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Synaptic Plasticity - Science topic

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Hello everyone,
I am performing some fEPSP recordings to study LTP on hippocampal slices. I am using a submerged chamber to maintain my hippocampal slices.
However, my baseline is very unstable, with constant fluctuations of fEPSP size (see figure attached). These changes are rather slow.. gradually oscillating from minimal to maximal values in 15-30 minutes.
Does anyone have ever experienced something similar? Do you know what can I do to solve this issue?
I don't think this is a problem with the stimulator electrode/box because it happens in both my two chambers and I also changed the stim box and the problem is still there..
Thank you for your feedback!
Diego
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Hi Diego,
I think the issue could be due to some sort of physical instability.
I've found with electrode and slim box issues normally theres more of a pattern to the fluctuations and not random changes in FV/EPSP amplitude. And when I've had issues with slices its more of a constant rundown issue.
So I'd try looking at things like are your manipulators moving in any way, is your suction constant, is there any tension in the cables, does the table feel like its floating, does using a different perfusion pump help.
How do you perfuse the slices? Is there a drip system to prevent oscillations from the pump?
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The brain remains dynamic even in old age and can benefit from mental exercise. Thus, it is important to understand the concepts of positive neuroplasticity and negative neuroplasticity and how these mechanisms support or decrease cognitive reserve.
Neural plasticity, also known as neuroplasticity or brain plasticity, can be defined as the ability of the nervous system to change its activity in response to intrinsic or extrinsic stimuli by reorganizing its structure, functions or connections. A fundamental property of neurons is their ability to alter the strength and efficiency of synaptic transmission through a large number of activity-dependent mechanisms, generally referred to as synaptic plasticity. Research over the past century has shown that neural plasticity is a fundamental property of the nervous system in species, from insects to humans. Indeed, studies on synaptic plasticity have not only been an important driver in neuroscience research, but they also contribute to the well-being of our societies because this phenomenon is involved in learning and memory, brain development and homeostasis, sensory training and recovery. brain damage. However, despite intense research into the mechanisms governing synaptic plasticity, it is still unclear exactly how plasticity shapes the morphology and physiology of the brain. Thus, the study of synaptic plasticity is clearly still important if one wishes to fully understand how the brain works.
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I wish you well with your research and look forward to your findings! From extensive experience working with movement as medicine from infants to the elderly, we confirm the ability to alter the strength and efficiency of synaptic transmission through a large number of activity-dependent mechanisms. We confrim that it works, we look forward to collaborate to investigate HOW it works.
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I am currently doing a project on long term plasticity in the amygdala, particularly the BLA. As part of preparing for my own experiment, I have been looking at some of the recording techniques and stimulation paradigms that others have used to induce LTP there.
From my understanding, changes in EPSPs recorded in current clamp are one way that LTP can be quantified. What I'm confused about is that some papers say they use "current clamp", but are still fixing the voltage. For example, this paper
states in the methods
"Cells accepted for analysis had resting membrane potentials more negative than −60 mV.(The majority of neurons in lateral amygdala resemble pyramidal neurons [31, 35].) In most cases, cells were held at about −80 mV by DC current clamp to prevent action potentials, except during tetanization or pairing procedures."
I've always been told (and have read from multiple sources) that you only fix voltage in voltage clamp, whereas voltage is allowed to change freely in current clamp.
Could someone clarify what is going on here? Is this perhaps a recording convention that extends beyond LTP experiments? Many thanks in advance.
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For this case, the main reason to hyperpolarize the cell is to reduce the likelihood of the cell firing action potentials except during the tetanic, LTP induction protocol. The closer the celll sits to threshold, the more likely you may get action potentials spontaneously or in response to evoked EPSPs. Random spikes during the recording and while evoking EPSPs can be problematic for inducing LTP and for measuring EPSP amplitude.
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I am trying to study synaptic plasticity in hippocampus in urethane anesthetized rats (in vivo). Following are the coordinates recording electrode (Bregma −4.4, lateral 2.0–2.25, depth 2.0–2.7 mm), and the stimulation electrode (Bregma −3.4, lateral 2.5, depth 2–3 mm).
Is there any procedure to optimize the amplitude of the population spike. What are the factors to be considered for optimization.
Thanks
Pradeep
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Pradeep Jayarajan Where are your recordings from within the hippocampus?
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Hello everyone!
In your experience, changes in the expression of which genes better represent synaptic growth and activity? I look for a marker of vesicular pool (quantity of synaptic vesicles, more precisely) and a marker of synaptic activity itself - whether the synapse is actively working or silent. I'm thinking about SV2, VAMP-associated proteins and synaptotagmin.
Do you have any suggestions?
P.S. Method - real-time PCR
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Hi everyone,
for synaptic activity i would suggest for c-Fos or ARK. They are the best characterized IEG for monitoring synaptic activity.
Cheers
Luca
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Electroconvulsive therapy (ECT) as an established technique and technology in a variety of psychiatric and neurologic conditions may require reassessment with relevance to memory loss and brain damage. The therapeutic benefit of ECT will depend on heat stress induction and inactivation of the heat shock gene Sirtuin 1 (Sirt 1) with relevance to mitochondrial apoptosis and neuron death. ECT and neurostimulation in diabetes and neurodegenerative diseases should allow intact suprachiasmatic nucleus (SCN) function and activation to avoid sleep/ wake disturbances, heat shock response dysregulation and induction of circadian abnormalities. ECT technology should be used with caution (dose/frequency) in psychiatric individuals with synaptic plasticity defects with relevance to unhealthy diets and core body temperature dysregulation. ECT application may now involve plasma tests for various Sirt 1 regulated protein hormones with relevance to SCN function and circadian signals.
KEY WORDS:
Electroconvulsive therapy; Mental disorders; Screening tests; Heat shock gene; Neurodegenerative diseases; Suprachiasmatic nucleus; Sirtuin 1; Diabetes; Circadian rhythm
REFERENCE: Electroconvulsive Therapy and Heat Shock Gene Inactivation in Neurodegenerative Diseases. Ann Neurodegener Dis 3(1): (2018). 1028.
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Brain stimulation therapies include Electroconvulsive Therapy (ECT), Vagus Nerve Stimulation (VNS), Deep Brain Stimulation (DBS), Transcranial Direct Current Stimulation (tDCS) and repetitive transcranial magnetic stimulation. Brain stimulation therapies such as ECT should be reassessed with relevance to dose and frequency for the treatment of psychiatric and behavioral disorders. The major concern with ECT is associated with excessive heat generation and inactivation of genes required for neuron survival
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Hi dear colleagues I need to record AMPA and NMDA currents of CA1 neurons in p21-p32 mice but I do not know what internal solution to prepare. There are papers that use CsCl, other Cs-Glu and other K-Gluc and differ in the use of QX314 (besides that they use different reactants). I really do not know what internal solution is better for this type of records and I am starting in the patch-clamp world. Also, is it possible that you can recommend a publication that supports the use of your internal solution? What care should I have when preparing the internal solution (ATP / GTP) and during the electrophysiological record? I appreciate your help very much
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Hello!
For voltage-clamp recordings the Cs-Methanesulfonate based solution is used in our lab.
127 CsMeS, 10 NaCl, 5 EGTA, 10 HEPES, 6 QX314, 4 ATP-Mg, and 0.3 GTP; pH adjusted to 7.25 with CsOH
It allows to record synaptic currents in pyramidal neurons in slices for prolonged period of time. As it contains the blockers of potassium and sodium channels, the input resistance in whole-cell configuration is rather high (about 250-400 MOhm). I would recommend to voltage-clamp the cell at -50 - -20 mV most of the time during the experiment. Prolonged recordings at more negative voltages decrease the cell viability during the recording. This solution can't be used for current-clamp recordings.
For current-clamp recordings the K-Gluconate based solution is used:
135 K-gluconate, 10 NaCl, 5 EGTA, 10 HEPES, 4 ATP-Mg, and 0.3 GTP (with pH adjusted to 7.25 with KOH)
It allows to record membrane voltage and action potentials in pyramidal neurons in slices. However it does not perform too well in voltage-clamp mode, as the input resistance in whole-cell configuration is quite low (60-180 MOhm depending on cell type). If you use it in voltage-clamp recordings I would recommend to clamp the cell at -90 - -60 mV most of the time during the experiment.
Both of the solutions are prepared in the same manner. First we dissolve all the components in water, except ATP and GTP. After that we rougly adjust pH. Then we rapidly add ATP and GTP, make a final adjustment of pH (ATP decreases the pH) and freeze the solution in 1 ml tubes. The osmolarity of the resulting solutions is about 300 mOsm (for better patching it should be a little lower than the osmolarity of the extracellular solution).
