Good luck with your experiments!
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WHat do you think is the correlation? It's impact? and the possible transmission route?
Neurologic Features in Severe SARS-CoV-2 Infection
DOI: 10.1056/NEJMc2008597
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Hello to everyone,
my name is Stylianos and these days I have focused on pairing recordings.
I cause 5 APs (5 pulses 20Hz) to a SOM-Martinotti neuron and I record IPSCs in a principal neuron in cortical slices at 0 mV in presence of DNQX and CGP. I found depression of the synaptic transmission (pulses 2,3,4,5 were smaller in amplitude compared to the 1st). This finding is in agreement with the literature.
BUT.....When I did the same experiment by optogenetically activating many SOMs with CAG.ChR2 and recording IPSCs in principal neurons, I found facilitation of the 2nd pulse compared to the 1st (in these experiments I applied the PPR protocol with 2 pulses at 20Hz).
So, I am wondering if there is an explanation about this discrepancy.
I thank you a priori for reading this.
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Interesting question.
I don't KNOW the answer. But I have a testable hypothesis.
My theory is that with optogenetic stimulation you are basically seeing temporal integration in SOM cells. That is to say, with your first light flash, you are causing say, 20% of SOM cells to spike, and the rest to be depolarized. On your second light flash, you are causing 40% of cells to spike, the increased number caused by residual depolarization left over from the first optogenetic exposure (i.e. the membrane is still charged up).
If this is the case, you should notice a change in the paired pulse ratio with varying light intensities, i.e. with very weak light intensities, you should see more facilitation than with very high intensities. Furthermore, the facilitation should not behave to changes in extracellular Ca2+ concentration in the expected way (though the first test is easier to perform), i.e. if you increase the extracellular Ca2+ concentration you should see no significant change in the paired pulse ratio.
Interested to see if anyone else has a better idea.
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I have noticed an increased in the asymmetrical synaptic excitatory junctions to be increased in the diabetic brain but could not find any discussion of this in the literature at present.  If you have any information or publications regarding this question please share with me when you have the time.  
My hypothesis is that in younger diabetic brains this increase in asymmetrical synaptic excitatory junctions may result in an overuse phenomenon and result in a later or older models in their attentuation and or loss as in accelerated aging in the diabetic brain.  Thank you for your time and consideration.
Sincerely,
M.R. (Pete) Hayden, MD 
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Dr. S. Liu,
Your answer was very much appreciated and I have learned a great deal due to your paper in PNAS.  I am going to be studying the db/db K strain model in the coming months, while I will be focusing my TEM  studies of this model primarily on the neurovascular unit I will also be evaluating the  dysmorphic myelin white matter and due to the better understancing of you PNAS paper I will be keeping a close observation regarding the assymettrical excitatory snapses as well.  You paper really helped and I am so thankful for your earlier response to my questions regarding an observation of increased excitatory synapses in the type 1 model and now will be alert to observe similar changes in the obese T2DM of obesity insulin resistance hyperinsulinemia  and persisitent elevation of glucose levels well above the diabetic range for mice  - approximately 300-400 ranges. 
Sincerely ,
M.R.(Pete) Hayden MD 
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Two papers are using the method.
One attached and the other cited here: "Single rodent mesohabenular axons release glutamate and GABA" Root et al  2014
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Supplemental figure 3 and supplemental text from "Cho JH, Deisseroth K, Bolshakov VY. Synaptic encoding of fear extinction in mPFC-amygdala circuits. Neuron. 2013 Dec 18;80(6):1491-507." have a very nice explanation of the logic behind using TTX and 4-AP isolating monosynaptic inputs. They also show a positive control in which disynaptic inputs are not rescued by 4-AP. Here is a link to their supplemental materials: 
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is this theory right?
Pathway 1:  the fast increase of intercellular [K+] produced by the spiking activity of the postsynaptic neuron.
Pathway 2: the slow production of a mediator (Glutamat) triggered by the synaptic activity.
