Science topic

Hippocampus - Science topic

A curved elevation of gray matter extending the entire length of the floor of the temporal horn of the lateral ventricle. The hippocampus, subiculum, and DENTATE GYRUS constitute the hippocampal formation. Sometimes authors include the ENTORHINAL CORTEX in the hippocampal formation.
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What is the dimension and weight of ventral hippocampus?
Is there is a paper support the difficulty of separation of both ventral and dorsal hippocampus
Or any paper support biochemical analysis of hippocampus not require isolation of certain hippocampal region?
Thanks
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Please read this paper you may get insights.
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We are interested in isolating the hippocampus from frozen brains (flash frozen with isopentane and stored in -80C). Does anyone have a recommendation on how to best isolate the hippocampus? Do we need to thaw the brain and then isolate or should we section frozen and then isolate? We are interested in then running PCR once the hippocampus is isolated.
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Hi Rose,
The method is to briefly thaw the frozen brain sample for 2-3 mins and then conveniently place the brain onto an inverted petri plate under which some ice is placed, further you can cut the brain into the respective hemispheres and just pull back the cortex area and isolate the hippocampus
A video is attached below
I hope this helps you
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I have these squiggles that are appearing in my GAD67 IF stain and I'm not sure why. I've used this secondary antibody (Cy5 goat anti-mouse) with another primary antibody and never had this problem. In some areas, the squiggles aren't bad, but in others like the hippocampus, they're more prominent.
Any ideas?
EDIT: I don't know why the image is coming out blurry on here.
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Marcia Chavez, I've read that Sudan Black B treatment can help with this kind of autofluorescence, although I have never tried it myself. Here is an article in which the authors compared different techniques to reduce autofluorescence and found that a 20-min incubation in 0.1% Sudan Black B dissolved in 70% Ethanol was the most effective. They did it in combination with FISH though, not IHC : https://core.ac.uk/download/pdf/37464562.pdf
There is also a commercially available compound named TrueVIEW, which acts as an autofluorescence quencher, and is supposed to be effective on red blood cell endogenous fluorescence : https://vectorlabs.com/products/blocking/trueview-autofluorescence-quenching-kit
I've tested TrueVIEW, and in my hands (brain slices from perfused mice), it decreases the background, but also greatly reduces the intensity of any specific staining. If you use Sudan Black B though, I've read somewhere that it can also increase the background in the red and far-red channels specifically, so beware of this.
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I am working on a project entitled 'How exercise protects against the neurological impacts of stress.' I can see that during the 1980's there was a flurry of research suggesting that because circulating levels of β-endorphins increased during running that this was responsible for the 'runner's high.' However, later on, researchers dismissed this, arguing that β-endorphins cannot cross the blood brain barrier and this phenomenon should be attributed to endocannabinoids. But now I see rat studies indicating that β-endorphin levels do increase in key parts of the brain, including the amygdala. And I can also see that it may contribute to neurogenesis in the hippocampus. Could I argue that yes, β-endorphin levels do increase during exercise and do mitigate the effects on stress by specific changes in the hippocampus by creating a 'runner's high'?
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Does anyone ever done cell sorting of microglia from 1 whole mouse brain? how many total cells do you obtain and how many specific cd11b after cell sorting?
What about 2 hippocampi?
I'm trying to isolate microglia cells from adult mouse brain and from specific area such as hippocampus and i'm interested to understand how many cells I can get from the total and after cell sorting!
Thanks all :) :)
Elena
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Hi Elena Possemato,
Have you figured out how many microglia we can get from the hippocampus?
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If I use only 1 hippocampus to measure certain neurotransmitter using ELISA, I am afraid that it is not sufficient. Is it possible to measure 3 items from rat's hippocampus using ELISA?
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Thank you very much
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I am working on primary hippocampus neurons from mice. I am trying to look at the effect of amyloid beta on the neurons. I am looking at parameters like cell death, number of neurons in a field of view, neuronal length. I also want to try to analyze the neuronal coverage or area occupied by neurites to understand how amyloid beta affects the neurons.
Does anyone know how to measure the neuronal coverage or area occupied by neurites?
Thank You.
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The neuronal culture can be either live or fixed but would prefer to determine the coverage of neurites in live conditions.
Thanks in advance.
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We are currently performing this protocol in tissue obtained from mice (Cortex and hippocampus)
following the next steps:
MSH Buffer :
230 mM Manitol
70 mM Sucrose
5 mM HEPES
pH: 7,4
+protease inhibitor cocktail and a phosphatase inhibitor
+ 1mM EDTA (0,2M)
1 hemisphere of hippocampus in 400 ul of MSH buffer
Centrifugate to 600 g x 10 min 4ºC
-Rescue supernatant and centrifugate to 8000 g x 10 min 4ºC
Rescue pellet ( mitochondrial fraction).
The problem arises when we made western blot to the determination of mitochondrial purification and we still have citolosic proteins ( as observed by b-actin) in the mitochondrial fraction.
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Maybe try a sucrose gradient to isolate them better, or just add an extra wash step on your pellet (recentrifuge pellet at 8000 xg and resuspend it again)
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I homogenize my sample with PBS and then I add solphosalcylic acid %4 in same mount in the supernatant. The sample incubated for 30min in 4c and then centrifuged in 4000g for 10min and I add prepared Ellman reagent the samples and incubated for 15 min in room temperature but i dont see yellow color. I am sure my Ellman reagent is active and I think that my sample preparation has problem. Does anybody have any experience in GSH measurement to help me??
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I conducted a comprehensive study, in part it can help (I think we need to collect a fraction and it can be converted into a quantitative volume using statistical programs).
Blood was quickly removed from the brain, separated from the meninges and the studied pieces were placed in liquid nitrogen. Then, they were ground in liquid nitrogen to a powder and homogenized in a 10-fold volume of the medium at (2°С) containing (in mmol): sucrose, 250; TrisHCl buffer, 20; EDTA-1 (pH 7.4). At a temperature (+4°C) by the method of differential centrifugation on a refrigerated centrifuge Sigma 3-30k separated the cytosolic and mitochondrial fractions (17000 g).