Good luck with your experiments.
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The latest antidepressant vortioxetine has a new mechanism of action that combines direct 5-HT receptor modulation and SERT inhibition. In fact it is a 5-HT3, 5-HT7 and 5-HT1D receptor antagonist, 5-HT1B receptor partial agonist, 5-HT1A receptor agonist and serotonin (5-HT) transporter (SERT) inhibitor. The result of this target profile seems to be the modulated control of neuronal activity in key areas of the brain involved in MDD but also the enhancing of synaptic plasticity and cognitive function.
Any clinical experience of  effectiveness in the treatment of cognitive disorders?
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Please look in the Attachment some information from my library.
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I currently perform fEPSPs recordings in the stratum radiatum of dorsal CA1 while stimulating Schaffer collaterals in mouse hippocampal brain slices. I cut 300 micrometers thicks sections at 45 degrees angle from the coronal/sagittal plane, in order to preserve the fibers in the dorsal hippocampus. The cutting speed is 0.06 mm/s. I use cold choline for both cardiac perfusion and cutting and at 32 degrees for recovery (10 min). I then move them in normal aCSF.
Unfortunately, the amplitude of my fEPSPs is only as large as the fiber volley.
I am afraid that this is mainly due to the survival of my slices (from decapitation until the last slice it takes me more than 30 min) but I've been suggested also to use bicuculline to increase the excitatory events. Could this be helpfull?
Thank you!
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Hi Vincenzo,
Two things came to my mind are the location of your stimulation electrode and your recording electrode.
1) If you have a bipolar stimulation electrode, in order to stimulate as many fibers as possible it is better if the two feet of your stimulation electrode are not parallel to Schaffer Collaterals but span them as in the figure attached (from Power JM et al.,1997, J. Neurophysiology). Another reason would be that your stimulation electrode is too far away from your recording electrode.
2) As Stylianos mentioned in his 5th point, your recording electrode may not stand exactly on str. radiatum but lay more apically (close to the str. lacunosum moleculare). The 2nd attached figure (B)  may give you an idea (from Kloosterman F. et al., 2001, J. Neurophysiology-though the one of the stimulation electrodes is on CA3b in this paper). You can also check the 3rd attached figure from Isomura Y et al.,2002, J. Neurophysiology
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Does anyone know of any intracellular solutions or methods for reducing or preventing LTP washout after going whole cell? Canoncial thinking suggests that after 5 minutes dyalisis of the cell resulting from the pippette solution starts to "washout" key molecular components of LTP. I would like to extend this time window if at all possible to give drugs in my pippette solution time to diffuse throughout as much of the cell as possible.  Along with this, are there any studies that have measured the diffusion kinetics of drugs introduced into cells via patch pipettes? This would be in pyramidal neurons, area CA1. I'm sure people have done this with florescent labels etc. just not sure if the kinetics would be the same for a drug of interest.  
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Personally, I think the dogma of "5 minutes" is a bit over sold, as I've seen 50% LTP after 15-30 minutes of whole cell. That said, some people still get pretty worked up about this concept. What I would typically say is "patch with high resistance pipettes", but seeing as you want to diffuse in a drug, I think you should try another approach. Go in, patch the cell, hold it for a couple of minutes, then pull off. Wait for half an hour/45 minutes, then go in and patch it again. I know it's not perfect, but I think it's a good happy medium. Give it a try with some Alexa to see how it compares to just holding the cell for 45 minutes.
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Here we are working to get consistent results in rats hippocampal LTP in-vivo following some of the research articles. But we are facing some trouble shooting issues like we could able to find fEPSP and we could achieve steady state Population spike for at least 10 min.
And the problem is that we couldnt induce LTP with theta-burst of the Schaffer collateral pathway in every rat we tested where we could see only in 1 out of ten tested rats, so what could be the reason.
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Following are the major points which you should consider. I think one of these factors might be causing not to induce LTP.
1. Tip of your electrodes (recording as well as stimulating). Bad tips can damage neurons and you cannot get long lasting recordings. the major reason of failure in electrophysiology experiments.
2. Use of too much stimulation power/current.  You should be using 40-60 % of the maximum spike amplitude for baseline recordings.
3. Too much disturbing movements of the electrode tips and movement. this can cause damage in the fibers and would result in poor recordings. There should not be viration in the area and on the table.
4. Check that you are injecting proper stimulation current. Over stimulation can cause damage.
5. Also make sure which drug are you using for the anesthesia. It also matters.
6. Age of the animal is important.
I hope this helps.
Best wishes
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Can you use 4-AP to assess excitability in dendritic FIELD POTENTIAL recordings from medial perforant path to dentate granule cell pathway?
I am trying to assess excitability of MPP-DGC (medial perforant path-dentate granule cell) synapses and have 4-AP available in lab. I am already doing other field experiments animals and therefore cannot do whole-cell yet to answer this question. I was wondering if I could get some prelim data in the mean-time by getting a baseline field recording at MPP-DGG (1 stim, every 30 sec, 100us duration) and then apply 4-AP (100 uM) to assess changes in excitability when Kv channels are blocked. 
Just need to know if I am even in the ballpark for assessing excitability at these synapses in each group. 
I am already doing separate experiments with picrotoxin and therefore am already looking at inhibitory transmission in this regard. 
Please help!
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Thank you for your timely response William! This does help a lot. Information about how well the cell can get signals to the the soma is still pertinent information in my study. 
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Usually, quantal size and release probability were determined from the amplitude and frequency miniature EPSCs(mEPSC), I wonder why does NMDAR-mediated EPSCs by MK-801 can be used to examine basal release probability?And what is the mechanism?
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It is very difficult to measure release probability.   I would not consider mini frequency a reliable indicator.  You can assess release probability indirectly using the paired-pulse ratio of AMPA receptor EPSCs, but it depends on the characteristics of the receptors a little (do they desensitize, do they saturate), and it might be nice to know whether an effect is presynaptic. 
Using the NMDAR EPSC is an approach.  The basic idea is that MK-801 is a use-dependent blocker.  It gets stuck in the pore when the channel is activated, is my understanding.  The logic is that when release probability is high, lots of glutamate is released in response to a presynaptic action potential, lots of NMDA receptors activate, and a high proportion get blocked by MK-801, so the next time you look at the NMDAR EPSC amplitude, it will be smaller.  When release probability is low, less glutamate is released, not that many NMDA receptors activate, and only a small proportion get blocked, so the rundown of the NMDAR EPSC will be slower.
What you compare then is the rate of rundown of the NMDAR EPSC.  Faster rundown = higher release probability.  Then you don't have to worry about baseline differences in synapse size, or saturation.  However, it will not report to you an actual value of release probability, just a relative number for comparing synapses.
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Dear Researchers,
We are working on acute hippocampal slice preparations for electrophysiological studies using MEAs' (P28 Wistar rats).
We are getting good viable slices with spontaneous and evoked signals.
We wish to know various drug testing protocols using acute hippocampal slice preparations for electrophysiological studies in view of increase in spike rate, amplitude, burst analysis, LTP, LTD etc.
Would you please share your expertise with any standardised drug testing protocols using MEAs'
Thanking you,
Best Regards,
Dr. Grandhi V Ramalingayya
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I guess you only have the option of bath perfusing a drug and washing it of after sometime.
However, you would have some circuit level artifacts ie due to the activity on certain neurons which might be connecting to and hence activating some other neurons on the dish.
But to start with its not a bad idea.
Regards,
Debanjan
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Under a blue light stimulation on ChR2-expressed interneurons, which is not decided whether through a pre- or post- synaptic mechanism modulating the neurotransmission of a disynaptic transmission close to it, the first phase of the EPSC was reduced, and PPR was found increased compared to without the light stimulation on those interneurons. 
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Essentially paired-pulse stimulus measures the short term plasticity characteristics of neurons. It is a characteristic of the probability of release of the presynaptic neuron and the type of receptors present at the postsynaptic neuron. PPR can be in both directions (facilitation or depression) and there are various explanations for what that means (as Bryan Krause mentioned).
A nice paper that goes over mechanisms for short term plasticity is: Fioravante & Regehr Current Opinion in Neurobiology 2011, 21:269–274
The postsynaptic receptor population should not have time to change during a paired pulse stimulation and if the treatment causes an effect by binding to the post-synaptic receptors, then the PPR should remain stable. While the amplitude of individual currents may be inhibited or enhanced by the treatment, the release of NT should be stable during the pulse and NT interactions with receptors should be equally effected by the postsynaptic change - thus the PPR is unchanged.
