Synaptic Plasticity - Science topic

For an initial period (minutes to hours), an explicit or declarative memory, such as a name or event, is registered and stored in the hippocampus, where it can be retrieved by relevant cues. Here, the memory may fall silent and sequentially erased without frequent recalls. Frequent recall attempts turn the memory to long-term (days to months) by a process in the hippocampus called long-term potentiation. Long-term memory is stored in the cortical areas linked to the sensory modalities used in that memory, for example, visual memory will find a space in the visual cortex. In the cortical areas, the memories can remain stored for longer (months to years, called remote memory) or dormant depending on intermittent recalling attempts. The implicit memories, such as driving skills, are registered and stored for the long term (if thoroughly practiced) in the striatum or other basal ganglia components and linked cortical regions. The key to turning memory for the long term is that you value it and keep recalling it.

Zeeshan Mushtaq Thanks for your answer. I also think that is the adapter. Maybe A-M sytems have the original cable, but I doubt it. I will try it anyway. Thanks a lot

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?

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.

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.

Pradeep Jayarajan Where are your recordings from within the hippocampus?

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

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

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.

Hi, I am not sure what pipette solution you are using at the moment, but I am wondering if you have some calcium in there? I know standard pipette solution usually do not include calcium, however, calcium can be important in terms of signaling cascade. I have found that with physiological calcium concentration (~ 100 nM), you can get pretty good patch recording without compromising patch quality. 

Please look in the Attachment some information from my library.

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

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

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. 

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.

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

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.

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  

Local increase includes 

1, K+ release from axons as a results of APs.

2, synaptic transmission, which is a major source. 

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.

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.

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.

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!

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

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

please refer the following article:

 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. 

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? 

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

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. 

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

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

Dear Mathew

In addition to the above comments, please consider this simply rule that: if you are recording from sink the slope will be the best parameter of synaptic strength and if you are recording from the source region, you have to measure the population spike amplitude.

Good luck 

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

Ideally, you can infuse over hippocampus, because as mentioned by Giovanna you can induce gliosis in hippocampus.

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.

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!

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.

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. 

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.

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.

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.

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.

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

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

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.

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.

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.

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.

Hi at least you need confocal microscopy and two-photon microscopy, of cause good antibodies and sensitive calcium marker. :)

Hello

It may be that you trigger an LTD with your low frequency stimulation. I pulse each 15s is not much but I would try to switch to one pluse each minute, or wait 15 minute to test if the response still decrease

Good luck!!!!

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.

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

Figure 2

Well, there is a term called metaplasticity as well too which further complicates the plasticity.

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.

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.

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.

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!

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.

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.

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.

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

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.

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.

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?

Hi Timoteo,

Here are some links might be useful for you

thanks

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.