The supernatant was discarded and stored at –80°С. The state of the thiol-disulfide system of the brain was assessed by the content of reduced glutathione (GSH) and oxidized glutathione (GSSG) in the cytosolic fraction fluorimetrically using o-phthalic anhydride on a Quantech fluorometer. Level of free
SH-groups, activity of glutathione reductase, glutathione peroxidase and glutathione transferase in
the cytosolic fraction was measured spectrophotometrically on a Libra S 32 PC spectrophotometer. The content of cysteine ​​and methionine was determined by thin layer chromatography in the cytosolic fraction followed by spectrophotometry on a spectrophotometer. The total content of reduced and oxidized sulfhydryl groups in the cytosolic fraction was determined using Elman's reagent spectrophotometrically on a spectrophotometer. The protein concentration was assessed by the Bradford method.
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As there are two methods for the intrahippocampal injection for the establishment of SE.
Kainic acid and pilocarpine.
which one is better?
As far as I know, the kainic acid is a typical model for TLE, while the direct injection of kainic acid will trigger the excitoxicity which make me have some questions upon this model. Like, the theory of this model is that inducing the epilepsy then result in the degeneration or the excitoxicity triggers the epilepsy?
Hope anyone could discuss with me about that interesting question. THX.
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Read this paper, you may get insights;
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Good day scholars.
Please I want to extract RNA from two brain regions of rats - striatum and hippocampus. Rather than using commercial kits, I want to use conventional method of extraction so as to reduce cost of extraction from about 300 samples. Please I will appreciate if anyone can help me with a step by step protocol that can be used for this RNA extraction. Please the RNA will be used for Gene Expression and other downstream applications. I will appreciate a reliable protocol from anyone who has been into such.
Thanks very much.
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Here it is the protocol I used with chicken hypothalamus
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I'm working on taking LFP signal from the hippocampus of mice brains for my project, So I wonder what percentage of the baseline signal you would consider as LTP? and what is the best period of time to take a signal for recognizing LTP after rapid and sequential stimulations?
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any percentage that is significantly different to your baseline. typically 50% will be considered mild LTP and 150% strong LTP
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I am studying the post-synaptic protein quantitatively in ventral hippocampus and have problem to identify postsynaptic soma/dendrites with a reliable marker, at immunohistochemistry level. Would be most grateful if some colleague could give me a hint... Thanks much.
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MAP2?
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In time past it was believed that the human brain cells do not produce new cells and that our brains remain static throughout life and any damage to any brain cell is a life long damage. But recently it has been brought to our knowledge that the brain does produce news cells continually at the hippocampus and the sub ventricular regions of the brain and that this regeneration process can been enhanced through physical exercise and that it plays an important role in memory and stroke rehabilitation. What are the characteristics of the hippocampus and sub ventricular zone that aids this peculiar characteristic and how can this be applied to our knowledge of neuron regeneration in other parts of the brain?
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Hi Benedict, I am a psychiatrist and recently published an article on how important GABA is in the mode of action of ECT. It is hypothesised that both GABA and glutamate are important in neuroplasticity and neurogenesis in recovery from depression, with the hippocampus, wider limbic system, and dorsolateral prefrontal cortex being the geographical area of interest. Can I direct you to looking at GABA in hippocampus and subventricles as a component of recovery in stroke rehabilitation?
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I have a Cre-DTA mouse which Cre is specificity express in brain, and i have used 400ppm
tamoxifen diet to induce Cre express, but the efficiency was too low, so want to use 4-OHT improve the cre recombination efficiency, but i find that there are so little information about 4-OHT use in specific tissue, such as brain, my question is how to use 4-OHT in brain (concentration\dose\diluent)?and what is the disadvantage of 4-OHT use in brain?
I have seen a paper which use 3-5ul 2mM 4-OHT in 4%EtOH in hippocampus, which induce brain parenchymal recombination, but mo figure about the result , so if there anyone else also try 4-OHT inject in brain? Thanks~
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I have used Jax's IP protocol (https://www.jax.org/research-and-faculty/resources/cre-repository/tamoxifen; IP inject 75mg/kg body weight for 5 consecutive days) for efficient recombination of floxed genes in the brain. However, this is highly dependent on both the gene and the cre driver. Some work MUCH better than others.
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I want method in estimating the thickness of the granular cells by histology.
Is estimating the thickness of the granular cells of great value ?
Thanks
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بقى سلائف الخلايا الحبيبية في منطقة تحت حبيبية تصبح أرق تدريجياً مع نمو التلفيف المسنن، ولكن يتم الاحتفاظ بهذه الخلايا السليفة في الفئران البالغة
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Good day scholars. Please can anyone help with a video or procedure on how to isolate hypothalamus from rat's brain?
All I have been seeing is that on hippocampus.
Thanks.
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Thanks Jens Pahnke
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For isolation of mitochondria from rat hippocampus, chilled isolation medium (0.25 M sucrose containing 10 mM Tris–HCL, pH 7.4, 1 mM EDTA, and BSA 250 lg/mL) is made.
Can we make this solution and store it for long? If yes, kindly confirm how long, at what temperature would we can store and use this already made chilled isolation medium ?
or it should always be freshly made ?
AND
If we place our rat hippocampus in this solution, how long can we keep the tissue in it until mitochondrial isolation from the tissue ??
Can we preserve our isolated hippocampus tissue in it for several weeks ? If no, suggest the alternative.
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You can keep the mito isolation buffer in 4degC no problem. I don't know exactly how long you can store it but I think at least 1 month should be okay. However, the BSA should be added fresh, same day and then double check the pH after adding the BSA. If possible, BSA that is "fraction V, fatty acid free" is best. If you plan to do any proteomics, consider that there is BSA protein in these isolated mitochondria samples. BSA helps protect the mitochondrial fraction from oxidative damage during the isolation procedure.