However, if the drug was interacting at the presynaptic site and changing the way the presynaptic neuron acts when it receives an action potential - its probability of release - then the way the presynaptic terminal responds to a paired pulse stimulus should also change - and the receptors see something different from before  - and PPR changes.
Its not conclusive, but a change in PPR after a drug treatment, indicates that the drug may be acting at the presynaptic side of the terminal.
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I am looking for a genetic or pharmacological tool that allows me to block the occurrence of synaptic plasticity in a specific class of neurons. Any ideas? Thanks
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Hi Michele,
Knocking out or reducing CaMKII activity is the best bet for inhibiting synaptic plasticity in glutamatergic neurons, as it is well established that CaMKII knock out mice do not show input specific plasticity. However the bigger question is do you have a marker that can be used to target your neuron of interest. Although excitatory neurons are classified based on physiology, but unlike inhibitory neurons there is not much literature about molecular markers based sub classification of excitatory neurons. 
Best,
Anurag  
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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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I have read a lot of papers from neuroscience ... psychology ... Biological sciences... they talk about the roles of astrocytes in synaptic plasticity? Is there any evidence to consider astrocytes as the main player of plasticity not the neurons?..is it possible that the plasticity definition based on changing of astrocytic strength by synapses, as well as altering synaptic strength by asrtrocytes? 
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Dear Bassam:
"Neural" includes neurons and glia.
The concept of "plasticity" as the strengthening of synapses apply only to neurons, because the astroglial network does not have synapses (astrocytes communicate by means of gap junctions). 
Neuron-astrocyte interactions is a new, integrative field in neuroscience. There are some models attempting to interpret this interacting system. Vera Maura has done a good job of understanding these complex interactions using the retina as a model of the whole brain.
I have built a model in which neurons execute cognitive operations (processing sensory signals, selective attention, memory, motor control), while the astroglial network instantiates feelings (all kinds of feelings - from basic sensations as hunger and thirst to perceptual 'qualia' as the feel of color, sound, taste, smell, also emotional feelings as joy and sadness and social feelings as love and hate).
In this model, neurons induce hydro-ionic waves in brain tissue, having the astroglial network as the master hub that propagates the small waves to the whole system and allow their interferences. For instance, when we are facing dangerous situations the amygdala triggers a wave of fear in brain/body tissue. The feeling of fear is not inside the amygdala, but in the living tissue, having astrocytes as the master hub that broadcasts it to the whole brain and body.
Constructive interferences in the astroglial network produce global waves that instantiate feelings; these waves feed back on the neurons and modulate their activity. The feedback of astrocytes on neurons is part of the neural plasticity phenomenon. According to the model, the wave of feeling reinforce the neuronal patterns that feel good, and depress the patterns that feel bad. There are several well known mechanisms that execute this modulation, for the details please read the paper below.
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If, and eventually, when, the model of neural spikes and synaptic strengthening will be undermined and replaced by the Aur and Yog Neuro-Electro-Dynamics?
Are there new studies/experiments confirming the neural memory stimulus in the form of deformation and conformation of synaptic proteins, dendrites and axons under the influence of local electrical fields?
Aur and Yog pointed to the possibility of developing a new paradigm of memory stimuli reaching neural synaptic fields. These stimuli are in the form of distributions of micro-electric fields interacting with the local micro-fields of peptide chains constituting the protein structure of each neuron. Short- and long lasting deformation of these proteins may underlie of short- and long-term memory. This mechanism called Neuro-Electro-Dynamics, helps us browse processing sufficient amount of information in the process of pattern recognition, planning, decision making and other higher mental functions. This revolutionary hypothesis could be verified experimentally. However, it seems that it does not raise much interest of experimenters, which means that the neural spike model and synaptic strengthening continues to function as a current paradigm.
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Dear Wieslaw,
My opinions are:
1) Without taking into consideration the roles of ions and water, and the interactions of neurons with glial cells, extracellular matrix and blood, there is no future for NED;
2) The model of neural spikes and synaptic strengthning is not wrong, and needs not to be replaced, but extended to include the dynamics of ions and water in brain tissue;
3) Memory phenomena is well accounted by the LTP paradigm, which is part of the synaptic strengthning model. What is there to be explained is not learning and memory, since these phenomena are well explained by the current dominant paradigm. The issues to be explained are those related to basic sensations (as pain, pleasure, hunger, thrist), feelings and conscious gestalts, which are not explained by that model.
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I am doing BLA LTP field potential measurements, and I use one time 100 Hz stimulation.
I guess I should leave it out, as this might affect the measurement, but it is also bad to just leave out data. What is the best solution?
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Misapplied protocol is as legit as a reason to leave the data out as any :-).  By the way, douling the number of 100 Hz from 1 to 2 will change the size of LTP.
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Right now, I am doing transverse hippocampal slices. The acsf I use is: 11mM glucose; 120mM nacl; 26mM NaHCo3-; 3mM KCL; 1mM NH2PO4; 1.2 MgSo4; 2mM 147.01. I cut the slice in calcium free ACSF and put them in Ca containing acsf. I use mice 3-6 weeks old. The basal response I am getting is stable (at least for 30 minutes). I stimulate at 40% of maximum response. I stimulate from the schaffer collateral and record from the CA1 (not a population spike). The experiments are done in room temperature.
The problem is that I cannot induce a decent LTP. I've tried theta burst and multiple trains of high frequency protocols (100hz for 1second; 20 second intervals between trains). I only get 20% increases in fEPSP not like the huge 50% in papers. I also checked the protocols and they were correct.
Does anybody have any idea? This has been driving me crazy.... 
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Dear Tony,
You are recording at room temp, that could be the reason of having low-magnitude LTP. You could try recording at 32C (that is what we are doing in the lab). 
Good luck!
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While working on acute hippocampal slice electrophysiology using non perforated MEAs, we have used self made grids using washer type (metal alloy rings) and cotton threading as mesh.We have used McILwain tissue chopper for making hippocampal slices. We are not getting the spontaneous activity at all.
Will the metal alloy have a great impact on recordings using MEAs? Please help us in using the grids for non perforated MEAs.
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Hi Grandhi,
Selecting the right cutting solution, specially in the case of hippocampal slices, is a critical step, as it will dramatically affect the health of the slice. The exact composition varies from group to group and also depending of the age of the animals used. In general, a sucrose based ACSF works well with young animals whereas is not so great with older ones. A similar approach is to use a sucrose based but with not a full substitution of sodium. When working with older animals NMDG or choline based ACSF tends to work better. I would recomend you to find a paper that does the type of experiments you want to do and at the same age and try to mimic what they use. Alternatively you could try the "recovery method" proposed by Jonathan Ting that is supposed to give you very healthy slices even from aged animals.
Hope this helps,
Ezequiel
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I am a very new user on IGOR and neuromatic and wish to know how one can do NSFA in there.
The biological question I am interested in is to estimate whether there is increase in AMPAR number or AMPAR conductivity that underlies the synaptic potentiation I am looking at in the hippocampus??
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I've done NSFA on mIPSCs and I suppose it's similar for the EPSCs. You can perform the calculations in Igor by writing a simple procedure to (a) scale the amplitude of each event to that of the average mEPSC and (b) plotting the mean variance against mean current and fitting a parabola. A good example can be found in this paper: http://www.jneurosci.org/content/22/4/1328.full.html
Activity Deprivation Reduces Miniature IPSC Amplitude by Decreasing the Number of Postsynaptic GABAA Receptors Clustered at Neocortical Synapses
Valerie Kilman, Mark C. W. van Rossum, and Gina G. Turrigiano
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I have time dependent practical data of neuron system. This is a spiking data. I like to calculate synaptic weight with time between two neuron. I have input signal and output signal, which are experimentally measure with time. Any one can suggest that how i can calculate synaptic weight and is there any way that can tell me about synaptic plasticity?
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Dear fellow researchers,
I just started doing field stimulation in the CA3 region of wild-type (WT) SVE129 mice (2-4 month old) to record long-term potentiation in the CA1 region. However, I have major problems seeing LTP in young sve129 mice slices. Instead of an instant excitatory postsynaptic potential (EPSP) elevation, which is maintained at least 30min (typical LTP), I see a slow incline over 5 to 10 min. I already tried to improve my slicing, double checked my solutions and changed batteries in the stimulator. I would be grateful for your help and advice how to fix it.
Thanks!!
