Synapses - Science topic

The rate of glucose consumption by the neocortex is reduced by over 80% during anesthesia (Sibson et al. 1998), which disables the synapses (Richards 2002) that are supported by glial tissue (Engl and Attwell 2015). It is the synapses that provide the brain with its computational power (Hebb 1949). Disconnected (pig) neurons on life support (e.g., Sestan 2018)[1] have no ability to transfer information, and some might argue that such cells have been reduced to having a computational power below that of a single-cell organism, the amoeba (Saigusa et al. 2008), since they have been taken out of their ‘social’ environment for the expected programming between individual neural members. The organizational life of a multicellular organism is no trivial matter, requiring that each cell be subjected to some biological constraints (Albert et al. 2002) in exchange for the energy efficiency obtained per cell, which scales as the 3/4th power of an organism’s body mass (DeLong et al. 2010; Kleiber 1947; Wells 2007). This process has been shaped by 500 million years of evolution. We are a long way away from merely dumping a bunch of disconnected neurons into a dish that self-organize into a superorganism that supports consciousness, a well-studied topic by entomologist E.O. Wilson (Wilson 2012). Indeed, this calls for taking evolution seriously so that one day we might be able to really engineer a superorganism, which is not trivial (also see ‘converting rodents into humans’[2]).

Footnotes:

[1] The Yale researcher, Prof. Nenad Sestan, has managed to keep pig brains that were detached from the body and on life support (i.e. a warm blood supply mediated by pumps) alive for up to 36 hours (Regalado 2018). This result created quite a sensation at the National Institutes of Health with some even suggesting that this could yield the possibility of studying consciousness and the brain in the absence of the body. A notable observation was that the EEG activity of the pig brains was flat. What is clear from this is that a body is necessary to give the brain life through feedback (Tehovnik and Chen 2015). The challenge now is to determine how much of the body (or its prosthetic equivalent) is sufficient to provide function to a brain. A similar misthinking, as that which motivated Sestan (2018), has occurred by investigators who have hooked up two brains via wires to create the illusion that significant information can be transmitted between them (cf. Pais-Vieira et al. 2013 & Tehovnik and Teixeira e Silva 2014).

[2] Converting rodents into humans: Brain tissues from humans, called organoids, have been implanted into the brains of mice raising the possibility of having human brain cells incorporated into the rodent biostructure (Mansour et al. 2018). Some have speculated that this could endow rodents with an enhanced cognitive ability if the number of human cells were numerous enough (Begley 2017). Note that there are some 71 million neurons in a mouse brain, so this would require a significant addition of organized tissue. A fear persists amongst bioethicists that such implants might give rodents some degree of humanness: i.e., augmented consciousness. But injections of neural tissue into a foreign body are riddled with incompatibilities such as problems with blood supply, immunity, and functional connectivity.

The bird brain, unlike the human brain, regularly injects itself with new neurons via neurogenesis, and therefore it might provide clues about the challenges of adding new neurons into another’s nervous system (Barnea and Pravosudov 2011). Cell proliferation, cell migration via glia, and cell replacement are some of the steps that make up neurogenesis. Riding the brain of old cells is also part of the process (as anyone who has ever received chemotherapy understands). To add, this process is tightly regulated. For example, in the canary, neural augmentation occurs in the vocal control nuclei during periods of song and mating (Goldman and Nottebohm 1983). The point behind emphasizing this detail is to show that for the new neurons to contribute, one might need to reprogram the existing tissues—neurons, glia, epithelia—so that the new neurons are accepted and utilized effectively. At this point, injections of human neurons into a rodent brain may be more prone to producing cognitive deficits than cognitive enhancements.

We have mice brains that were over-fixed due to old PFA used during perfusion. Thus, the synapses are no longer being labeled by the Synaptophysin (SY38) mAB. which works perfectly every other time.

I have already tried antigen retrieval, but that has not been helpful either.

It is really important for us to label the synapses and we have no option but to use the over-fixed mice brains we have right now.

I am planning to try other synaptic markers like PSD95 and Synapsin next.

But I am open to more suggestions or troubleshooting ideas.

Would love to hear if someone has faced similar issues.

Thanks a lot.