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Local increase includes 
1, K+ release from axons as a results of APs.
2, synaptic transmission, which is a major source. 
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Neurons are grown on coverslips in culture after some time they form connections and synapses (very basic networks). What kind of stimulator (digitimer) do i get to look at synaptically stimulated APs? I have used a voltage stimulator like the DS2 (digitimer) with a bipolar electrode in brain slice neurons, but I'm not sure if that's the best kind of stimulation for neurons in culture. Is current stimulator better or biphasic and what difference does it make? Also what kind of electrode would work better? Would a bipolar electrode fry my neuron cultures?
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Hi Dina,
I think it's most practical to stick with a bipolar stimulating electrode, not sure what other configurations you were considering - like a concentric (single-point) bipolar for example? The reason being that the synaptic connections are not going to be organized in such a way as they would be in brain slice - certainly not like in laminar structures like hippocampus or cerebellum. Actually for cultures, the bipolar electrodes can be much more widely spaced than ones typically used for brain slice. Essentially you can have the poles straddle both edges of the coverslip, if you wanted to maximize chance of stimulating inputs to the cell you're recording from.
As for the stimulator, I'm not sure if you're referring to the output (V vs I), but I've used ones that are easily switched from constant voltage output to constant current output - I typically use constant current output. Haven't tried constant voltage output when stimulating cultures, but I know in slices, both output modes produce effective stimulation.
Best,
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If one uses antibodies against VGLUT1 and synaptophysin to stain in WT cultured hippocampal neurons, one expects the immunosignals to co-localize, since both are synaptic vesicle specific proteins. how to explain the finding of solely vglut1 signals? I understand if I have only synaptophysin signals, maybe I thereby detected vgat expressing neurons, but synaptic vesicles that are positive for vglut1 and not stained with anti synaptophysin antibody?
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I am not surprized by negative syn terminals
for example VGLUT2 and VGLUT3 (on which we work) very poorly colocalize with syn
and only half a gaba terminals are syn-positive
with VGLUT1 I would estmate that around 20% of VGLUT1-positive terminals are syn-negative
How to explain it ?? I don't know for sure
1) if you look at these synaptic proteins they are heterogeneous not only between synapses but also inside synapses
2) as far as I know the role of synaptophysin is not clear
3) and we do not have a generic marker of all synpases
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I’m doing in vivo electrophysiology in anesthetized mice where I record evoked field excitatory post synaptic potentials (fEPSP) in the hippocampus (electrical stimulation at CA 3 fibers; recording at CA1 stratum radiatum). I would like to learn more strategies to study short-term plasticity mechanisms and other parameters of basal synaptic function in in vivo anesthetized preparation, always keeping in mind that intend to study evoked fEPSP. So far I use classical input/output measurements and Paired Pulse analysis but I’ve seen other types of protocols although I’m not yet familiar with the principles behind. If someone has any suggestion/advice, I would be very grateful. Thank you.   
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Good answer Debanjan,
We've also used short higher frequency trains (40Hz). I'll upload a recent publication on some of our work. 
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In our scenario, we let an Axon grow in Vitro over a distance of 500µm before it will find Dendrites to connect to.
How many Synapses are formed by that Axon when coming in contact with those Dendrites?
  • The elapsed time will be 7 Days
  •  The Cells are embryonic Hippocampal cells from Rats
A second Question would be related: What would be the spatial spreading of the most distant Synapses?
I need to know this to solve a problem we have when connecting single Neurons to Clusters.
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For a niche set up like this probably best to test it yourself? eg. dye filling your 'presynaptic' cell and post hoc staining for postsynaptic proteins, eg PSD95, then count the number of juxtaposed contact points? 
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Hi guys,
I'm a little bit in trouble with LTP on CA1. I performed some experiments inducing LTP after HFS (1 for 100 stimuli at 250Hz). This protocol is able to induce a strong potentiation of the synapse. My problem is that the potentiation after the induction is not stable and the response continues to increase without reaching a plateau. What do you think the problem is? I recorded extracellularly fEPSPs from hippocampal brain slices (400uM thickness; flow rate of about 2 mL/minute).