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 Dear Flo Hi, please send me your e.mail address so that I can send, in privet, a dedicated paper concerning to your question or difficulties. 
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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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What could be the reason behind synaptic enhancement by altered testosterone levels? I found increased level of testosterone by pre-treating rats with Danazol alters the post synaptic calcium release which finally alter the plasticity of the synaptic neurons and increases the glutamate release in synapse. Is there any possible role of testosterone in learning and memory function?
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Dear Dinesh,
Testosterone can affect both intrinsic membrane properties and synaptic transmission in the nervous system. There are many ways and potentials routes how it may cause these changes. Among are the effects on nuclear receptors or gene expression, and the transformation of testosterone to estrogen via aromatase enzyme and than act on estrogen receptors. For details see my following publication.
best wishes, Refik
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Is there a way to interpret an increased frequency of mEPSC while both presynaptic vesicle number and dendritic spine density are decreased?
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Dear Huxing,
First of all i dont know which neurons you are talking about and what is causing the spine density to drop. If you tell me a bit more I may be able to give you a bit more detailed answer. Usually, there is a direct correlation between increased mEPSCs with increased synapse and spine numbers. How confident are you that the spine numbers are decreased? Spines can be very dynamic, for instance, sometimes spines can get very short-stubby or shift from proximal to peripheral dendrites with maturation in motoneurons that I study. See my paper below for example.
best wishes, Refik
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One of the most accepted theories for synaptic plasticity is STDP, but it fails when the frequency of neural firing is very high or very low, since it works in medium rates , my question is: is there any model for plasticity that consider more than one factor?
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Both the Clopath and the Sjoestrom models sound important. However, the word "plasticity" has now taken on so many meanings that I doubt that any single model, particularly one limited to spike-timing, can handle all the instances of 'plasticity." I am not aware of a math model of the old work by Mersenich et al on cortical allocation of finger regions in the macaque. The range of intracortical oscillatory activity known today varies from <1 to 200 Hz, with faster peaks up to 600 Hz. Hippocampal episodic and retrieval is linked to theta-gamma oscillations, where "gamma" may range from perhaps 40-200 Hz. As Dehaene and colleagues have shown, learning of conscious visual stimuli (plasticity) is also associated with cortical binding and propagation in visual cortex, showing resonant oscillatory activity of high amplitude and long-distance activity spread. Spike timing could be linked to population oscillations via single unit phase-linking to a dominant waveform. Pop oscillations in turn can change single unit firing rate by adding to regional depolarizing voltages. Thus unit activity and population oscillations could be closely linked. Steriade (2006) has indeed suggested widespread slow-to-fast oscillations, yielding complex waveforms that may peak in many places in cortex. 
Do we have models of plasticity? Only of some kinds of plasticity.   This is a major challenge, and we would need to know more about neuronal transmitters and receptors, glutamate and NMDA, etc. 
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Hello, 
The redox signaling and the activation/nactivation of proteins by specific oxidation is a currently hot topic of research. Nox2 KO mice, resembling the human chronic granulomatous disorder, are available and present some plasticity modifications and mild cognitive decline in addition to the immune problems.
However, I was not able to find any pathways relating LTD and LTP to redox signaling cascades, with elucidation of the envolved pathways etc.
Do you know any work that has been done on this or anyone who is currently working with that? 
Thanks for your time!
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Hi Tiago,
In addition to the papers suggested by Tatyana, I can recommend a quite recent review on this topic:
Reactive Oxygen Species in the Regulation of Synaptic Plasticity and Memory http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078504/
Hope this helps. Norbert
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I am trying to understand how depolarisation induced with KCl or glutamate/ NMDA or any such stimuli leads to the balance of actin polymerization: actin depolymerization in neurons. Is it possible that actin depolymerization precedes actin polymerization as an immediate step post depolarization. How does it fit the spine dynamics post depolarization. This relates to temporal dynamics of actin organization in the neurons post depolarization. I think if actin depolymerization precedes polymerization for few minutes (30-1 hr) post depolarization it will help the spines to be flexible and let the movement of receptors and function of other spine machinery possible. It might also help to let the new dendritic mRNA and newly synthesized proteins to enter the spines and take their appropriate functions. Does this concept sound logical? Or are there other possibilities?
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Hi Apurva,
 I think this paper will help your conceptual  questions but this one is more focused and single molecular study but the references will  be more helpful.
Neuron. 2015 Aug 19;87(4):813-26. doi: 10.1016/j.neuron.2015.07.023.
A Temporary Gating of Actin Remodeling during Synaptic Plasticity Consists of the Interplay between the Kinase and Structural Functions of CaMKII.
Kim K1, Lakhanpal G2, Lu HE3, Khan M2, Suzuki A1, Kato-Hayashi M4, Narayanan R4, Luyben TT5, Matsuda T6, Nagai T6, Blanpied TA7, Hayashi Y8, Okamoto K9.
Best
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I am recording in vivo, from anaesthetised rats, trying to assess changes in synaptic plasticity or at least cortical excitability. 
The logistics of these experiments requires that I use metal electrodes, which can be bent out of the way to have a clear skull surface for later stimulation via another method. 
Currently I am stimulating primary motor (M1) cortex and recording contralateral M1. The necessity of bendable metal wires means I have had to rely on field excitatory postsynaptic potentials to measure any changes in excitability/efficacy. 
My question is which component of the field response would be best to measure for cortical fEPSPs? I am measuring the initial slope, the second slope and the peak-to-peak amplitude. 
Additionally, if anyone thinks this is a fools errand or has advice on another method (single unit perhaps?) I should pursue, I would be greatly appreciative. Cheers. 
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Hi Matthew,
I'm an old alum from the Anatomy Dept Otago (in fact, I get the feeling my Old boss might be your supervisor). So one thing I'd say straight away, is go talk to Cliff Abraham, if you haven't already (it seems hard to believe you haven't, but...).
That aside, cortical fEPSPs are messy at the best of times, and worse in vivo. Go look at Agmon and Connors (1991) if you want to see the best that they can possibly be.
This brings me to my next point, The exact placement of your electrode, and to a lesser degree its impedance, is going to vastly alter your fEPSP waveform. So in a best possible world, you'd have a silicon probe that gave you reliable recording sites through the cortical layers, a la Swadlow and Gusev 2000.
That said, if you can place your field electrode accurately, and hence get stable waveforms within animals, and vaguely similar waveforms between animals, then the slope of the EPSP waveform (not fiber volley!) seems as good as anything. The negative going area under the curve might be good too. Play around. For instance, stimulate contralateral M1 harder and harder (i.e. run an I/O curve) and see what changes. You would expect your measure to vary in a monotonic fashion with your stimulus current (though if it's too linear, then be worried it's just a fiber volley). Wash some CNQX onto the surface of the cortex to be sure you're really measuring a synaptic event. Also try a normal plasticity protocol, i.e. normal HFS. I wouldn't expect real LTP, but you should see a reasonable boost in EPSP amplitude for a few minutes. 
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Hello everybody.
I have seen through publications that some labs have been routinaly able to induce very long-lasting in vitro LTD in young and adult animals. I would like to ask you if you can give me some details of the methods to achieve it.
I am trying to get LTD in my hippocampal slices from ~10 weeks old mice, but so far it has been almost impossible. I have been using both electrical stimulation (single 900 pulses at 1 Hz) or the application of mGluR2/3 agonist LY354740. I am stimulating the medial perforant path and recording fEPSPs in the dentate gyrus, at room temperature.
Does anybody have some advice to get stable LTD?
Thank you very much in advance for your contributions.
Diego Fernández
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Hi Diego, as you want to focus on chemical mGluR2/3 dependent LTD, together with all suggestions from our colleagues, I suggest you to have a look on the papers below. Both papers explore the roles of mGluR2/3 in the cortical – Temporo-ammonic path to CA1 region.
1.    Ceolin L et al., Study of Novel Selective mGlu2 Agonist in the Temporo-Ammonic Input to CA1 Neurons Reveals Reduced mGlu2 Receptor Expression in a Wistar Substrain with an Anxiety-Like Phenotype. The Journal of Neuroscience, May 4, 2011 • 31(18):6721– 6731 • 6721
2.    Hanna L et al., Differentiating the roles of mGlu2 and mGlu3 receptors using LY541850, an mGlu2 agonist/mGlu3 antagonist. Neuropharmacology 66 (2013) 114-121
Good luck.- Zuner
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I am planning to perform a pharmacological study to assess hippocampal neurogenesis via IGF-1 receptor inhibitor. But most studies were performed  in vitro or in vivo (systemic). So whether this inhibitor of IGF-1R (such as AG-1024 or picropodophyllin) can be  intracranially injected by stereotaxic localization and what is the optimal dose? Thank you!