The neurology of learning to ride a bike is illustrated in the left panel (this is not a proof but rather a heuristic, Fig, 3 of Gallistel et al. 2022). The cell is a Purkinje neuron (that is being classically conditioned). Imagine that after months of conscious, declarative training a child by day 600 (trial 600 in the figure, left panel) is finally off on his or her bicycle. This behavioral change should be reflected as an abrupt modification in neural firing within the cerebellum and the neocortex (Gallistel et al. 2022). Furthermore, the ‘consciousness’ of neocortex is immediately informed about the accomplishment once the child rides off on the bike (Ikeda et al. 1994; Libet 1985; Soon et al. 2008). Curiously, once learned. all records of the months of learning to ride seem to disappear which is why we all think (wrongly) that learning to ride a bike is a procedural, motor accomplishment rather than a conscious, declarative accomplishment. But we (including all parents that assist in the learning) all know better (see Tehovnik, Hasanbegović, and Chen 2024).

Contrary to what many assume (e.g., Duhamel, Goldberg et al. 1992), the efference-copy coding circuitry is located at the level of the Purkinje synapses, the same synapses that are modified during classical conditioning (Bell et al. 1997; De Zeeuw 2021; Gallistel et al. 2022; Giovannucci et al. 2017; Loyola et al. 2019; Shadmehr 2020; Tehovnik et al. 2021; Wang et al. 2023); the coding circuitry after being configured anew following a first successful bike ride, will require updates throughout life. For instance, such adjustments are made when a child’s vestibular system needs to undergo change due to body growth and further adjustments are made when the body begins to decline due to old age.

We know that the brain stores data in neurons or networks of neurons. The data exchange center is called a synapse through which the data passes and is coordinated. The neurons are connected to millions of sensors. The question is can we run analytics on them or can we visualize our dreams?

Are there any published studies? Is the question sensible?

Consider:

Why Neurons Have Thousands of Synapses, a Theory of Sequence Memory in Neocortex

Front. Neural Circuits, 30 March 2016 Volume 10 - 2016 | https://doi.org/10.3389/fncir.2016.00023

I’ve been having numerous issues with achieving stable baselines recording from the TA-CA1 synapse from juvenile (P12-P24) rat hippocampus slices. In addition, when applying drugs such as antagonists/inhibitors which should not show any effect on baseline, I have been seeing gradual increases in synaptic transmission that differ from what other students have previously shown in my lab.

I cull my rats by cervical dislocation and slice in ice cold sucrose aCSF and allow the slices to rest for 1 h at RT in regular aCSF. I then stimulate and record from the TA-CA1 and my first slice usually takes 2-3 hours to stabilise. I oxygenate my aCSF for at least 40 minutes prior to putting a slice on the rig and I use a platinum harp to hold it down in the bath. My rig uses a gravity feed system and the flow rate is 2.5 mL/min. My recording electrode is filled with aCSF and I bleach the silver wire every few days.

When the slice eventually stabilises for 20 min, I add my drug which has been oxygenating for at least 10 min. I can often see strange increases caused by the drugs that have not previously been seen. I thought it might be down to changes in oxygenation but I’ve been keeping all of my solutions in similar sized cylinders and have increased my oxygen so that everything is saturated.

Can anyone advise me how I can improve this and shed some light onto why I am seeing such instability and increases when switching drug?

Any help would be much appreciated, as I feel as though I’ve exhausted all ideas at this point.

Thank you!

I heard that a group did a 3D EM reconstruction showing that some neurons could form both excitatory and inhibitory synapses, but I have been unable to find it.

I was thinking about VGlut2 (presynaptic) and PSD95 (post-synaptic) for the excitatory synapses, and VGAT (presynaptic) and Gephrin (post-synaptic) for the inhibitory ones.

Should I define as a synapse only the areas of colocalization between pre and post synaptic marker? Or should I consider also the isolate VGlut2/VGAT and PSD95/Gephrin ?

Is there any experimental data for the amount of energy transfer involved in registration of a bit of information at synapse?

Is there any experimental data for the amount of energy transfer involved in bit storage and bit release (erasure)?

How many synapses in visual pathway?

We are recording field EPSPs in CA3-CA1 synapses from 2-3-month old WT mice. We can get good EPSPs (size trace in blue) but during the baseline the EPSP gets small (yellow trace) and then large again and cycles over the course of the baseline.