Thanks in advance for your advices.
Luca
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Under the microscope how does your slices look, bright and shiny with nice cell line?
In the CA1, if the GABAergic tone is damaged, you do get epileptic EPSPs that look spikey (increased amplitude or slope and could explain what you observed) and the EPSP duration is also increased. In normal condition,  following HFS your EPSP should increase. Do you have any traces recorded in these slices? One stupid question have you checked your temperature probe (battery)?  Don`t put picro or bicculine it will make your EPSP epileptic, not in the CA1. It works in the dentate but not in the CA1   
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It is quite well established that DHPG perfused in acute hippocampal slices causes a transient decrease in synaptic transmission in hippocampal CA1 region (DHPG 100 micoM is perfused for 10 minutes in Palmer et al. 1997) but i haven't found the molecular mechanism that is proposed to underlay this short depression (i did find a lot of mechanisms by which mGluR-LTD could occur, including retrograde transmitters hypothesis)
It seems to be related to a reduction in calcium influx in pre-synaptic terminals but i don't know how this can happen upon group 1 mGluR activation induced by DHPG....
Thanks!
Celia
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Dear Celia, may I suggest you check the paper:
Switch from facilitation to inhibition of excitatory synaptic transmission by group I mGluR desensitization.
Rodríguez-Moreno A, Sistiaga A, Lerma J, Sánchez-Prieto J.
Neuron. 1998 Dec;21(6):1477-86
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Hello Everyone,
First I do apologize for posting my question here. I just signed up and am quite new. I just started recording fEPSP from hippocampal CA1 region and the problem is that the signal amplitude I get is very small (between 0.2-0.5 mV). I do sagittal sectioning (300 µm) in cold ASCF and incubate for 1-1:30 h at 30 degrees. I work with 2-4 month-old mice. When I put the slices under the microscope, all the layers can be easily distinguished. The fiber valley is very very small which I think can be good sign.
I have been struggling with this problem for a couple of weeks and tried many things to get bigger signal but nothing seems to work. The last thing that recently came to my mind was that maybe there is something wrong with the stimulation protocol. This is my first recording experience and I have no idea about how to make a protocol. The one that I am currently using is the one used by one of the students in other lab for recording EPSP from cortex and is apparently working perfectly for him. 
Can anyone explain what each of the following is:
First level, delta level, first duration, delta duration, digital bit pattern (#3-0) & (#7-4), train rate...
Maybe I will be able to make a protocol for hippocampal fEPSP if I have a better idea about each of the above term and the best possible range they can be within.
Also, does anyone have any recommendations for the positioning of the stimulating and recording electrodes? Is there any particular area to get the best possible response?
I do appreciate anyone's help in this regard.
Thank you all in advance
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digital bit pattern (#3-0) & (#7-4): this is trickier. it's asking you which output to send the stimulation through. look at your digital outputs as a grid: 3210 top row, 7654 bottom row. the bit pattern is a 0 for outputs you do not want to use, 1 for those you do. 
so for channel 2 it would be 0100 and 0000
for channels 1 and 7 it would be 0001 1000
for channel 5 0000 0010
or try * to enable a bit if you use train and it can keep up with it
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Hey,
I want to do some calcium current measurements in dissociated hippocampal cultures, to make an I-V plot. I suspect the mutated protein I express, influences the VGCC-conductance. In order to isolate the calcium currents I plan on blocking the sodium-currents with TTX and use a cesium based intracellular to block the potassium currents.
My problem is choosing the cesium based intracellular medium. After doing a quick the literature I have seen several mixtures. I have seen recipes with either: Cs-glutamate, Cs-sulfate or Cs-methanesulfonate.  Can someone help me by explaining these compounds?