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Ideally, you can infuse over hippocampus, because as mentioned by Giovanna you can induce gliosis in hippocampus.
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The question is based on the wide use of neuronal activity markers including Arc to dissect neuronal circuits activated in certain behavior or to certain stimuli. Does anyone have any idea of whether changes in these markers indicate changes in synaptic activity (i.e. short term or long term synaptic potentiation) or intrinsic neuronal activity (i.e. up-regulation of hyperpolarization-activated current, Ih)? Any litterature on this topic are welcome! :)
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IEGs are surely not simple activity markers, but reflect "above threshold" significant stimulation e. g. a sensory stimulus like a taste or smell do not elicit IEG expression when familiar but do so when experienced for the first time. So the novelty adds the significance. It does not make sense to alter gene expression for every "normal" signal transmission, but only if something exceeds basal activities like coincidence of stimulation.
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I am having trouble to replicate one of the main postulates about the expression of late LTP, concerning its dependence on de novo protein synthesis. I performe fEPSP recordings on the Schaffer collateral to CA1 synapse in rat hippocampal slices. Stimulate with 4xHFS (100 Hz, 1 sec), every five minutes. However, I cannot see an effect using anisomycin 30 µM on late LTP, even if I preincubate the slices in anisomycin for 2 hours before the experiment.
Villers et al. (PLoS One. 2012;7) already reported no effect of anisomycin or cycloheximide on late LTP. In this paper they argue that the protein-synthesis hypothesis for development of late LTP should be reconsidered.
What do you think? Do you have any idea about why these experiments are not working?
Thank you in advance for your help!
Diego Fernández
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Building on to what Gabriele mentioned:
How often are your test pulses? That matters too, see Fonseca studies listed below.
At slow rates of test stimulus, I had to wait many hours. Others get faster effects with more frequent test pulses.
Redondo RL, Okuno H, Spooner PA, Frenguelli BG, Bito H, Morris RGM (2010) Synaptic Tagging and Capture: Differential Role of Distinct Calcium/Calmodulin Kinases in Protein Synthesis-Dependent Long-Term Potentiation. J Neurosci 30:4981-4989.
Also, I advise that you keep a non-stimulated pathway as control at all times so you can monitor overal slice excitability/health.
Fonseca R, Nagerl UV, Bonhoeffer T (2006) Neuronal activity determines the protein synthesis dependence of long-term potentiation. Nature Neuroscience 9:478-480.
Fonseca R, Nagerl UV, Morris RGM, Bonhoeffer T (2004) Competing for memory: hippocampal LTP under regimes of reduced protein synthesis. Neuron 44:1011-1020.
Fonseca R, Vabulas RM, Hartl FU, Bonhoeffer T, Nagerl UV (2006) A Balance of Protein Synthesis and Proteasome-Dependent Degradation Determines the Maintenance of LTP. Neuron 52:239-245.
Good luck!
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I am looking for a trans-synaptic anterograde tracer - does anyone know if wheat germ agglutinin conjugated to an alexa fluor is able to cross synapses?
I would like to do an in vivo injection into the ventral Hippocampus and expect to see labeling in the mPFC... I know that this is a distance, but I only truly need WGA to get to the second order neuron, so I feel like a viral system (such as HSV129) is overkill. 
Has anyone tried WGA-fluor for a similar application? Thanks in advance! 
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Just to add to the information mentioned above about WGA:
- It apparently tends to induce inflammatory responses in the injection site moreso than other tracer chemicals.
- It is allegedly taken up by some glial cells between first and second order neurons.
- Not only does circuit length (distance the tracer must travel) make things difficult in estimating survival time, but WGA transcytoses on a rather fast but variable time scale, ranging from one to four days. This effect seems to be concentration-dependent. You'd have to test out time courses and concentrations in several mice to determine the optimal transsynaptic spread. 
With its bidirectional spread, I'm not sure that WGA would be appropriate. You wouldn't be able to isolate transsynaptic anterograde labeling from transsynaptic retrograde labeling. You may want to look into using recombinant Tetanus Toxin C Fragment (TTC), which is preferrentially retrograde and transsynaptic, and tracing from PFC back to vHipp. The main issue there is that recombinant TTC is prohibitively expensive to purchase.
One last general thing - using IHC staining will provide superior visibility of labeled cells compared to visualizing the fluorescently-conjugated tracer as-is (in most cases). This may be important if transcytosis substantially dilutes your tracer over synapses.
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Hi all, 
I am treating dissociated hippocampal neurons at 10DIV with 30 uM glutamate and 1 uM glycine for 10 min before washout and the aim is to look at plasticity events (up/downregulation of receptors).
However, I am concerned that this treatment might lead to excitotoxicity and cell death although it is my impression from the litterature that excitotoxicity protocols usually use concentrations and treatment durations that are much higher/longer i.e. high uM to mM range and hours. What are your opinions on this? And if it is too harsh is there a better way to go?
Many thanks and best wishes,
Tau     
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Hi Tau,
I have not used neuronal cultured but I have done similar experiments on glioma cells and have used 10uM of L-Glu for 5 mins. Cells die but timings matter. For my 5 mins, it was fine. But pH maintenance is very essential.  It becomes very acidic the moment you add glutamate. So if you are making a buffer, you need to check pH post glu addition and keep it physiological. 
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Traditional golgi cox stain method can be used in brain tissue slice. However, I did not see in cell culture. Thus, does it need any modifications before applying to cell culture?
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Please see below:
Neurochem Int. 2013 Jul;63(1):35-41. doi: 10.1016/j.neuint.2013.04.009. Epub 2013 Apr 22.
A novel, Golgi-Cox-based fluorescent staining method for visualizing full-length processes in primary rat neurons.
Koyama Y1, Tohyama M.
Author information
1Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan. koyama@anat2.med.osaka-u.ac.jp
Abstract
The Golgi method, a well-known method used for staining whole dendrites and axonal trees of neurons, has been used widely for studying dendritic growth in vivo. Although detailed structural examination of neurons and their processes stained by the Golgi method has elucidated the complicated neuronal circuit, application of the method in cultured neurons has been unsuccessful to date. Here, we report the development of a stable, highly sensitive Golgi-Cox method that allows visualization of full-length processes, including the dendritic spines and the growth cones, of cultured rat neurons. This modified staining method requires: (1) rat cultured neurons fixed with a mixture containing 4% paraformaldehyde and 12.5% glutaraldehyde before impregnation with mercury; (2) rapid freezing of the fixed neurons using dry ice; and (3) immersion of the fixed neurons in Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin-G antibody (Molecular Probes, Eugene, OR) for visualization after impregnation.
Copyright © 2013 Elsevier Ltd. All rights reserved.
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NOTE: This is a continuation from a previous post regarding the issue of fEPSP depression upon induction of LTP. (https://www.researchgate.net/post/Why_is_fEPSP_depressed_upon_induction_of_LTP)
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I have an additional query with regards to this issue of fEPSP depression.
Sometimes, a 1st attempt at LTP leads to immediate fEPSP depression. However after some time, the fEPSP returns back to baseline, and I redo the I-O and successfully manage to induce LTP on this 2nd attempt on the same slice. Is LTP valid in this instance?
In another scenario, I manage to induce LTP on the 1st attempt, but I wait until it falls back to baseline, then I repulsed the slice with HFS for a 2nd LTP induction, which similarly exhibits robust LTP (that drops gradually just like the 1st LTP). Is this 2nd LTP induction also valid for inclusion?
I am aware that LTP induction leads to the synthesis of plasticity-related proteins, hence, I have a hypothesis that it is easier to re-induce LTP on slices that have successfully had a 1st LTP.
Here are also some additional queries I hope to seek for your help in.
Additional Questions
  1. Should the electrodes be placed within stratum radiatum or stratum moleculare (as both have Schaffer Collaterals) in CA1? I was also told that both the recording and stimulating electrode should be on the same Schaffer Collateral pathway (but there's no way to confirm for that visually when lowering the electrodes onto the slices isn't it?)
  2. What is the physiological mechanism behind this LTP induction-induced fEPSP 'depression'? Discussions with my lab mates suggest either stimulation of inhibitory interneurons or overstimulation of glutaminergic neurons, but I don't find them convincing.
  3. Has anyone compared how the use of Morris ACSF (for slice recordings) vs Sucrose ACSF (protective cutting method with supposed better preservation of neurons, used in my lab only for patch clamping brain slices) would affect their findings?