As you can see in the traces, the EPSP changes in size but the fibre volley remains the same, so I don't think the electrodes aren't drifting. We also wrap our recording electrodes with cotton so droplets of aCSF do not form and land on the slice.

Please let me know if you have any suggestions of other things we can check. Thanks!

I want to measure STDP for biological synapse but I am stuck on

1. how to give write and read pre-post synaptic pulses.

2. Data can read (read voltage) after pre-post or pre and post individually

Please anyone can help.

Good day,

we would like to see/count projections of 5-HT neurons and their synapses in the mouse/rat brains - mainly in cortical areas and the hippocampus - to see if they are affected by our experimental treatment. For that sake we would like to use immunohistochemistry on PFA-fixed free floating brain sections.

We already found that TPH2 immunohistochemistry (IHC) effectively visualizes 5-HT neuron axons. Right know we are looking for a marker of synapses to combine with TPH2 IHC so that the synapses stained with TPH2 are doublestained with this marker. We already tried synaptophysin+tph2 combination but sadly synaptophysin signal was too abundant (unspecific). So now we are looking for a marker more specific to synapses of 5-HT projections to pair with TPH2 IHC. Any ideas we can try? Thanks in advance for any input.

Parallelism is said to be an important difference between digital and human brain computation? If brains can use dimensional capacity to obtain computational advantages (if they do) then can computer architecture be modified to emulate brain architecture?

Reference

Nagarajan N, Stevens CF. How does the speed of thought compare for brains and digital computers? Curr Biol. 2008 Sep 9;18(17):R756-R758. doi: 10.1016/j.cub.2008.06.043. PMID: 18786380.

My research is about memristor-based synapses. Spike-timing-dependent plasticity has been routinely reported in different papers.

Here is a description from the paper "The spikes from both preneuron and postneuron arrive at the synapse occasionally in the opposite direction [7]. In STDP, the change of the synaptic weight is the function of relative neuron spike timing ∆t (∆t = tpre − tpost), where tpre is the time when the presynaptic neuron spike arrives and tpost is the time when the postsynaptic neuron spike arrives [4]. In a typical STDP behavior, if postsynaptic neuron spike arrives after presynaptic neuron spike (∆t < 0), the synaptic weight increases. If postsynaptic neuron spike arrives before presynaptic neuron spike (∆t > 0), the synaptic weight decreases." (10.5772/intechopen.69535)

I do not know how to get ∆w. Here ∆w should be the change of conductance (G). according to my understanding. ∆w=G (before pulse)-G (after pulse). My question is "what is the G (before pulse) and how to chose the G (before pulse)". In published papers, they get many data points of ∆w determined by ∆t. shall they use the same G (before pulse) and monitor the G (after pulse) after pluses with different interval times? But the resistance of memristors is tunable, how can they confirm the same G (before pulse)?

Thanks

Hello everyone,

Is it possible to calculate the E/I ratio on single-cell levels only based on miniature signals (Excitatory and inhibitory)?

If yes, then what parameters should I take to calculate it? and how?

Currently, I can get lots of parameters from mIPSPs and mEPSPs such as the number of events, decay and rise time, area, baseline, noise, half-width, 10-90 slope, etc...

Which one of these can i use to calculate the E/I balance and the calculation mathematical formula?

Thank you!

I need a marker protein that is expressed only in the post-synapse, not outside the synapse, preferably with a reference, thank you. PSD95 is not suitable for my project .

Does it form networks in 3D cell culture (e.g. when grown in collagen matrix)? What other options for neural cell lines (PNS related) exist that show network connectivity? Can the network connectivity be tested without co-culturing with other cells? Literature shows that NG108-15 forms synapses when cultured together with muscle cells.

Dear all,

I would try for the first time the DiI crystals for tracing neuronal pathways. I'm wondering if the Dil labelling, applyed post-mortem and post-fixation period, should pass the chemical synapses or not, as biocitine does. Just because I'm interested in labeling only the first neuron of the neural pathway, and not the second or the third etc.