Once you place the fresh hippocampus in the solution (on ice), you continue quickly with the homogenization, centrifugation, density gradient isolation procedures which may take awhile.
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Samples were homogenized with a mortar and pestle and then passed through a syringe but at the first centrifuge step the sample is getting stuck - we can't see anything on the membrane and only about half is coming through.
Has anyone got any experience with this kind of tissue and these columns? Thanks!
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Helen Marshall-No problem! Best wishes :)
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Glutamate activates synaptic ionotropic NMDA receptors present in the post synaptic densities or PSDs, which sequentially activates the CAMK2 and CREB (cAMP-response element binding protein) that actively participates in neuronal plasticity, learning, and memory formation in adult brain, Then what is the exact role of amyloid beta 42 oligomers with respect to NMDA receptors a in causing dementia ?
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I am currently preparing a paper on feeding regimes in some European syngnathids. I am highly interested in data on mouth and snout (especially mouth gape )biometrics in adults of the following species: the seahorse Hippocampus guttulatus, and the pipefishes Syngnathus acus and Entelurus aequoreus. The aim is to assess any relationship between those biometrics and the composition of the diet in the wild. I would greatly appreciate any information. Thank you very much in advance.
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Dear Sule,
Thank you very much for the papers.
Are you currently sampling syngnathids or planning more surveys in the field?
My best wishes,
Miquel
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I am working on estradiol neuroprotection impact and measuring its concentration in hippocampal female rats in their diestrus phase by 17beta-Estradiol ELISA (Diagnostics Biochem Canada). To extract estradiol from hippocampus the tissue was homogenized in 150µl PBS and centrifuged at 15,000g for 30 min at 4ºC and supernatants were collected. The problem is almost no level of estradiol was detected through this method.
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I will try to explain it better. The average concentration of estradiol in brain is of little value. Then, even having an excellent method of extraction of estradiol from brain tissue the data will tell you little. Unless yo are measuring pharmacokinetics/biodistribution after estradiol administration.
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I need to target PV interneurons in 1 and 2 months old mice.
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It might be useful to check a mouse brain atlas as well,
The Allen Reference Atlas (available for P56 and P14 mice):
An interactive tool for stereotaxic coordinates is also available:
Good Luck
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Hi everyone,
Do you have any experience in preparing sample dilutions to measure the concentrations of inflammatory cytokines (TNFα, TGFβ, MCP-1, IL-1α, IL-1β, IL-6, and so forth) for an ELISA when it comes to any rat brain tissue, especially hippocampus. I'd prefer not to perform a pretest, and waste any well-strips beforehand. Based on your experience from different labs, what would be the expected protein concentration ranges of these cytokines in a supernatant saved from tissue homogenate prepared from a healthy rat (Sprague Dawley) brain tissue (hippocampus)?
Kind regards,
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with the aim of doing RT-PCR, we homogenized hippocampus and took the aquatic phase. Now we need to do western blot analysis but just the organic phase (included protein) is left. does somebody know the protocol?
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Hi Mohammad, if you have used TRizol for RNA extraction, please check page 4 of this manual for isolation proteins: https://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf
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I have H&E stained hippocampus sections and would like to look at thickness and number of degenerated cells of hippocampus at different areas in different studied groups to confirm neuronal loss after injury. Thank you
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Try to stain your neurons with an anti-MAP2 antibody.
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I've recently begun collecting hippocampus tissue from mice brain for a project of mine but as it is my first time working with mice brains, I was wondering if there's a good marker or stain for hippocampus to validate that the tissue I am extracting is actually hippocampus.
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Hippocampus is usually distinguished from surrounding tissue anatomically through its distinct appearance, even unstained and with no magnification. It is resolvable on the cut surface while sectioning with a cryostat, where it appears as two interlocking Cs (make C shapes with your hands, move them so that the fingers of one hand are between the fingers and thumb of the other, and you've got it). However, this shape curves as it moves through the brain and it can take some training and consultation with mouse brain atlases to appreciate the way its appearance changes as you section through and is dependent on the mounting orientation during sectioning).
Because of its defined anatomic borders, most histology has focused on distinguishing hippocampal subregions. Rusty Gage did publish a fairly comprehensive analysis of the transcriptional profile of subregions (see below) that may also point you to some targets that might be expressed more strongly in hippocampus than in surrounding tissue (e.g., with PCR).
If you are taking the entire hippocampus (not sectioning) then get someone to show you how to dissect (usually a biochemist or electrophysiologist)- in each hemisphere, it should come out as a distinct lima bean-shaped piece (pay careful attention to the removal of meninges/ choroid plexus tissue). Across the long- axis, the surface closest to the CA region should be convex and coated in a thin layer of myelin (alveus- looks like onion skin), the other surface (closer to DG) should be flatter and have two darker grooves that run its length. The dorsal aspect should taper to a white matter tip, and the ventral aspect should be thicker and rounded at its end.
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I'm looking to validate my hM4Di expression in GABAergic neurons of the hippocampus through acute section recordings. Attached is what we have so far but my PI argues that it did not silence the neuron entirely. Could someone share resources or experiences with hM4Di recordings to help me contextualize my results? Is this typical or should I be troubleshooting my DREADDs expression?
Thanks
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You can record the hM4Di+ neurons for a long time. For instance, 5 min for baseline, 10 min after administration of CNO, and 10 min after washout of CNO
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Hi there!
I'm trying to patch Purkinje cells but I always get -200 pA of leaky current after I break the giga seal. It reaches -200 pA about 5-50 sec after I break the seal.
I'm using a classic K-gluconate based solution. I already checked for osmolarity, pH and so on. I also tried an intracellular solution containing EGTA. However, it always go back to -200 pA, even when patching on another setup.
Never had this problem when patching pyramidal cells of the cortex or hippocampus.