Furthermore, I have seen some use barium instead of calcium to assess the conductance of the VGCC. Why use another ion?
Thanks in advance,
Marvin Ruiter
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Hi Marvin,
Nicolas and Norbert gave you the information you needed, The only thing I would advice is that if you have a nice large Ca current then it's better to use external Ca as a carrier as your experiment will be more physiologically relevant, Just remember to buffer internal Ca at low level with EGTA (e.g. use 0.5mM CaCl and 5mM EGTA).
Good luck
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Hi,
I am new into electrophysiology. I am studying LTP via a STDP protocol in whole-cell configuration, recording currents. After the LTP induction my Rs badly increases (more than 30%) but there is no change into synaptic currents. As far as i know, when Rs increases the currents should decrease following ohm law. If my currents don't decrease can be due to a potentiation into synaptic transmission?
I am using clampex 10.2 and multiclamp 700B to record from the cell, with a pipette resistance of 5 mOhm.
I hope I explained myself well,
Thanks
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Hi Francesco,
I agree with Fabien, these experiments are most likely lost. Now there are a couple of things that you can check.
if you repeat your experiment, without the stimulation, do you also see these changes? If yes, you should check your solutions (internal most likely) or see if you don't have any drift of your pipettes. Check also if your slice is correctly fixed in the chamber and if it doesn't get swollen (if your solutions are not correct that might happen and move your cell away from your pipette)
If it doesn't happen without the stim, then you might want to make sure that your stim electrode is not too close from your recorded cell. Also always try to use as less current as you can, cells don't like it usually...
Hope this helps,
Good luck
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I just get very puzzled when reading some paper results about brain slice work, when the author stimulates one intracortical region and records the response of another intracortical region, how can I know whether it is direct connection between these two regions or may involve other subcortical regions, eg the thalamus? I didn't find any evidence in the paper about preserving the thalamocortical pathway or not. How can I understand the results in these kinds of paper?
Hope anybody expert in brain slice work can help me with this. Thanks
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Hi, If I would attempt to study thalamocortical circuitry I would start with reading paper attached. It does evaluate preservation of thalamocortical pathway using both electrophysiology and Dil. Best!
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It seems the dual virus-based monosynaptic tracing, i.e.G-TVA plus EnvA-pseudotyped deltaG RABV, is a widely used technique for seeking presynaptic partners of host cells. See (Bergami et al., 2015, Neuron 85, 710–717) and (Garcia et al., 2014, Dev Cell 30, 645-659). Is there any possibility of leakage? 
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The RABV infected cells are alive 2-3 weeks after the infection and leakage is not an issue since budding occurs at the synapse. The picture you'll get with mGRASP will be different (seeing synapses) and you'd probably use it to see if region A and B (or cell type A and B) are in general connected, but not to see which cells exactly project to your cell of interest.
So it depends a bit on the question at hand, which method to chose or if its necessary to combine both.
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As mentioned by Jim Al-Khalili in 'Life on the Edge':
The EM field, pulling together all those coherent ion channels in disparate parts of the brain to generate synchronous firing, could play a role in the transition between unconscious and conscious thoughts.
Can it be that the synchronous firing can make something visible what is not visible by asynchronous firing.  
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May be I can ask it more directly: should there be a connection between quantum biology and synchronicity?
If the coherence keeps on, on the macroscopic level or if there is a continual feedback nano-macro, in certain special circumstances, if the order at the unconscious level is well structured, theoretically the order out of order can be taken in consideration in concurrence with the Newtonian mechanics.
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To test pre- versus postsynaptic mechanism of drug action, I am using the paired-pulse protocol. I am curious about the rationale behind altering Ca2+/Mg2+ ratio to better detect small changes. Can anyone help? How does increasing or decreasing this ratio affect PPR? I would love to know the mechanism (residual calcium hypothesis etc) behind it. Also how would this relate to synapses that show control paired-pulse depression like the medial perforant pathway in DG?