Thank you in advance

Riccardo

Matlhworks provides ANN Toolbox for constructing and training ANNs. However, one come across situations where e.g. MLP with a single hidden layer will not predict accurately enough the outputs one is expecting. Trying to add more layers may end up with endless training sessions. My question is two fold:

First: Does Python truly address this issue ? Any examples and references?

Second: Is there any visualization tools that shows the dynamics of the ANN during training or utilization after training? In other words, are there any tool that shows values of weights (synapses) and neurons dynamically?

Chemical synapses are clearly necessary to the operation of Earth brains as they have been conserved omni-presently and refined and differentiated for hundreds of millions of years.

1) The synaptic gap is only on the order of ten times the size of a neurotransmitter molecule, and anchored to hold its dimension. This means the gap is a tightly-controlled busy place with dense chemistry going on. That supports the possibility that the "neural" correlate of consciousness is entirely disconnected from the neuron, and an effect of molecular goings-on in the gap. We know that some neurons correlate to consciousness and some do not, and that different neurotransmitters are used by different neuron types, and that some neural circuits produce pain and others, pleasure.

2) The actions between the neurons means there will also be electric potential swings across the gap (which also influence the closely-spaced molecules therein).

3) The scales and densities involved also suggest the possibility of multimolecular quantum effects.

For consciousness to both arise from and affect electrochemical brain activity, there must be a locus of rich enough physics and with sufficient opportunity for continued evolution.

Hello everyone,

I'm currently studying a certain synapse and, apart from the functional characterization, I want to show that a receptor is present in a certain type of cell.

Recently, 4 different papers have been published that perform single-cell RNAseq in that kind of cells with slightly different controls and all of them have made available both the raw data and the pre-processed data with the normalized and corrected relative expression levels.

As my only intention is to show that 2 genes are being diferentially expressed in my cells of interest, I thought about showing the log 2 fold-change in expression for my genes of interest vs. the corresponding control for each study.

Is this acceptable or I have to inevitably perform the analysis from scratch with the raw data from each study?

thanks in advance!

We have several fixed coverslips of NK cells synapsing to cancer cell lines, which we were going to use in an experiment with demo equipment. The cells are labeled with Dil, but during the experiment, it became apparent that this dye was bleeding through into the green channel. I'm not sure whether the instrument just had poor quality filters or if this is just the dye itself, but in any event, we'd like to possibly use these slips for confocal imaging to look at the immune synapse.

Even if the Dil will stay in the red channel on a better imaging system, most of our conjugated antibodies are bound to Alexa 488, 568, or 594. We'd need to stain with 2 colors, and the dil will interfere with both 568 and 594 on our system. Does anyone know of a way to remove the dye? Or can we just blast the slides for a while/leave them out under the lights to bleach the old dye, and re-stain with whatever we want?

I'd like to avoid having to buy a blue fluorophore-conjugated Ab if possible, as we'd like to keep using DAPI for counterstaining. If anyone has any advice, it would be greatly appreciated!

P.S. I'd like to avoid making new coverslips at this point, as we've been having trouble culturing these (transduced) NK cells and our supply is dwindling -- otherwise the obvious solution would be to just re-make the coverslips!

I suspect the stimulation intensity of paired-pulse stimulation will affect whether it will be PPF or PPD, or the percentage of enhancement or depression. Please advise what the best the intensity of paired-pulse stimulation to correctly reflect the pre-synaptic release probability of given synapse.

I'm trying to find examples of EM being used in rodent brain tissue for quantification of synapse density and ultrastructure. If this has been done, are there special considerations that have to be taken into account when preparing the brain tissue at these early stages. The earliest I've seen is P14, but I'm hoping to find examples before this; P0-P12, if they exist.

Hi there,

I'm currently working on a project that aims to classify synapses into three distinct classes.

Class One: Containing only PSD95

Class Two: Containing only SAP102

Class Three: Containing both PSD95 and SAP102

I am looking for any open-access software that would allow me to quantify both individual synaptic puncta and those that are colocalised.

Any help would be really appreciated.

Autism spectrum disorders are a group of neurodevelopmental disorders, one of the main characteristics of which is impaired social communication. But what happens in patients' brains that disrupts their social skills?