Any help, tips or advice are more than welcome :)
Cheerio!
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Oh okay, thank you! Yes I will check.
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I am recording local field potential from stratum radiatum at CA1 by stimulating Schaffer Collaterals. I wanted to get a better idea of the size of SR as I do not want to get too close to the pyramidal layer or hit the lacunosum-moleculare layer. Literature search did not return any measurements. Also, how far do dendrites extend from the cell body at CA1 pyramidal layer into the deeper layers of the hippocampus?
Thanks
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Thank you Max Anstötz and Paula Parra .
I will try improving the illumination. The size guidance are helpful. Thanks
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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.
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Hy to stain neurones and synapses for electron microscopy it’s necessary to use antibodies against 5HT labelled with colloïdal gold or HRP revelled by DAB or TMB. Good luck
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The brain tissue sample is stained with NeuN. I want to be about to automatically count neurons especially of the dense area of the DG region of the hippocampus.
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I agree with the workflow suggested by Gabriella Marin where I want to add that during thresholding you can use either 'Huang' or 'Yen' as logic which will give more specific segmentation for selection. Further, in the same line, it will better if you can make binary of the image, which may again give more specific and automatic detection of the NeuN positive cells. Apart from the mentioned pipeline you can also go and check out a plugin of ImageJ/ FIJI called an image-based tool for nuclei detection (ITCN), where you have to mention the circularity of one NeuN positive signal in any of your unknown sample. It works well with immunofluorescence images.
Best wishes,
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I'm measuring fEPSP in the CA1-CA3 hippocampus region and lately I have had presence of bubbles in the bath where ACSF comes in. This leads to unstable recordings and from what I understand the bubbles are caused by the in-line solution heater. I have been thinking pre-heating the ACSF in a beaker where it's perfused before it goes to the in- line solution heater and was wondering what kind of heater you guys might be using out there. Preferably one that is not know to cause any electrical noise and small so that it fits/gets completely submerged in a 500ml beaker. Thank you!
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Hi Deogratias,
We have used by many years the Warner Instruments SHM-828 with multi inline heater and it goes very nice and very easy to use.
Regards
Enrique
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I found contradictory results for this question.
Few research groups reported high ChAT levels in hippocampus while others say hippocampus lacks ChAT activity. Please provide relevant answer with references.
Thanks
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I'm going to analyze GSK-3 concentration in rat hippocampus homogenate but I don't know how correctly should I calculate this concentration per total protein level examined by Bradford method?
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Depending on the kind of assay that you would like to use to detect GSK-3α or GSK-3β you can use immunohistochemistry or activity assays. Please see the following paper to know more about the details of the protocol and to see the calculation:
See the methods of the following papers as well:
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Hi! I would like to isolate synaptosomes from mouse hippocampus but I already have my sample homogenized in RIPA buffer. Is there any way to isolate synaptosomes from that? Thank you!
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Thank you very much!
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If damage occur in certain area of those region , different presentation will occur ?
For example CA1 is reach in place if damage occur in it what is the consequences?
Thanks and best regards .
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Currently I have some FFPE embedded mouse hemispheres. However, I wish to analyse solely the hippocampus and a region analogous to the DLPFC. Is this possible? And if so, should I melt the block, dissect and re-embedded? Or is it possible to punch tissue cores without the need to re-embedded? (I have an array puncher)
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You can slightly melt your block at 35C for 30 minutes to make the wax soft, take a punch of your region of interest and re-embed your punch in a new block. This will create a hole in your parent block but you can continuously section through a new block. You can also melt, dissect and re-embed but in both ways you would need a new block.
Without the re-embeding, I suggest scoring your block lightly and slightly with a new sharp razor blade and collect the area of interest as you section. This method will get you 3-4 sections at a time before you need a new score to collect more tissue. This way you will still have an intact parent block and you are only collecting small sections of your area of interest.
Alternatively, you can look into laser micro dissection. This way you take a whole slice on your slide and only dissect your neurons/area of interest. The last would require an expensive microscope.
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Does anyone have any good tips on how to distinguish between hippocampal pyramidal and basket cells?
I am studying the morphology (dendritic branching and spine counts) of hippocampal neurons in golgi-stained mouse tissue. I was initially looking at pyramidal neurons, but my PI suggested I could look at basket cells too. However, when I looked at the morphology of basket cells in the literature/on neuromorpho,org, they looked similar to some of the neurons I had been labelling as pyramidal neurons.
I know that typically pyramidal neurons have a singular long apical dendrite that extends for some distance before other dendrites branch off it, while basket cells have multiple branches from at or close to the soma. Also that neurons with soma clearly within the pyramidal layer are more likely to be pyramidal neurons, but neither of these criteria feel clear cut enough for me to be confident. Sadly the basket axon doesn't seem to be labelled in any slices as far as I can tell.
I've attached a few images of cells I have included. They are collapsed z-stacks of about 20-40 um in z-axis. If anyone could give their opinion on what cell type they are and why, that would be very helpful.
Thanks in advance!
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Hi Craig,
Trying to differentiate between basket cell and pyramidal in golgi-stained sections may not be the best. Also, golgi works best for spine analysis in my opinion and not easy for dendritic analysis either and axons usually don't get stained with golgi (I never saw in my sections).
Having said that, the first image is a pyramidal for me, the second one likely, the third seems so but it's difficult to say. Also in your images, it's not clear which layer are you in. There seems to be less. The staining of adjacent cells in the hippocampal layers make it easier to categorize the neurons as pyramidal cells as they look quite alike with their somas packed next to each other and in similar orientations. But basket cells, as you already mentioned are not usually arranged in the same axis and orientation, if you can use that fact to differentiate between the two (maybe? I am not sure).
Here is a paper from our lab with some nice golgi images from CA1,CA3. Idk if it may help
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I've been trying to lesion dorsal hippocampus after surgically implanting bilateral cannulas in wistar rats. other than manual restraining, Is there any other way?