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To answer your 3rd question first---Collingridge's group found that slices prepared in sucrose-based aCSF show reduced LTP compared to normal aCSF:
On your other questions...first, I would say that repeated attempts at inducing plasticity within the same slice are difficult to interpret. LTP is usually thought of as 'de novo', but if you have already given bouts of HFS, then you cannot assume the synaptic state is identical to the way it was prior to the stimulation. There may be some underlying meta plasticity or homeostatic scaling that has occurred, so you have to interpret your data with those caveats in mind.
As far as the best way to induce LTP---what kind of slices do you have? Typically with a transverse section that preserves more of the Schaffer collateral pathway, you can place the stimulator farther away from the recording electrode. This in turn allows you to use lower stimulus intensity during both baseline and HFS, which might prevent 'over stimulating' the tissue (which could explain the short term depression). You might also try an arrangement where you place two stimulating electrodes (one on either side of the recording electrode), to see if the plasticity is pathway specific. There have been numerous papers demonstrating heterosynaptic vs. homosynaptic LTP using this type of approach.
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Is there any relationship between DNA methylation and synaptic plasticity in the brain?
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The attached publications are just a few examples of literature on the role of DNA methylation in memory, as it's been shown to strongly contribute to memory-related plasticity and LTP particularly in the hippocampus, but in general, DNA methylation has been implicated in synaptic plasticity associated with a wide variety of topics from aging to addiction to psychosis.
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Hello,
I'am using a planar multielectrode array system to record field potentials in acute hippocampal slices. In particular I am trying to study synaptic plasticity, but I encountered several problems.     
After having setted my baseline (30-50% of max), I start a protocol, usally 1 train of HFS ( 100hz in 1 second, duration 200 micr-second). After I , unfortunately, find out that LTP goes in rundown. Very often. It is very disappointing.
3 point more in the story:
1) without induction protocol my baseline seems stable for about 2 h.
2) I obtain some LTP with plateau or slow( acceptable) deacay. I mean, it seems that in some conditions ( but I don't konw why ) the induction protocol works.
3) My conditions : about 30 °C; 3,5-4 mil/min ( but often a little bit less beacuse lower flows seem to prolong slices viability); titanium electrodes (64 channels, interdistance 200 micromt. , diameter 30 micromt.) with standard chamber ( electrodes are placed on the bottom of chamber and the slice is over them, submerged, so could be some problems of oxigenations) ; amplifier 1060 series. (Multy channel system ); I use a light mesh to hold the slices and my solution is the same that has  working for years for synaptic plasticity . 
I hope that somebody could help me. In particular I refer to those people  used my planar MEA system.
Thanks in advance
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I don't think the problem is necessarily in your mea. To rule this out , have you tried to induce LTP, within the same conditions, in the "classical way": 1 stim electrode in stratum radiatum and a recording between cell layer and radiatum?
If you can induce LTP in this way then the problem is definitely MEA related. 9 times out of 10 is that your stim electrode is not stimulating appropriately because it's dirty or worn out or the isolation unit is not delivering the right amount of current: this would be compatible with the fact that you can induce LTP only certain times, unreliably but, when you do, it is for good. Do you use an isolation unit? If yes check the batteries. If the isolation/stim unit works fine, it might be the electrode being/dirty worn out: ask the producer to send you a new mea to check out: usually they do.
If the problem is not mea related:
Looking at your conditions I'd say you might be a bit too cold: I'd work around 32-34, especially for LTP experiments, to thermically facilitate the cell signalling necessary to LTP induction and long term maintenance. Also the flow-rate might be a bit high: I'd stay on 2-3 ml/min. Also, what is your Ca2+ and Mg2+ conc?
You could get around the problem changing stim protocol, for example applying 4 trains of 1 sec 100 Hz bursts, 15 sec apart. That is really strong and induces a very reliable ltp. But if the problem is in the stim electrode  being worn out/isolation unit out of batteries, soon or later you will have the same problem again. I hope this helped.
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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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It is said that dendritic spines can naturally occur among primary mouse neurons and their density or morphology can be thoroughly assayed. So can anybody provide any information?
At the moment we want to evaluate effect of some chemicals at synaptic level, but we wonder how valid such effect on cultured spines is, given that synapses need extra modulation in vivo.
Thanks a lot if you could help!
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If you have access to a good microscope (confocal, 2-photon) you can try to measure the number of spines over some period of time. From this you can extract data such as spine density (spines per micrometer), fractions of dynamic spines (new spines, lost spines, transient spines) and their evolution over time etc.
You could also measure intensities which are found to correlate with actual spine volume, as validated with electron microscopy (see link).
One step further is to construct models based on these measured spine parameters in order to make predictions and extract fundamental characteristics of your system.
As you eluded to it is known that primary cultures do make an excess of connections as compared to the in vivo situation, so ultimately any results will have to be validated in vivo. It stills makes sense to start with cultures if your lab is adjusted to those methods, as setting up in vivo studies can take a considerable amount of time (years).
I hope this helps!
Vassilis
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I want to perform a small intervention study with magnesium supplementation on cognitive functioning in patients with eating disorders. In addition to erythrocyte magnesium levels, I want to assess protein levels, epigenetic variation and gene expression levels of genes/pathways that are associated with magnesium.
Animal studies have shown that magnesium enhances synaptic plasticity and cognitive functioning through blocking the NMDA receptor, but this takes place in the brain. I can only get blood samples. Are there any relevant markers or variants that can be measured in blood for this process?   
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The best way? Hmmm...you may look at levels of the following for magnesium: total, ionized, free, red cell, and 24-hr urine (of magnesium).
PTH functioning, sodium, alk  phos, potassium, and calcium deficiencies are common in patients with low Mg.
Gene mutations may include TRPM 6 (deficiency usually discovered in infants), CLCNKB, ROMK, NKCC2, NCCT.
Other ideas include HgA1C, CBG, and tests related to alcohol consumption.
Here's a good article: 
Swaminathan, R. (2003). Magnesium metabolism and its disorders. Clinical Biochemistry, 24(2), 47-66.
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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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For my art model I want some data of synaptic plasticity.
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That depends on the type of plasticity data .. for Spike timing-dependent plasticity (STDP) you should be able to find the papers by Mu Ming Poo & Bi as well as Froemke.  There is experimental data there but you need to  extract it from the figures which they appear in.
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I am looking for a reliable and powerful compound to validate a positive effect enhancing LTP in Schaffer collateral-CA1 experiments (rat hippocampal slices).<br /><br />
I am performing both E-LTP (HFS 100 Hz, 20 stimulations) and L-LTP (4 times HFS 100 Hz, 100 stimulations) experiments at room temperature. However, so far I could not find a compound clearly increasing LTP. I have used nicotine 100 µM and an alpha 7 nAChR agonist. I put the compound half hour before HFS, and wash-out half hour after. However, the slices in presence of the compounds do not seem to be different from the controls.<br />
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So if I have understood correctly, you intent to use a drug that by itself alone does not have any effect but in the presence of suitable LTP induction protocol, is able to cause enhancement of LTP.
Phosphodiesterase PDE4 inhibitors such as Rolipram and Ro-20-1724 are some drugs that by blocking PDE activity cause enhancement of LTP by normal LTP stimulation protocols. Also, as far as I remember, they have no effect on baseline activity. May be you can try these drugs too and verify for yourself in your experiments.
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I am trying to find a suitable model to study certain questions. 
A. Dynamics of molecular subcellular localisation under different conditions.
B. Calcium Dynamics.
To study these two objectives under live- imaging conditions, I would like to know which system will be better. Hippocampal organotypic slice cultures or hippcampal dissociated neuronal cultures from embryonic stage. 
If hippocampal organotypic slice cultures are good model for these objectives, is it easy to observe such subcellular localisation dynamics of molecules under live imaging/ after PFA fixation and immunostaining in slice cultures. I am just doubtful because of the tangling and very close positioning of the neurites in the slices which might be a problem for analysis in slices if there is any change in subcellular localisation of the molecules after certain treatments.
Anyone with experience with this line of work, please help.