Hello

I would like to isolate synapses from whole brain lysate. We have a ultracentrifuge with Sorvall SS-34 rotor. Does someone recommend centrifuge tubes that fit in the rotor and that are puncturable so that I can suck out the different phases? I found some Seton tubes online but I am not sure if they fit or whether they are too narrow. Are Nalgene tubes thin enough to pierce? First time doing this so any advice greatly appreciated!

Thanks!!

Hi,

I am a second year graduate student and have been doing flow cytometry on embryonic neurons as a part of my experimental procedures. The neurons were cultured in vitro and allowed to form synapses for 10 days after which I used them for flow cytometry. In order to understand the percentage of live and dead cells, I used 7AAD as a marker. Ideally, I would have expected two distant peaks indicating 7AAD positive (dead cells) and 7AAD negative (live cells). However, my graph has three peaks.It looks like a negative peak and two positive peaks. I am however a little confused about this and wanted help to interpret this data.I am attaching the histogram plot in order to get an idea of what it looks like. If anyone has any inputs please do comment. I would really appreciate your help.

Thank you.

Hello! I try to stain some synaptic proteins (synaptophysin, synapsin, SNAP-25) by immunofluorescence in synapses on mSOD1 mouse diaphragms, but I have faced 2 problems - first, sometimes the background fluorescence is high too much. Second, sometimes specific fluorescence of the proteins absents absolutely.

So I am wondering does anybody have a good, well-working protocol? Could you please share it with me?

If so, can we use electric as stimulus to increase the IQ?

I wish to test the synapses in the ASD brain vs those with N typical. Specifically immature neurons within the e/i networks targeting glutamatergic/GABAergic alongside their contributing networks. which program(s) could do this at preferably lower cost?

Is there any software for drawing synapses and neurons?

I am trying to plan an experiment in which I am trying to silence neurons in a circuit-specific manner. I would like to target neurons in a certain nucleus that project to the cortex and synapse onto neurons that descend to the spinal cord. I'm looking for either an anterograde AAV or a mouse that expresses DREADDs (particularly hmd4i) in a Flp-dependent manner. I've found mice that express DREADDs but require both Flp/Cre recombinase, but because of my experiment design (I don't want to burden you with details) I can't express Cre in my neurons of interest. Any ideas? Thanks!

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

I am working on a experiment to analyze the immune synapse between T cells and macrophages. In our protocol to stain the cells with fluorescent markers, we have quite a few pipetting, washing and centrifugation at 300G steps, before and after fixation.

I am wondering how many immune synapses get disrupted by all these mechanical forces. Does anybody have an idea how strong and rigid this complex is?

Thanks in advance!

Hi All,

I was wondering if anyone knew of any correlation analyses comparing the size of CA1 pyramidal cells and frequency of spontaneous activity. Doing the eye test, it seems like I’m getting increased rates of sIPSCs onto CA1 pyramidal as the cell size increases (measured by capaticance).

Additionally, I was curious about the best way to interpret miniature (with TTx) IPSC frequency data. If the frequency is increased compared to control, can you conclude that there are increased numbers of synaptic connections or that the vesicular release probability is increased or both? My gut tells me it is both and I’d have to do other experiments to tease apart the exact mechanism...ie paired-pulse for release probability and some sort of synaptic labeling and counting for increased synaptic number.

Thanks,

Chad

I'm looking for references to justify max and min conductance values of synapses containg AMPA and/or NMDA receptors in a biophysical model using the Neuron simulation environment. If I can be really specific I'd like values from stellate cells in the medial entorhinal cortex. But general values would be fine. I have some general numbers I'm playing with that I've found from different sources but those sources don't have any citations. So for AMPA I've found a range form 0.1-1.5 nS and for NMDA 0.05-3.9 nS.

I'm trying to come up with an LTP protocol induction(TBS) in CA1 pyramidal neurons which reliably lead to firing of neuron.

I'm using a theta glass for bipolar stimulation and the diameter of the tip is approximately ~40-50um. I'm interested to stimulate proximal synapses of SC.

when I set the baseline EPSP to be around one third of the difference between firing threshold and resting membrane potential, in most cases I won't get a spike although I can see the potentiation effect.

I tried different position for stimulating electrode when it's distant from the neuron (more than 600um) I can't usually get big EPSPs.

What is the best strategy to have spikes during induction?