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A well handled and habituated rat should need very little restraining. We often spend several minutes a day for several days or more handling them after surgery, playing with them to get them habituated to experimenter touch. At that time we will practice loosening the dummy cannula but not taking it all the way out. It also helps make sure the dummy doesn't get stuck in the guide.
Regarding injection, if you are trying to inject straight from a Hamilton syringe in to the cannula then that would be very tricky. We inject with a Hamilton syringe connected by FEP tubing to the injection cannula, which has been pre-implanted stereotypically.
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Hi everyone,
I'm consistently having small fEPSP's of approximately -0.01 mv. I've been moving the position of my electrodes and increasing the stimulation intensity but the response doesn't change much. I'm looking at the CA1 region of the hippocampus and would like to do an LTP experiment.
I'm following a protocol that was done in our lab 10 years ago. However, I recently set up the rig after it had be unused for the last 10 years. I'm not sure how to rule out potential problems for the rig. This is a consistent problem that happens each time I record. Does anyone have suggestions for what to check for?
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Hi Don, did you resolve this issue? I just found it while looking for something else. I had the same issue and after going crazy for a wee or two I realized the problem was the amplifier parameters. Check that there are no filters that are hiding your signal. My waveforms looked exactly the same as yours so it might be the same! Feel free to message me and I'll explain better.
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According to the vertebrate literature, I got the impression that head-direction cells are always anchored to an allocentric reference frame. This can easily be tested with freely moving animals. But what is about tethered animals that can freely rotate around their body axis but can`t change their actual position. Are neurons encoding the animals heading automatically defined as allocentric ones? Or how could you test with a tethered approach, if the neurons encode heading in a allocentric reference frame or in an egocentric one?
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Please correct me if I am wrong ,but the neurons (of the parietal cortex) reported in the PNAS paper are not classically circumscribed as head-direction cells. Sure, they show heading tuning but for me this tuning looks more like a sensory-based peripheral tuning. Although not being an expert in that field, I Interpret the parietal neurons to encode more general aspects of body-posture relative to surrounding objects and not necessarily to be specialized to purely encode aspects of navigation like cells of the hippocampus network. I guess this may be also the reason, why the authors do not explicitly termed the parietal cortex neurons as head-direction cells. But it is valid to say that there has to occur a transformation from egocentric to allocentric reference frames somewehere in the brain (and obviously also a retransformation from allocentric to egocentric).
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Hi everyone.
I bumped into the difficulty that I couldn't get any signals from my brain section in the DG region of hippocampus. However, I have standardized the BrdU protocol in my brain positive control. Any possible reasons to have that issue??
thanks!
My BrdU protocol:
DNA Hydrolysis with 2N HCL at 37oC for 30min, and the neutralised with 0.1M Borate buffer for 10min, and washing with 1X PBS and go for the normal immunostaining step.
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Hello Louis,
What are you trying to quantify using BrdU staining? Is it cell proliferation or cell survival? And how many BrdU injections did you perform before sacrificing the animals?
Sometimes the reason for not detecting any signal could be a non successful BrdU injection, regardless of your immunostaining protocol. Also, if your treatment conditions could potentially deplete adult neurogenesis, then you'll expect to get very few stained cells (especially if you're trying to evaluate cell proliferation). You'll need to examine your slides really carefully under microscope to be able to see those very few stained cells.
I hope this helps.
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Dear all,
for my Master thesis (not a publication) I compare Astrocytes of Alzheimer Hippocampus brain slides with (more or less) healthy ones.
For that I use k-means in Fiji to generate a ROI around GFAP flourescent signal to get the cells. Then the ROIs are put on the original acquisitions to measure the integrated density (mean grey value * area) to compare the expression of GFAP with a second protein of interest.
The ROIs quite precisely get the cell bodies and I'm happy with it.
Questions:
1. Do I have to do something in addition like a citation or validation to proof the method is doing what its supposed to do?
2. How do I best measure the colocalization/correlation of those two proteins? I'm not sure if measuring the intensity under the ROI is enough to understand the correlation of expression of these two proteins. Do I have to set it in relation to the global pixel value or something like that?
I would be very glad to get some support :) Hope you are all fine :)
Regards,
Simon
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You can find detailed instructions on how to perform colocalization analysis using the Coloc 2 package of ImageJ/Fiji here: https://imagej.net/Coloc_2
Also, you can find more explanations on the different methods and pitfalls of these analyses here: https://imagej.net/Colocalization_Analysis.html#What_is_colocalization.3F
Good luck!
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I'm setting up an electrophysiology rig examining LTP in the CA1 region of the hippocampus. What is an appropriate tip diameter size for a stimulating electrode to generate fEPSP? Also, I'm planning to use Tungsten Concentric Bipolar electrodes.
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we also use FHC - total tip size is 250 um but the center - active stimulation tip is 50 um - you can get good focal stimulation with those bipolar tungsten from FHC.
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I am trying to start a protocol involving bilateral cannula implantation in specific brain regions in mice (mPFC, hippocampus). For that, I need to buy an anesthesia system. I found many alternative companies to buy from, but I am particularly interested in MINERVE 1301303. I am also considering WPI EZ-B800 or Kent Scientific SomnoSuite® Low-Flow Anesthesia System. Can anyone give me feedback about these anesthesia systems? Maybe you have other recommendations? I will be grateful for all suggestions.
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Thank you!
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Hi,
I am injecting AAV2-Retro virus (AAV2-retro-EF1a-DIO-FLPo-WPRE-hGHpA from Addgene) that carries cre-dependent flp recombinase, in the mouse dorsal hippocampus and I need to verify that the spread is limited to the hippocampus. When the virus infects the cells in the hippocampus, there will not be any fluorescence expression because the viral construct has no fluorophore. I am of course exploring alternatives to this virus so there will be fluorophore expression. However, I was wondering if there is any other easy way to check the viral spread. Has anyone tried any antibodies against AAV2 proteins?