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Hi at least you need confocal microscopy and two-photon microscopy, of cause good antibodies and sensitive calcium marker. :)
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Good Afternoon, I’m doing in vivo electrophysiology in anesthetized mice. I’m recording evoked field excitatory postsynaptic potentials (fEPSP) in the CA1 stratum radiatum after the electrical stimulation of CA3 axons. Since I moved a couple of weeks ago to a new setup I’m experiencing a weird response. Basically, after setting the electrodes in the place where it is possible to find a clear evoked response, this response tends to decrease over the time without inducing any specific protocol (Recordings of 120 minutes; evoked response corresponds to 1 pulse each 15seconds). In this experiment I use Isoflurane (5% during induction, 1.5-1.25% during the recording, Iso delivered with compressed air at a rate of 1L/min). I use also concentric bipolar stimulation electrodes (I get a clear response with a minimal electrical artifact with an intensity of 300-500μA and duration of 20-40 μseconds). To record I use glass microelectrodes with a tip of 1-2 μm diameter filled with Pontamine sky blue in sodium acetate. Since this is a limitation to my work, I’m trying to find a possible explanation or issue in my procedure/setup. All the possible hints are very welcome. If you need more details I can send right away. Thank you very much.
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Hi Jose
It may be because of your new set up. Stimulating electrodes may have current leakage or even short circuit and so you will not have a fine stimulating media. That is why you should check the output of stimulating electrode on a oscilloscope.
My other guess is that your new electrode driver may have drift and during time it moves and cut the Schaffer fibers gradually, then your field response will be result of fewer responses and you will judge that as LTD.
Best wishes
Mahmoud
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I need help with some aspects the extracellular recordings. I use mouse hippocampal slices (350microm) and mainly my experiments consist of stimulating CA3 shaffer collaterals and record from dentrides of CA1 pyramidal neuron. I have a problem when I try to induce LTP in particular when i try to stabilize the signal for ten minutes for the baseline, sometimes the field potential continues to increase. I try to reduce the stimulation but in some slices there is no way to obtain a stable baseline, (increases or decreases in other cases). Do you think that the problem could be the stimulator? Or do you have some other suggestion? Thank you very much
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How long do you incubate the slices before the recordings? Try to increase the incubation time, the metabolism will stabilize. Some people incubate the slices for 3-4 hours for the same reason for the late LTP recordings. Also, I would record the baseline for much longer time than 10 min. We usually record 30-60 min of the baseline. In some slices it is simply impossible to get the stable responses. How good is your perfusion and oxygenation? What chamber your are using, submerged or the interface? I have noticed that the responses are much stable in interface chambers. Also, try to reduce the tissue polarization as much as you can - use platinum or platinum/iridium electrodes, and try biphasic stimulation instead of the monophasic one. The stimulator could be tested by measuring the stability of the voltage (with the digital voltmeter or the scope) on the resistor connected to the stimulator output instead of the electrode.
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According the effects of astrocyte in synaptic plasticity and neural function, it's not out of mind that astrocyte may play an important roles in formation and retrieval of spatial memory. But I’m looking for the related mechanism(s).I'll be appreciate who help me to finding my answer.
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Hi Hossein:
Nice question... Well, astrocytes have been involved in synaptic physiology as energetic suppliers. They also have an important role in NT clearance, which is a crucial function for neuronal signalling and all the underlying processes (cognitive or not) for review see: http://www.ncbi.nlm.nih.gov/pubmed/16261178. A more cannonical point of view about synaptogenesis and astrocytes is provided here http://www.ncbi.nlm.nih.gov/pubmed/21068831
An interesting function of astrocytes over synapse physiology is the so-called synaptic engulfement, in which they mediate synapse elimination, more than formation. Is a kind of refinemennt of a pre-existent network that coul be involved in memory formation (through synaptic elimination) http://www.ncbi.nlm.nih.gov/pubmed/24270812
However it's difficult to translate these results into behaviours, although there is a recent paper in which authors link a cognitive improvement with astrocytic function in hippocampus http://www.ncbi.nlm.nih.gov/pubmed/24727294. There are also papers in which changes in astrocytic function/morphology are correlated with behavioural tasks performance or environmental enrichment http://www.ncbi.nlm.nih.gov/pubmed/19374889
Hope it helps,
cheers
Jorge
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Tripartite synapse was studied and modeled by many researchers such as Pereira, Hydon, Nadkarni, and Postnov, but how is the network really structured?
What is the ratio of neurons to astrocytes in this network?
How do astrocytes connect with each other using connexins (Cx43,26,30)?
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'Plastic' as defined, is a irreversible property of a material. One form once attained, can not come back to original form. On the contrary, synapses not only get Strengthen but also get weakened depending upon the processes going on.It means they are highly flexible system. So why do we use word Plastic to define this bidirectional property of synapses?
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Well, there is a term called metaplasticity as well too which further complicates the plasticity.
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I am trying to validate LTP experiments in 7 weeks C57BL mice.
Hippocampal slices (400 µm). Measurements of the slope of the fEPSP (Schaffer collateral-CA1 synapse).
I am not sure about how well I am performing these experiments.
Maybe you could give me some feedback?
I attach a PDF where I show selected signals for 6 slices and the global result for the experiment.
Three of these slices (1, 2 and 5) were potentiated after 3 hours.. and the other 3 are almost back to baseline. Do you see anything wrong with the signals which can account for these inconsistencies?
In each slide, the signals in the top show selected examples of the input-output (the one in the right is the maximal response I got). I set the 30% of this maximal for the LTP experiment. Do you think they are overstimulated?
In the bottom line I show a selected example for the signal before HFS (control), just after HFS, and 1 and 3 hours after HFS.
Now, I am also trying experiments to get early LTP (using 20 stimulations instead of 100).. but after 1 hour the difference with the baseline is not significant. The problem is that if I try more stimulations... I use to have late LTP in many of the slices (I already tried 25, 30 and 50 stimulations).
ACFS composition (mM): D-glucose 10, NaHCO3 25.6, NaCl 124, KCl 2.45, KH2PO4 1.2, CaCl2 2.25 and MgSO4 1.2. Recordings are made at room temperature.
Thank you very much for your help!
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Congrats, you got a nice record, and you have done good experiments, just go ahead.
it is normal to have some slices without LTP. You can also report the percentage of slices that show LTP.
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PTP is normally considered to be dependent on pre-synaptic activity (result of calcium accumulation pre-synaptically as a result of the stimulation). How can the drug cause changes in PTP values in one protocol (200Hz LTP) while no change in another (theta burst LTP)? What does this mean? Please help me with this query. Thanks a lot in advance.
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Hi,
We ran into a similar question back in 2004, Dumas et al., Hippocampus, 14:701-709. Some of the responses already submitted seem to have summed up the issue. Different rates of presynaptic activity produce different levels of presynaptic Ca2+ accumulation. Check Figure 4, calbindin overexpression affected 50 Hz but not 100 Hz PTP. Hope this helps.
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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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I got confused when reading the mechanism of Synaptic plasticity - (Glutamate neurotransmitter Physiological role) - necessary of learning & memory.
In some literature, they are telling that NMDA will get activated, leads to Ca2+ entry, which phophorylate the AMPA receptor. AMPA removes the Blackade of Mg2+ from NMDA & thus more NMDA will activated which multiplies the response - LTP - synaptic plasticity. As well as for the activation of AMPA, NMDA activation is must required.
While other material suggests that, During resting potential AMPA are in active stage while NMDA are blocked by Mg2+.
So can anybody suggest me the exact mechanism of synaptic plasticity???
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Just in a nutshell, AMPA is activated first, because it doesn't have a block (such as Mg+), and therefore it can make a depolarization that might be enough to remove the Mg block from NMDA. AMPA has a fast time-scale, with fast activation and inactivation. NMDA is activated in a much slower time-scale and it inactivated in almost a sec. So if AMPA can acitave NMDA, that for a second AP, you will get a much higher response, because, you don't need to remove the Mg+ block, as NMDA is still active. So this is the high-school case of plasticity. But for a bigger picture you need to see into the actions of mGluR-s, because they can facilitate of act as depressors for synaptic transmission. I hope it helped!
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According to the standard model of spike timing-dependent plasticity (STDP), when presynaptic spiking precedes postsynaptic spiking, the synapse undergoes potentiation, and when presynaptic spiking follows postsynaptic spiking, it undergoes depression.
Have people studied what is the effect of variability in the pre-post delay time? In other words, for the same mean pre-post delay, is a narrower delay distribution more effective in driving synaptic modification than a wider one? I would be mostly interested in experimental results, but theoretical studies would be welcome as well.
Thanks much in advance.
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Hello,
Here you have a review on this topic by DE Feldman:
and also one of my favorite articles about STDP by T Nevian and B Sakmann:
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I'm a bit reluctant to toss this out there, but I could really use some insight. I have researched and found both quantitative and qualitative studies to support chemical and physical changes (increase in oxytocin, brain structural changes viewed by MRIs, for example) in both animals and humans during animal-assisted therapy and training, but I have yet to find any research that supports a possible relationship between HAB/AAT and cognition and memory.