Should I try blocking interneurons?

Hi there,

I've recently started recording fEPSPs from coronal sections (incubating with bicuculline), focusing on the CA3-CA1 synapse. I put my stimulating and recording electrodes both in the middle of stratum radiatum of CA1, about 100µm apart. I'd like to confirm some uncertainties I have about the recordings. in Image A, is that the presynaptic fiber volley? As I increase the stimulation intensity, I get Image B. What is the small peak at the beginning of the trace? Also, other peaks aside from the fEPSP start to appear, what are those? Is that normal? Finally, when I start my paired pulse ratio protocol (ISI = 50 ms), I get these strange looking peaks in the trace. What are those? I'm saying these look strange because in representative traces from Kaeser et al. 2009, they look very different (refer to image D). If there may be something you notice that is wrong, please point it out as that would be greatly appreciated.

Finally, I wanted to confirm that during stimulation, the membrane of the neurons being stimulated are depolarized, which will induce glutamate to be released at excitatory synapses and GABA to be released at inhibitory synapses, which leads to influx of sodium and chloride, respectively. The purpose of bicuculline is to block the GABA currents, and ensure that the output is purely from excitatory synapses?

I have tried to stain the GABA neuron marker GAD67 in the hypothalamus paraventricular nucleus (PVN) brain region. After trying some antibody condition, I still can't see the GAD67 signal expressed on the neuron soma, but it seemed that some signal expressed on the synapse in the PVN. However, I can clear see the GAD67 neuron soma signal in the cortex and other brain region such as hippocampus. The attachment is the results. I am looking forward to some suggestions. Thank you so much.

I am using SHSY5Y cells to develop neuroinflammation. I wanted to use MAP2 as a marker, but MAP2 only indicates the cells are differentiated. Is it necessary to ensure the cells are mature (eg: have synapse (synaptophysin as marker) to validate my in vitro model of neuroinflammation?

Hi all,

Recently I have been working on analyzing the immune synapse formation using imaging flow cytometry. I co-culture mouse APCs and T cells and subsequently perform fixation/permeabilization and different staining steps with fluorescent antibodies.

I am wondering how strong the immune synapse is, and if it can withstand external mechanical forces like pipetting and centrifugation. I have been successful in finding cells that are crosstalking with each other, but I don’t know if this number is reduced due to the steps of my staining protocol.

Thanks in advance!

I'm making a model of cortex with an excitatory layer (e.g. layer 2/3 pyramidal neurons) and an inhibitory layer (e.g. gabaergic interneurons). Right now I'm refining the connection matrix. I have the option to eliminate reciprocal connections where neuron A connects to neuron B and B back to A. This would affect the extent of positive feedback in the system. I would like to base this on biology. I did a quick search for this and didn't find exactly what I was looking for. Hoping you might know answer or a relevant paper offhand.

I know that identifying a synapse between two cells can be done in various ways with various trade-offs. Serial electron microscopy would give the most detail, but serial EM reconstructions haven't even been achieved of much more than a single neuron, much less a group. Fluorescence based methods often require some interpretation. Dual patch clamp electrophysiology might be the best approach but is low yield.

Also, if this isn't known, that's good to know as well.

Thanks in advance.

Currently I use “Reconstruct Synapse Web”, made available by the Harris lab at the University of Texas at Austin. Although I am very familiar with this software and do enjoy using it, it requires me to segment all of my TEM series manually. I am looking for a segmentation program that is at least somewhat automated in order to increase my efficiency in taking blocks of tissue to the reconstruction stage. I have heard that the Lichtman group at Harvard utilizes a program called ”Mojo” to do this but I have been unable to get the program to work for me from its link on their website. If any of you know a suitable program that you have used could you please let me know?

cheers

Ian

I am looking for a way to localize energy use in a synapse during endocytosis of the synaptic vesicle cycle.

I'm trying to find a method to study the number of active mature synapses of purkinje cells (PCs) in the critical week of cerebellum development. I thought about mesuring AMPA-mediated mEPSC (whole-cell patch clamp). However, I'm not sure if I can compare different ages between P10 and P15 considering that the arborization of PCs is critically different at these ages and I can have space-clamp problems. 

Do you think you can help me?