Thanks
Mani
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Thank you Matias.
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I am looking for a skilled personnel in Kuwait University that is familiar with antioxidant studies using ELISA kit or other techniques on rat's brain homogenate, particularly the hippocampal and anterior cortex regions.
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Hi Ahmed. I used to work with antioxidant assays on brain homogenates but not with commercial kits. The assays that I used are affordable and pretty much reproducible.
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Hi,
I have done a seed to voxel analysis recently using conn toolbox. I have a basic question about how to report the result. My seed was a hippocampus structural mask and I compared its functioncal connectivty with the whole brain between two condiions. The result was a netwrok that included several clusters. but I'm not sure how to report this result. For example if the netwrok contained a special cluster (e.g. DLPFC), can I say that hippocampus showed a significant change in connectivity with that cluster (DLPFC) in one condition compared with the other condition? Can I infer about the connectivity of one of the clusters separately or I can only infer about the whole netwrok?
Many thanks, Haleh
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If you want to report statistical results this link might be helpful:
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I have to determine the effect of some phytoconstituent on MARK4 and microtubule of neuron. How can I do it. I mean do I have to first extract the MARK4 from hippocampus and cortex by RT PCR and then check its expression? Or what else. Please suggest.
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yes you can purify it first and then check the effect on the activity
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Hello to all.
I am performing a thesis on Cognitive disorder modeling in rats and the effect of
Pharmacological Treatment on the disease and the expression of certain miRNAs.
I am seeking guidance from any researcher who has experience in "Rat brain tissue miRNA analysis" to help me understand more about my project so I would be grateful for even the smallest info and tips based on your experience.
I would also be really thankful if anyone could help me with informations about U6 as a housekeeping gene and primer design for this sequence.
Thanks in advance.
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Well, I may not have very specific points for "Rat brain tissue", but widely accepted and feasible to do steps are.
-You can even isolate total RNA using the Trizol method and use Qiagen miScript II RT Kit, which converts all miRNA in a sample to miRNA cDNA and then use those cDNA directly for qPCR to check the miR level itself as well as other GOI.
--Kindly prefer at least 3 endogenous control.
***Never rely on only one endogenous control***
---U6 is miRNA endogenous control but along with it includes two more.
----miRNA primer has universal sequences. The miRDB database provides stem-loop & mature miRNA sequence. Prefer predesigned primers of your interested miRNA from the standard company. Nowadays, Companies do a slight modification or say a technology, which enhances the stability and avoid unwanted binding. Qiagen has LNA tech. based on miRNA primers, it has a readymade, pre-mix(FP+RP) pre-designed primer of most of the miRNAs.Just dissolve and use 1ul for per rxn.
Cheers!!!
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Hello everyone, I wanted to ask you if you know any good paper that dig into the connection between medial Prefrontal Cortex and the Dorsal Hippocampus. Also if you knew some paper related to shizofrenia would be great.
Thank you :-)
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Hello;
In terms of learning and memory, do you think that proinflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6 may act differently in the prefrontal cortex and hippocampus?
I've some data that shows TNF-alpha and IL-1beta are reduced in the prefrontal cortex but increased in the hippocampus in the animals with impaired learning and memory scores in Morris Water Maze and Novel Object Recognition tests. In my opinion, it may possible that the neurotoxin that I used may reduce live cells and cause to detect a lower amount of proinflammatory cytokines. However, since the level of them are higher in the hippocampus (as expected) I can not make any connection with that difference between prefrontal cortex and hippocampus.
Of course, there are some papers reporting the diverse role of those cytokines in different conditions, however, I could not find the proper and clear answer for my case.
Thank you for your help.
Kind Regards.
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Hello,
In may be due to the glial difference between the two regions. The ratio neuron/glial cell is lower in hippocampus than in prefrontal cortex.
Inflammatory reaction may also be different between region depending on neurotoxin injection mode. However, if there are papers on the opposite role of these cytokines in brain regions I am also interested!
Best Regards
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Hello,
I want to prepare brain tissue for western blotting. I have the hippocampus extracted and frozen in liquid nitrogen. I tried to crush it using a tissue pulverizer but I was afraid that would lead to contamination. What's the best way to prepare the tissue and lyse it in RIPA buffer? Also, is there a specific volume per gram of buffer I should follow?
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Hi,
I always extracted my proteins by using the pestle in the 1.5 mL eppendorfs, celllytic buffer from sigma and usually a protease inhibitor cocktail. The pestle I used was glass and was cleaned between each sample to prevent any contamination.
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Hello.
I have a question about the shelf life of Corning's NuSerum I that we use for the first days of primary culture of neurons from the rat embryo hippocampus. How long do you keep it at -20°C before it starts to degrade? We have been worried about premature death since this summer and we still have this solution to check. There is no expiration date, it is simply noted storage superior or equal to 3 months, which does not mean much. Thank you.
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Thanks, but it is not what is written. They say storage >= 3 months. I think they tested at 3 month and not more, that's all. Usuallt serum like FBS can be stored for years. We buy a full lot that works, and avoid to change too often.
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These images were the immunofluorescence of my protein in the hippocampus. The images represent the merged signals with DAPI signals (blue),my protein(red), astrocyte marker glial fibrillary acidic protein (GFAP,green).thanks.
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Hi,
Although -in my opinion- the IF are some subjective experiments, I can see a notorious colocalization of green and red markers in some cells, but I am not sure if is enough to call it a special location. I don't know so well the shape of astrocytes, but it seems express in the cytosol. Also, it is important that you review the pattern expression of your antibody, sometimes the manufacturer specifies the location where the antibody labels,
Best,
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Hello,
Does anybody know a program in which I can enter data from biological replicates, with an untreated or treated category to plot as bar graphs? I know this feature exists in GraphPad, but as I am a student, the license is too expensive for me at the time.