My ultimate goal is to discover whether use of the HAB and AAT can positively increase brain plasticity, and use this form of teaching for elderly, and those who may have suffered brain injuries, to increase memory capabilities. I've included some of the references I've gathered, so far.
Insight, anyone? Thoughts?
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I agree. There doesn't seem to be a literature out there which suggests that AAT specifically improves learning or memory. However, given it's effects on emotion and motivation, I wouldn't be surprised if it did have a positive effect on learning and memory (which can be triggered by emotional associations) in the elderly - if just by increasing their motivation to perform the task (using AAT as a reward - or teaching the dog a trick as the motivator). I'm glad you are thinking about doing this study and am excited to hear about your results.
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I am performing fEPSP recordings (Schaffer collateral-CA1) in slices from rat hippocampus, and I find so much difference in the PPF ratio from one experiment to another. Why is that?
Sometimes I do not see PPF, or even a weak PPD. But normally my PPF ratio values range from 1.50 until 3.00, with a average of 1.87±0.05 (n = 80).
Moreover, in some of my experiments, after having a stable baseline, when I evoke PPF protocols, the baseline somehow starts to potentiate. Is this usual?
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Stimulus strength can be an important factor, but there is a common misconception as the why this can be the case. One needs to understand that the stimulus strength does not matter at all to transmitter release of an individual synapse within your recorded population. The action potential that is evoked by your stimulus is all-or-none, and it doesn't matter how 'hard' you have to stimulate to evoke it. You don't get any change in release probability because an individual presynaptic action potential was evoked with a stronger stimulus. Since the field potentials that are typically recorded for PPF and other measures are population responses, turning up the stimulus has the main effect of increasing the size of the population of axons that are stimulated, and thus increasing the NUMBER of synapses activated. In a homogeneous population of synapses, this will have no effect on PPF directly. If turning up the stimulus strength is having an apparent effect of PPF, then this is likely coming from an indirect effect. Possibilities include evoking larger EPSPs (with a greater number of axons) that more closely approach the reversal potential (and thus have less 'head-room' to get larger, or increased synaptic inhibition that may influence the response to the second stimulus. Both of these factors can be minimized by recording only the initial slope and only in the dendritic layer (EPSPs recorded in s. pyramidale are more susceptible to influence by synaptic inhibition, and besides, are an indirect reflection of the synaptic current). I would upgrade the advice that many have given about recording only the initial slope of the EPSP from 'important' to 'essential'. The population spike is NOT a synaptic potential, and besides, it behaves quite differently then the fEPSP with regards to PPF and other factors.
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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 was wondering if anyone has any suggestions for antibodies that they have used to stain for glutamatergic cell bodies (and preferably not terminals) via IHC in tissue slices?
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Hi Sam. Let us know if you don't find a suitable antibody. We specialize in discovery of novel DNA aptamers as antibody replacements. We working on protocols for selecting aptamers against FFPE and related samples. We'd be willing to work with you on a collaborative basis if you're willing. Please see www.BasePairBio.com for more info. Best, Bill
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I'm doing in vivo electrophysiology (LTP protocol - 3 strains, 100Hz*1s) in CA3-CA1 synapses in mutant mice. In an electrophysiology experiment, sometimes (1 in 5 times) I fail to induce LTP in WT littermates and this results in considerable changes in my final results (when compared with the KO). Before changing the approach to induce LTP, I would really appreciate some feedback concerning this issue. How can I reliably exclude an animal? Is it valid to exclude a WT littermate that doesn't induce LTP by the protocol that I use?
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Hi José,
This failure to induce LTP is part of the physiological response. It is not an outlier.
It may depend upon the angle for the slice, how old the slice is etc.
Hence, it is NORMAL to have LTP failure.
We have it everyday, more for mice than for rat for some reason.
What we do is be honnest; i.e. LTP failed in XX slice out of YY from ZZ animals.
Then, if you compare two conditions, you can not only compare the amount of LTP but also the failure rate, which is also a very good indication about modifications.
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Accumulating evidences indicated that synaptic plasticity underlies the mechanism of neuropathic pain. I want to use fluorescence stain and confocal microscope to separate the synapses undergoing plasticity from the ones not, so I need a tag of synaptic plasticity locating at the synapse. although I have done some research about this issue, but i do not decide which one should be choose .Do you think which one is reasonable?
F-actin(or the ratio of F-actin/G-actin);p-CaMKalpha/beta;the ratio of PSA-NCAM/NCAM;arc/arg3.1
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The best synaptic tag for plasticity events will depend on your timing. That is, when relative to the stimulus you plan to look. We have looked at a number of markers from phospho-cofilin which last minutes to longer one like pCamKII. F-actin is good because it tends to be there for at least an hour following stimulus. However, you will need good techniques to quantify because there is generally a good amount of it normally and you will not see any difference if you simply use F-actin labeling in fixed tissue. It works better if you apply the phalloidin in the live prep so that you label recently activated synapses. I suspect that may be problematic in a in vivo model of pain.
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Hi Jaun. BrdU is very useful as you can (for example) label which cells were proliferating at a specific time, and then leave the animals to age and fate map the cells and add various analogs such as EdU to see how proliferation changes over time. Ki67 is also useful, but can only tell you about the proliferation at the specific point in time you harvested the tissue (and it has been suggested it is also expressed by quiescent cells not just proliferating cells- J. Cell. Physiol. 206:6240635,2006). This paper has good materials and methods and the group uses BrdU a lot to characterise the effects of irradiation in their model, I used to work downstairs from them. What is the aim of your experiment and do you need advice on using and detecting BrdU, or quantification of the immuno?
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Can temperature (especially high temperature) play a role in this phenomena of neurotransmitter binding, internalization or receptor dynamics?
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There are a number of ways this can be done. If you mean receptor mediated internalisation, the first ways this was accomplished was to monitor total binding using radioactive ligand on live cells, the authors had noticed that the amount of total binding sites over time went into soluble endosomes instead of insoluble membrane. This can be done as well by looking at how much of the receptor was not dissassociable from repeated washing steps or after the use of extracellular digestive enzymes (or biotinylating the receptor and monitoring how much was not biotinylated before and after agonist administration for example). Otherwise more contemporary fluorescent methods might be easier, however less quantifiable. There are a number of fluorescent ligands that could be used, but I don't recommend using this approach since all live cells under imaging will internalise non specifically anything that is in the medium to some degree. Rather I would recommend looking at fluorescent receptor localisation. ImageJ can be used to look at punctated signals within cells of a certain dimension, while a receptor at the membrane would fluoresce around the plasma membrane, internalised receptors are seen as variable sized endosomes and lysosomes (if receptor goes towards recycling or degradation). Thus quantification of full endocytic agonist can be your comparative agonist readout, albeit it is difficult to determine the amount of molecules going inside your cell. Concerning reuptake (as in neurotransmitter restocking), you could use radioactive analogues to be incubated with the cells in question for a period of time, after a few washes all that is kept by the cells could be your amount uptaken. This is often used to measure neurotransmitter release following agonist stimulation, since these uptaken radioactive analogues will be included in the released vesicles.
My PhD thesis was based on temperature effects on receptor pharmacology, I can thus tell you a few things. Firstly, since binding is a factor of Koff and Kon, temperature affects both of these factors, nevertheless, when all other variables are taken into account, the temperature factor on binding affinity somewhat cancels itself out, meaning overall binding should be around the same. However, the rate at which equilibrium is reach could be drastically changed (normally this is proportional to temperature). Since the time your ligands are binding to the receptor could be crucial to receptor conformational stability (denaturation, protease activity, pH changes, oxydation and so on) longer incubation times are generally correlative to failed experiments. Temperature has an effect on all buffer pH so higher temperatures often mean acidifying effects, that could also affect your binding. As for cellular functions, they are generally slowed down at lower temperatures, this is true for activation and internalisation. Higher temperatures most often makes things go faster, up to the point of non functionality. Since a wide range of cellular mechanism work in concert during internalisation higher temperatures (over 37oC) often lead to reduced internalisation rate from denaturation of critical components (however the critical temperature where this tendency is maximised and then drastically reduced, I do not know). Conformational dynamics are also elevated as temperatures increase, but again as this goes over 37oC the receptor and its binding proteins can attain, non natural conformations and subsequently loose important structural functions. Since these proteins have all evolved to work around 37oC, evidently the maximal response of pharmacological characteristics is somewhat close to that temperature.