I have done some loss of function experiments in primary hippocample neurons. The neurons were transduced with a lentivirus having a GFP reporter. The neurons were further stained with postsynaptic (PSD95- Alexa 546) and presynaptic (Synapsin1- Alexa 633) proteins. We are trying to quantify synapse density in transduced neurons, considering one synapse as the colocalisation of Red, far-red and Green channel. I have tried it in Fiji (another version of ImageJ) but I am getting stuck in particle analysing step. Can anyone suggest of other ways to do it?

Does anyone have any experience using cyclothiazide (CTZ, 100uM) plus kynurenate (KYN, 300uM) in patch clamp recordings on mouse or rat hippocampal slices? We are trying to use KYN block (competitive glutamate antagonist on APMA receptors) from evoked EPSCs at the CA1 synapse as an indirect way to measure glutamate's timecourse within the synapse. 

I can isolate decent AMPA responses but the addition of CTZ is giving me some odd results. I first add 200uM APV and 10uM gabazine to isolate APMA responses (monopolar electrode, stimulating Schaffer collaterals in mice and rats aged P11-18).

In mice, I'm seeing APV, gabazine, CTZ, and KYN potentiate evoked EPSCs rather than inhibit/dampen them. To ensure our drugs worked, we performed two electrode voltage clamp experiments in oocytes expressing rat GluR1 and they worked just fine....CTZ potentiated due to relief of desensitization and CTZ plus KYN inhibited current to ~50% of CTZ plus saturating glutamate (100uM). 

Currenty working on the same experimental set up in rat slices (CTZ then KYN inhibition) as we thought there may be a species difference. Most, if not all, experiments using these compounds are in rat, not mice. 

Does anyone have any experience with these compounds, particularly when used together in slices? Any feedback would be greatly appreciated. 

Hi, I am doing extracellular field recording from CA1 region of hippocampus in rat. 

I usually have robust and big response (amplitude ~1mV) in older rat.

However, I could not get a big response in p7 rat (postnatal day 7) .

I need to isolate NMDA component from my response so it really needs to be big and stable. 

I slice the brain in ice cold sucrose solution and it works for older rat.  

Is p7 known to be difficult to get big CA1 response?

Are synapses not developed in p7 yet?

Anyone has a trick to share?

I have noticed an increased in the asymmetrical synaptic excitatory junctions to be increased in the diabetic brain but could not find any discussion of this in the literature at present.  If you have any information or publications regarding this question please share with me when you have the time.  

My hypothesis is that in younger diabetic brains this increase in asymmetrical synaptic excitatory junctions may result in an overuse phenomenon and result in a later or older models in their attentuation and or loss as in accelerated aging in the diabetic brain.  Thank you for your time and consideration.

Sincerely,

M.R. (Pete) Hayden, MD 

Anyone have a protocol written up?

what is the mechanism of neurotransmitters  be removed from synaptic clift of synapses ?

A linear summate and fire default model of post-synaptic integration would not require inputting axons to synapse at any particular place within the dendritic tree of the receiving neuron. However, if post-synaptic integration was non-linear and possibly pattern sensitive it might be essential that the site of each synapse bore a relation to the meaning or significance of a specific input signal. Thus for a multimodal high-level sensory neuron signals originating from different primary cortices might arrive at different domains of the dendritic tree.

I would be very interested to know whether there is already useful information on this topic or whether anyone hopes to collect such information.

Stimulation of apical dendrites (stratum radiatum) in CA1 region in hippocampal slices using MEA device induces a negative extracellular response due to Ca2+ and Na+ inward flow by activated-NMDAR and AMPAR at the synapsis. It also induces extracellular positive response in soma and basal dendrites due to passive return current.

I would like to know the ionic basis of this passive return current. Could It be due to Ca2+ waves and activation of BK and SK channels at the soma?

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.

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? 

For example, in olfactory neurons, EM has shown dendritic synapses between Mitral cells and Interneurons. But are there ways to detect such contacts using fluorescence microscopy? Are there any proteins unique to dendrodendritic synapses that may be molecular markers for IHC?