Is there any other free software that can deliver a similar result?
Very likely there is an option to achieve this using the R framwork, but I am very unexperienced with programming in R, so I would prefer a pre-made software solution.
My use case is counting specific cells in the hippocampus and plotting the different cells per area as a bar graph with n=4 different biological replicates. I want the graph to show the different data points for treated/untreated condition, the bar to be the Mean and to be able to show the error via normal error bars. (see image attached)
Thanks for your help! I would be very glad if there is a free alternative to GraphPad for my usage.
All the best,
Paul
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To Paul Wagner: perhaps you can try PSPP...
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What modifications do you make in ACSF for aged mice brain hippocampus recording?
Please if possible explain specifically for cutting and recording solutions?
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I would recommend checking this website for aCSF recipes, especially for older animals https://www.brainslicemethods.com/ .
Transcardial perfusion of cold cutting aCSF before slice preparation and incubating slices in an interface chamber might enhance slice health.
good luck!
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So, basically I would like to analyze the entire Hippocampus concerning the number of cells showing a certain stain. However, with the magnification I need to differentiate between the cells, the entire Hippocampus is not in one image. Is there an efficient way to merge the images of the different sections of the Hippocampus without them overlapping?
Thanks in advance,
Mate Abraham
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You can use stitching tools of Fiji (ImageJ). They are pretty robust. If you have whole slide images and you would like to stitch whole slide images, bigstitcher/big data viewer and hdf5 format would be useful.
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I'm doing segmentation of hippocampus in matlab using LAC method,i want to create mask for the segmentation of hippocampus..Can anyone help me in this regard..
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MedSeg (medseg.ai) has a model for automatic segmentation of the hippocampus on axial T1 scans. It is free to use and web-based.
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Hello, everyone!
I'm going to start a project with single-cell ATAC-seq for analysis of open chromatin regions from freeze tissue samples.
I'm lost about the nuclei concentration.
If I made a nuclei isolation protocol from the human brain freeze tissue sample (hippocampus), what would be the nuclei mean number from these?
2000 nuclei? 5000 nuclei?
There is any paper about that?
Thank you!
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Hello Jaqueline,
I would follow the work of Sebastian Preissl (now at UCSD). He has much experience on isolated nuclei sequencing and should provide the guidance you need in his publications. He recommends the far-red DNA dye DRAQ7 to label the nuclei to sort them free of debris, etc. He gave an excellent webinar recently (see Selectscience for a recording of this.
Regards and good luck,
Roy
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When I read articles I can only find the information "mostly surrounding PV+ interneurons but it doesn't help me... I guess it is found in the cortex but where?
In the cerebellum it's more clear. And I don't know about other regions like hippocampus, amygdala... So if you have any clue it could be very usefull. Thank you!
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Interneurons
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How long after the conditioning (control) rats remember the shock.
I know it depends on various factors (age of the animal, shock mA, etc.). What's the longest period you've come upon.
Thanks
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Hi everyone! I will isolate microglia from adult hippocampus by using Miltenyi protocol with Adult Brain dissociation kit and Cd11b microbeads. I will perform the success of my isolation with CD45 and Cd11b antibodies for FACS and then I will plate the cells on coated coverslips (30' UV and then washed with h20 for 3 times) with polylisine for immunocytochemistry experiments in DMEM-F12+GlutamaX 10% FBS 1% pen/strep, 1% gentamicin and 10ng/ml of MCSF.
I'm thinking to stimulate cells not more than 48 hours and changing at the same time of the stimulation.
What do you think about this protocol? Do you think it will work well?
It's the first time that i try to isolate microglia from a specific brain area :)
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This protocol is a time-tested method. But in my experience it should give you both macrophages and microglia. I used FACS using Iba-1 to sort out embryonic microglia.
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Our lab is interested in doing single-unit recordings in area CA2 of the hippocampus. We have a lot of experience with single-unit recording, but not in area CA2. Does anyone have experience trying to identify this area through histology? We are interested in both dorsal and ventral hippocampus.
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Thank you so much! Best regards
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I'm interested in isolating RNA from mouse hippocampus and prefrontal cortex. My lab routinely uses the Trizol method for large pieces of tissue (eg, liver and intestine), ending with dissolving the RNA pellet in 200 uL DEPC water. For hard-to-extract tissues (eg, adipose) we reduce the final step to 20-50 uL DEPC water. Is this an acceptable modification for hippocampus, or should I reduce the volume even more? We usually aim to get at least 250 ng/uL RNA for reverse transcription but I'm wondering if this is possible with such a small piece of tissue. Are there any other modifications to help with yield?
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Hello Jessica! Incubating of the supernatant after Trizol will tend to increase the yield since it may give you low purity, impure precipitation of the RNA. For the RNA work, efficient dissociation of the brain tissue is a must. I would suggest to try Lysing Matrix E https://www.mpbio.com/116914050-lysing-matrix-e-cf. You can use any kit, but I used this kit previously to get great yield especially on a single dorsal root ganglion https://www.mpbio.com/112721050-rapidpure-rna-tissue-kit, Spin Kits. Trizol is dangerous especially chloroform, so suggest to change to spin column. It will retain RNA better, your 260/280 should be close to 2.0. Another suggestion is to immediately place your brain samples in RNALater Buffer, let it sit until it sunk overnight at 4C, then take the brain sample, lyse with lysing matrix, and then spin column. You'd have great result with this methodology and avoid using harmful chemical, chloroform. Let me know should you have any questions.
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Hello everyone!
I'm currently working on my thesis, that is related with testing the anxiolytic effects of some substances in mice.
However, I believe I can enrich my project by adding histological assays, due to their low cost and relative simplicity.
Could anyone provide me with some advice/suggestion? Thus far, I've encountered analysis of addrenals and hippocampus, but I believe there can be more (especially with the heart tissue).
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I'm glad, if I can help you, Marjon Gjika !