Thank you
Nisha
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Calcium is a crucial element modulating the probability of neurotransmitter release, because its interaction with key components of the active site in the presynaptic terminal.
The increase of extracellular calcium will increase the equilibrium potential for this ion (ECa), so when the voltage gated calcium channels in the presynaptic terminal open when an action potential arrives, more calcium will enter the presynaptic active zones, evoking the release of a higher number of neurotransmitter vesicles. So increasing extracellular calcium increases neurotransmitter release. To preserve the ratio of divalent ions (a variable itself) constant, during experiments with changes in extracellular concentration of calcium, magnesium is accordingly changed. But the main character affecting PPR is calcium because its action onto the release machinery.
Classical explanation for paired-pulse facilitation is the residual calcium hypothesis. According to this hypothesis, during the first action potential there is an entrance of calcium in the presynaptic terminal. Most of these calcium ions will activate neurotransmitter release by its binding to the sensor for release (synaptotagmin), in a well-known cooperative manner. However, a small fraction of calcium ions will remain in the active zone as "passive ions", namely residual calcium. If there is a second action potential after some milliseconds (and assuming the same entrance of calcium), this new calcium income will sum up with the residual calcium (which needs more time to be removed as the inter-stimulus interval), thus activating an elevated number of vesicles to release. According with this hypothesis, if we reduce levels of extracellular calcium and so ECa, less calcium ions will enter the terminal after an action potential, but the same residual calcium is expected to remain. So, when we reduce extracellular calcium, residual calcium would become a more significant fraction of the local calcium signal after a second action potential. That means that the second action potential will be "more facilitated" with respect to the first one (>PPR) in this second “low calcium” scenario, than with higher extracellular calcium concentrations. This model predicts that PPR will decrease with higher extracellular calcium concentrations. However, it seems that for most synapses this hypothesis fails to explain paired-pulse facilitation, because residual calcium and PPF decay at different rates.
There are other models proposed to explain PPF. A "facilitator sensor" different than synaptotagmin has been suggested. This sensor would have slow kinetics of action, unable to work after a first action potential, but ready when a second action potential arrives, and somehow facilitating a greater amount of neurotransmitter release.
It has been also suggested that paired-pulse facilitation is dependent on endogenous calcium buffers. These buffers would have high affinity, so after a first action they would become saturated, but when a second action potential arrives after a short period, the kinetics of the buffer are slower than the inter-stimulus interval, so assuming that the same amount of calcium is entering the presynaptic terminal, less calcium will be "caught" by the buffers, so a greater number of ions will be available to active neurotransmitter release, and in this way facilitate the second response of the pair. An important prediction of this model is that PPR will increase with higher extracellular calcium concentrations.
In general, paired-pulse facilitation is a synapse-specific property. Mossy fiber to CA3 connections behave more accordingly to the "buffer saturation" hypothesis, because in this synapse, PPR does increase with increases in extracellular calcium concentration. However, this doesn’t happen in the Schaffer collateral to CA1 synapse, where PPR decreases with increases in extracellular calcium.
Hope this helps you to understand better the complexities of PPF, which even today, still remain very mysterious!
I would recommend you to consult the review from Regehr about short-term plasticity (Regehr Cold Spring Harb Perspect Biol. 2012).
Good luck with your PPF 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, I am trying to manipulate background synaptic release in acute brain slice.
Does anyone know how to increase background synaptic release without affecting postsynaptic dendritic active conductance too much (i.e. 4-AP increases presynptic releases but also blocks dendritic A-type channel)? What I have tried is to increase extracellular K+ from 2.5 to 3 mM, but the effect was not strong enough. 
Thank you!
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you can use up to 2mM 4-aminopyridine that would have a minimal effect on dendritic IA, and an intracellular Cs electrode (to prevent the postsynaptic effects of blocking other gKs)
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I'd be interested to know how subthreshold oscillations and noise of the inputs of a neuron are related. In particular, I would like to understand if there is some compensatory mechanism that may lead to neglect subthreshold oscillations with respect to the noise affecting synaptic inputs. This seems to me relevant to know in view of a simplified neuron model implementation for applications using very large spiking neural networks. 