Neurons are grown on coverslips in culture after some time they form connections and synapses (very basic networks). What kind of stimulator (digitimer) do i get to look at synaptically stimulated APs? I have used a voltage stimulator like the DS2 (digitimer) with a bipolar electrode in brain slice neurons, but I'm not sure if that's the best kind of stimulation for neurons in culture. Is current stimulator better or biphasic and what difference does it make? Also what kind of electrode would work better? Would a bipolar electrode fry my neuron cultures?

My understanding of direct vs indirect is that in the presence of TTX you can only measure activity at the synapse. However, I was discussing this with a colleague that thought it was only able to measure a postsynaptic effect. My thought is if you record in current clamp in the presence of TTX and apply a drug, if it releases a huge amount of neurotransmitter- such as glutamate- you could still get a depolarization of the post-synaptic membrane through the large influx of synaptic calcium. Is this wrong to believe? 

I am looking for a way to study the function of synapses on neurons in culture without having to do e.phys.

Calcium imaging, e.g. FLIPR?

Thanks!

I am developing a computational model of CA1, and I need to incorporate this parameter in the model. It has been surprisingly difficult to find within the literature. Thanks for the help!

Hi everyone,

I'm trying to induce chemical LTD at the CA3-CA1 hippocampal synapse using DHPG 100 uM as reported in literature but at the moment i'm not able to induce a stable LTD after 10 minutes of DHPG perfusion. I'm working on mice and i'm using 45 days old animals and i use 400 um brain slices for my experiments. When i applied DHPG i saw a reduction in the response but after about 20 minutes this reduction disappears and the response goes back at the control level (so the LTD is not induced). Do you have any suggestion or advice to have a stable DHPG induced LTD?

Thanks so much

Luca

Dear Community,

this is about Action Potential transduction speed over a Neuron!

Reaching a Neuron, an AP will first go through the dendrites, then reach the cell-body. Afterwards, it will go over to the Axon, then find a Synapse to give the AP to the next Neuron.

I need speed-estimates of every single one.

I found some estimates for conduction velocity to be about 1m/s for unmyelinated Axons.

The transduction over dendrites and cell-body might be in the same range!?

 The transduction over a synapse is fast, but I am interested in a cell-cluster, so I have about 10 Synapses.

It would help us a lot!

I wish to investigate activity independent measures of glutamate release at CA1-CA3 synapses in acute hippocampal slices.  I was wondering what is the standard concenteration of TTX people use for hippocampal slices. Thanks.

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?

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.

My dissertation is based on an interdisciplinary analogy on communication in diverse species. I am wondering if an expert would like to work with me on cancer cell communication?

Thanks

Ruben

Hi guys,

I'm a little bit in trouble with LTP on CA1. I performed some experiments inducing LTP after HFS (1 for 100 stimuli at 250Hz). This protocol is able to induce a strong potentiation of the synapse. My problem is that the potentiation after the induction is not stable and the response continues to increase without reaching a plateau. What do you think the problem is? I recorded extracellularly fEPSPs from hippocampal brain slices (400uM thickness; flow rate of about 2 mL/minute).

Thanks in advance for your advices.

Luca

I am investigating the roles of PKA and CaMKII following synaptic potentiation. Is there a way in acute slices in rats where I can image their phosphorylation activity with a single/few synapse resolution?  

I placed DiI crystals into the hilus of P10 mice horizontal sections (vibratome). I think the first image is from the CA3 region, and I can't tell if these are dendrites/axons or background, whereas the second image clearly has spines and does not belong near the CA3 region but is somewhere else in the cortex (can't tell right now where).

I would appreciate feedback (and protocols) on whether or not it is feasible to establish functional synapses between motor neuron and skeletal muscle cell lines.

Thank you!

P.

Dear all,

We know that each reflex involves a time delay between the stimulus and the reaction. This time delay is called reflex latency and It consists of three components:

I want to model the reflex latency of the stretch and miotatic reflexes in human upper limb (In particular,  I'm interested in biceps, triceps and brachialis muscles).

In your opinion which are the best values for Ta , Tc and Te?

After reading different papers and books, my ideas is that good values could be:

Ta= 10 msec;

Te= 10 msec;

Tc= 0.5 msec if we hypothesize that the motoneuron has just one synapse.

So, the stretch reflex latency is equal to 20.5 msec and the golgi tendon reflex latency is equal to 21 msec