There are some data about increasing of adrenoreceptors density in cardiac tissue, for example, look at this article: https://www.researchgate.net/publication/263433015
If you haven't got a lot of instruments, you can measure the thickness of left ventricle, or try to find fibrosis in cardiac tissue, but I think the result will be negative - these changes need a lot of time. How long you want to do chronic stress and treatment?
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Please, does anyone knows the best preservative that can fix brain tissue (hippocampus) for about a month before processing?
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Look into publications of H.K.P. Feiraband. Have publications not at hand. The article compares different methods of fixation and makes a clear choice. Succes Marani
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Our lab has noticed that after several tissue preparations for organotypic hippocampal slice cultures, some of our tissue is developing what appears to be large holes between the CA1 and dentate gyrus. Has anyone using this model noticed this or have any suggestions on how we might address this issue?
We have checked CO2 levels in the incubator, and no changes have been made in our procedure or culturing media preparations.
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Thanks for the details Caleb Bailey ,
Since you have mentioned that only some of your slices have such tears or holes, any possible problem with your sectioning and microslicer or vibratome blade can be eliminated.
Please see if the intensity of the vibratome and vibration of the blade is consistant and suitable to avoid any holes or mostly tears.
I am not sure if the age of the animals can be an issue (if someone else have any thoughts on this...)
The thickness is also good and cannot be an issue. Though I would recommend testing slices with different thicknesses (100 and 400 um for instance) to have a more clear comparison. Though this could be mostly due to physical uneven pressure during slicing, please consider the following too:
You may search and examine different dissection buffers as well (e.g. 1 mM CaCl2, 5 mM MgCl2, 4 mM KCl, 26 mM NaHCO3, 8% sucrose, 0.5% phenol red, and filter-sterilized and bubbled with 95% O2/5% CO2 (carbogen). Possibly ice-cold buffers.
Also, different culture media, like this one that I found: MEM with Earle’s salts and l-glutamine (Gibco 11095) supplemented with 1 mM CaCl2, 2 mM MgSO4, 1 mg/L insulin, 1 mM NaHCO3, 20% heat-inactivated horse serum, 0.5 mM l-ascorbate, 30 mM HEPES, 2.3 g/L d-glucose, and pH 7.3.
After the brain removal (as quick as possible), if you use ACSF to embed the slices, then check the temp to be around 5 degrees Celsius.
Store and hold them at 35.0 ± 0.5°C in carbogenated ACSF prior your IHC to see if this helps.
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I need to induce LTP in hippocampal acute brain slices (rodent) and measure extracellular fPSPs.
Different bipolar stimulation electrodes available on the market have different impedances (0.1 MOhm, 0.5 MOhm, 1Mohm, 1.5 MOhm, and 2 MOhm) and I am not sure about the most suitable (no info from the customer service…).
Could anyone recommend a particular impedance based on experience?
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Hi there. I use 0.1 MOhm tungsten electrodes. Using 0.5 MOhm or higher impedances didn't give good ltp results with my recordings. Good luck.
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I am working on scopolamine induced Alzheimer rat model. I want to keep the HC of one hemisphere for ELISA & WB. My Questions are:
1) does single HC will be enough for application of both tests?
2) is there a common buffer to keep the HC in for both tests?
3) which is it better to keep the samples; with or without homogenization in the recommended buffer?
I will appreciate if anyone can share his/her protocol with me
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Hi Abdelraheim Attaai,
I think it's totally possible. I use mouse HC from only one hemisphere to run WB using c400 Imaging System (Azure Biosystems). So, as rat HC is much bigger, it should be enough to do both.
I use dry ice-cold saline for washing and keep the selected brain structures in -80°C after dissection until further processing (without any buffer). Then, cut them on dry ice and homogenize using ice-cold 1 × Cell Lysis Buffer.
Good luck!
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The most accepted definition for RRP is " a small subset of the many vesicles in presynaptic bouton that is more readily released than other vesicles which are in the recycling and reserve pool", and RRP consists of "docked" vesicles. Well, can we increase the number of docked vesicles in the presynaptic terminal? If we do it, what is the physiological mechanism underlying this increment? Does this increment depend on the increase of intracellular Ca2+? Does the increment of the size of active bouton lead to the increment of the size of RRP?
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Actually I believe that the RRP size is just a result of the instant probability of vesicle exocytosis. If so, current Ca2+ concentration strongly impact RRP size by mean docking molecular machinery (SNAP-25, syntaxin, synaptotagmin and so on). Increasing the concentration of Ca2+ leads to more probable activation of Ca-sensors and more reliable exocytosis.
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I would like to quantify the number of dendritic spines in all the maturation stages by using a coronal section of a mouse brain. I will be looking more specifically in the hippocampus.
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In order to visualize dendritic spines, I utilized Golgi-Cox staining, which is a very simple and reliable method. To sort the spines in accordance with their maturation stage, you can analyze their morphology. In general, spine maturity progresses from thin filopodia type to wide-headed mushroom spines and the irregular branched spine. The conventional classes of spine shape are "thin", "stubby", "mushroom", and "branched".
The manual spines classification is a time-consuming and tedious task yielding varying results. Fortunately, there are several pieces of software, which perform a rapid spine analysis.
I worked with Neurolucida, which calculates in an automotive manner a large number of metrics, including volume, length, plane angle, and surface area, and includes a notation of whether the spine was classified and how it was classified. It is not a free program, but you can ask for a demo version and perform your analysis.
Currently, we use Imaris, which is a much more powerful piece of software (in my opinion).
Good luck!
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Dear all
I would like to use a platinum/iridium electrode for schaffer-collateral stimulations in a mouse hippocampal slice preparation.
I have good success using regular concentric tungsten electrodes, but for a specific purpose, I want an electrode that contains no stainless steel.
I have had no luck finding a commercially available twisted wire platinum/iridium electrode, so I was wondering if anyone had a good recipe for making them?
thanks in advance!
best
Nikolaj
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