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Alessandro,
I believe you are referring to phenomena described in the squid giant axon by Hodgkin and Huxley many years ago. My interpretation of these damped oscillations is that they reflect subthreshold kinetics of voltage-gated sodium and potassium channels and depend especially upon the constant, h, which describes the fraction of voltage gated sodium channels normally in an inactivated state at the resting potential level. At the termination of the falling phase of the AP the axonal membrane is slightly hyperpolarized at a level near the potassium equilibrium potential, leading to an increased readiness of a fraction of the voltage-gated sodium channels to become activated when the membrane returns toward the resting level. This may actually lead to a small depolarization due to net sodium influx  (depolarization) and, after a delay, to an  increased potassium conductance that repolarizes the membrane toward the potassium equilibrium potential, and so forth.Both effects (ion fluxes) diminish with time as the oscillations become damped out.
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Do you think the animals emotional state might be responsible? Ever tried publishing, what do reviewers say?
Thanks
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Hi, I worked with the DA system -behaviorally, electrophysiologically, pharmacologically and electrochemically at Univ Pittsburgh in the days when Dopamine was their main topic. Yes, many many methods and procedures to standardize responses.  Activation, habituation, handling, pressure to the tail, etc. all play key roles.  Happy to discuss this with you by phone - 317 276-5192  Nancy Ostrowski
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I need to block the glutamatergic and GABAergic vesicular synaptic release in cerebellar acute slices. I would apply Tetanus Toxin. Can I expect a complete blockage of excitatory and inhibitory postsynaptic currents (whole cell recording from Purkinje Neurons)? Is there a better alternative?
NOTE - I do not want to block pharmacologically the neurotransmitter-receptors or any ion channels.
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What animal will you use? You should have in mind rats don't respond to tetanus toxin, only botulinum toxin. Anyway, these toxins will act on SNARE complex, inhibiting the vesicular synaptic release.
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After having so many trouble with the signals I got in my rat hippocampal slices (small amplitude, poor inhibition, tendency for epileptic responses, often contamination with population spikes) I decided to try the already described protective cutting method substituting the NaCl for sucrose during the cutting procedure.
The results amazed me. The different laminar layers of the slices were clearly differentiable, and the signals were big and smooth, without any contamination by population spikes.
Now, I am naturally tempted to start working with this kind of preparation, but a paper from the group of Graham Collingridge warns that the level of LTP is lower in slices cut in sucrose ACSF, apparently due to an enhanced inhibition by GABAergic neurons (Kuenzi J Neurosci Methods. 2000 100(1-2):117-22); and raises the question I just set out. This enhanced GABAergic activity may come from a greater survival of these cells in the sucrose based procedure. However, looking through the literature, just few labs have been performing experiments under such conditions.
So, is it acceptable? Would the interpretation of my results be harder by the use of this protocol?
I rather think that this procedure better preserves both excitatory and inhibitory neurons, so it keeps the integrity of the network in a state which more closely resembles the physiological one.
I would love to know your opinion.
Thank you very much.
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Hi Diego,
You have pulled up an interesting paper about the sucrose aCSF protective cutting solutions. Indeed in the Kuenzi et al paper they show that LTP can't be induced with the normal protocol in slices prepared using sucrose aCSF, but when GABA-A receptor antagonist was applied the LTP could then be efficiently evoked--i.e. the inhibition in the slices was strong enough to constrain LTP induction. While at the time it might have been tempting to conclude something was wrong with sucrose aCSF, in hindsight I think most people would agree that the most logical interpretation is that interneurons are more vulnerable to deterioration in acute slices, and that the sucrose aCSF preserves neurons better than standard aCSF (prevents Na and Cl influx and subsequent swelling during the slicing procedure). You may also wish to see the original paper from Aghajanian lab (Aghajanian and Rasmussen 1989 Synapse) describing the sucrose aCSF method. Indeed the sucrose aCSF can preserve both excitatory and inhibitory neurons better, but perhaps the effect is more pronounced for inhibition--which can account for the finding in the Kuenzi et al study. It is arguable which situation is more physiological, but in my view you must already assume a non-physiological state once the brain is removed from the rest of the body and cut into slices. It is a model system to explore synaptic function and that allows better access for interrogation than in vivo techniques. Since it is not particularly helpful to study a few healthy cells in a sea of dead or dying cells, most people are happy to implement techniques that can provide better preservation and healthier neurons. Modified strategies offering better preservation are particularly relevant when comparing two groups (e.g. WT vs KO) since all steps with the procedure are equal. However, it is a tough call when you are actually trying to make measurements that you wish to claim are as close to 'physiological' as possible.
I would only offer up my personal view from many years of work on acute brain slices. Sucrose aCSF is not rare in the field and very likely is the most common method being implemented around the world. It has been used successfully in many contexts including for virtually all brain regions and cell types accessible for recordings in slices, and for juvenile and adult animals of many species. In addition to sucrose aCSF there are many other alternatives that can be highly effective at neuronal preservation (e.g. NMDG, Choline, Tris, glycerol...), and not all sodium substitutes are equal. Agreed that one should investigate in detail what effects (if any) are notable when switching solutions or preparation method, but perhaps it is most reasonable to think about this as a panel of options and not a one size fits all. What works for one person may not work for someone else--experimental context matters. If you are interested to explore more on the topic, I made a website a while back that goes into great detail on the topic and describes my methods for improving preservation of acute brain slices from adult and aging animals (namely transgenic mice). There are many relevant citations listed on this site including some recent ones from my own work. www.brainslicemethods.com
I hope this helps you. --Jonathan
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I have already tried adapting established methods for other animals with no success. I am using a polyclonal antibody (Sigma).
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Hi Giulia,
probably you want to try the line OK371 reported in the paper: http://dx.doi.org/10.1016/j.modgep.2005.07.006
the line is available in bloomington with the stock number 26160
Probably you want to cross it with GPF or other reporter and either visualize it in vivo or after IHC.
Good luck!
Abud
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I am currently planning an acute aerobic exercise study that is likely to increase circulating levels of BDNF. However, the increase in BDNF following this type of activity is expected to be transient, returning to baseline within a few hours following the activity. What I would like to know is how long the downstream effects of a transient change in BDNF are likely to be affected. Will the cascades initiated by BDNF-trkB binding continue to be modulated after the BDNF levels have returned to baseline? If so, for how long? Any articles that relate to this topic would be welcomed.
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While BDNF may transiently activate some signalling cascades others might undergo longer sustained increases in their activation. However, regardless of whether pathways are transiently or chronically engaged the resulting biology stimulated by BDNF will last much longer. In the synaptic plasticity field, for example, acute elevations in BDNF signaling while transient can facilitate the conversion of short-term plasticity into long-term plasticity which can last several hours to days. Are you planning on correlating your measures to blood BDNF levels? As for inhibitors, I'm not aware of any commercially available inhibitors for use in humans.
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I have read a study in which the authors labeled vesicles with quantum dots and suggested a number of times that a single vesicle can be used. What do you think about this?
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It depends - amongst others - how and where you look at synaptic vesicles; and obviously a question with a long(er) tradition, e.g.,
1. Alabi AA, Tsien RW. Synaptic vesicle pools and dynamics. Cold Spring Harbor perspectives in biology. 2012;4(8):a013680.
2. Blakely RD, Edwards RH. Vesicular and plasma membrane transporters for neurotransmitters. Cold Spring Harbor perspectives in biology. 2012;4(2).
3. Ceccarelli B, Hurlbut WP, Mauro A. Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. The Journal of cell biology. 1973;57(2):499-524.
4. Chung C, Raingo J. Vesicle dynamics: how synaptic proteins regulate different modes of neurotransmission. Journal of neurochemistry. 2013;126(2):146-54.
5. Hnasko TS, Edwards RH. Neurotransmitter corelease: mechanism and physiological role. Annual review of physiology. 2012;74:225-43.
6. Jahn R, Fasshauer D. Molecular machines governing exocytosis of synaptic vesicles. Nature. 2012;490(7419):201-7.
7. LoGiudice L, Sterling P, Matthews G. Mobility and turnover of vesicles at the synaptic ribbon. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2008;28(12):3150-8.
8. Ramakrishnan NA, Drescher MJ, Drescher DG. The SNARE complex in neuronal and sensory cells. Molecular and cellular neurosciences. 2012;50(1):58-69.
9. Saheki Y, De Camilli P. Synaptic vesicle endocytosis. Cold Spring Harbor perspectives in biology. 2012;4(9):a005645.
10. Sudhof TC, Rizo J. Synaptic vesicle exocytosis. Cold Spring Harbor perspectives in biology. 2011;3(12).
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I am making coronal slices and stimulating at 30% of the maximum response of my field potential.
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Field potentials? In my experience, as long as your conditions are constant (eg, temp, perfusion rate, etc.) instability issues usually arise from the slice not being sufficiently immobilized. Less often, it's declining slice health, especially at higher temperatures (lower oxygen tension). But usually, either the slice is moving, or the perfusion system is causing changes in fluid motion or level around the slice.
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Specifically from cortical Layer 2/3 pyramidal neurons using whole-cell patch configuration and from brain slices (2-3 weeks weeks old mice). But I don't mind any other brain area either or animal. I know of papers using excised patches but nothing in whole cell configuration.
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Brain-derived neurotrophic factor regulation of N-methyl-D-aspartate receptor-mediated synaptic currents in suprachiasmatic nucleus neurons.
Kim YI, Choi HJ, Colwell CS.
J Neurosci Res. 2006 Nov 15;84(7):1512-20.
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It is known that OGD supresses fEPSPs in CA1 region, and some reports demonstrated that presynaptic A1 (A2 or A3 in another way) and Ca++ flow regulation underlined this suppression. Intracellular signaling such as PKC was also suggested. Some researchers showed that the fEPSPs suppression is irreversible if OGD for more than 10 or 30 minutes (on acute brain slices). However, in my hand, fEPSPs still can be recovered to 60% of baseline level after 45 minutes OGD insult (completely recovered if it is 30 min OGD). Any opinion for this difference? In addition, some transgenic/mutant mice generated in our lab have shown either beneficial or deleterious effect, but I did not know much about the mechanisms that accounts for fEPSPs suppression. Can anyone provide advice?
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I agree with Dr Kohling . However, I would also further emphasize that the thickness of the slice, the type of recording chamber, whether you are using a submerged slice or a slice that is at the interface are all critical variables that will affect pO2 tension. Also how do you monitor the health of the slice before OGD? Do all the EPSPs have max amplitudes or is there a lot of variation. These variables all make a difference.
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I would like to try to block all synaptic transmission. TTX blocks the AP-dependent release at the terminal and so will Cd2+ be effecttive in blocking the VGCC. What about minis?
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Hi! Most likely to block it completely is impossible. Try EGTA in Ca-free bath solution/culture medium and as you mentione + TTX and VGCC blockers - that could be the most what you can get. You can not avoide some spontaneous release even in tetanus-toxin treated neurons (which cleaves SNARE comples responcible for vesicle fusion) because in the synapse there is some Tetanus-toxin-insensitive SNARE protein, which also can drive the vesicle fusion. And this is only one protein, but there are a lot of similar and you'd faster kill the cell than _COMPLETELY_ block exocytosis.
If you want just to block the postsynaptic responces, then the coctail of AP5, CNQX and bicucculine or picrotoxine will wipe it out, but the neurotransmitter release will be there. Your choise.