Neuroendocrinology - Science topic

Hi, arigo has a good Human Cortisol ELISA Kit for serum and plasma and is cheap also. Please refer to the website for the detail. https://www.arigobio.com/Human-Cortisol-ELISA-Kit-ARG81162.html

PLEASE READ THE SYS REVIEW

Hi. Did anyone manage to locate KRJ-1 cells? I am in need of some now but cannot find a supplier. Thanks.

Hi Yanuar

I've found lots of old papers in rats. Handling rats is always easier, their brain and bodies are x10 times larger, everything is easier with them. But since genetically modified animals are usually mice, they have become the species-of-use in the last two decades.

Optogenetics and pharmacogenetics can be done in rats as well, provided you get viral vectors designed for rats, which is not so difficult.

Both species are similar but not identical. Both are rodents, and good models for studying interesting issues for humans, I don't find advantages in mice or rats relative to their similarity to humans.

Good luck

Fernando

I am trying to find a way and equipment to measure non-invasively, stress and wellbeing. Any suggestions. I have already identified HRV as one modality.

Er....what?

If all you're using these brains for is western blotting, cell viability is irrelevant: all you care about is protein.

You'll be crushing the tissue under liquid nitrogen and then dissolving some tissue powder in some sort of lysis buffer (RIPA or SDS or similar).

You don't even need to use isopentane: you could just drop the brains straight into liquid N2, or place them on dry ice. The time taken to freeze solid is far shorter than the half-life of virtually every protein you might be interested in.

The only time isopentane is really necessary is for rapid freezing: to preserve cellular architecture for histology (liquid n2 alone freezes too slowly, due to leidenfrost effect).

If you want cell viability (!?) then you shouldn't be freezing them like this at all, you should be isolating and amplifying the cells of interest in culture, then freezing them as for normal cultured cells.

See this link of article

Prof Julio,

Thankyou very much for such adetailed informative answer.I do expect many of our RG colleagues will go through it,

Basically you need to know the hormone size in KDA. Once you know, run sds and confirm with western blot test by using antibodies. Once confirmed, you may run gel again and extract out the band that you have identified and confirmed. Then you will get your hormone. 

Fibrous adenomas are often described as "invasive" by the surgeon simply because they are difficult to remove from the cavernous sinus, carotid wall or optic nerve. As such many postoperative scans of fibrous adenomas show residual tumor in these areas, whereas most secretory adenomas are friable and allow complete resection.

Histopathology is a more objective measure of invasiveness. DA treatment does indeed predict some fibrosis.

Speaking from personal experience (and that of other researchers at the Yerkes National Primate Research Center), it seems that cortisol peaks 30-60min after acute social stressors as well.  I work with rhesus monkeys and typically use the human intruder paradigm (invited by Ned Kalin & Steve Shelton at the Uni. of Wisc) as my acute stressors (30min stressor). I've taken blood samples immediately before and after the stressor (Raper et al, 2013 Psychoneuroendo, Raper et al 2013 Horm Beh, Raper et al, 2016 Brain Beh Immun), as well as examined cortisol at 45 min post-stressor (unpublished data) and 24 hours post-stressor (Raper et al, 2016 Brain Beh Immun). I typically do these test at sunrise and/or lights-on, so the baseline/pre-stress cortisol levels are fairly high because of the awakening cortisol response. However, other colleagues and I choose to test at this time of day to avoid any other potential confound, such as the animal having a social conflict earlier in the day prior to testing that I might not be aware of (testing them first thing in the morning avoids that potential confound).   Even with the high basal cortisol I still see a significant increase in cortisol to the acute 30 min stressor, and in a manuscript I'm working on now I see a significant decline in cortisol at 45 min after the stressor has ended.

I'm not sure if the human intruder paradigm will work well for baboons, I know it doesn't work well in African Green Monkeys, they largely just ignore the "intruder" and don't react with cooing, freezing, or hostility that rhesus monkeys exhibit on the task. If the human intruder paradigm won't work for baboons, you might consider a social separation stressor, which is just separating them from their social group and/or cagemate and putting them alone in a novel environment for 30 min. Dettmer et al, 2012 (Psychoneuroendocrinology, 37, 191-199), published on using relocation as a stressor and examined cortisol levels in hair.   Lastly, you could also use pharmacological challenges of the HPA axis (Raper et al, 2014 J Neurosci).

If you have any additional questions please feel free to contact me.  I'm faster at responding if you send me a direct email jraper@emory.edu.

Hi Soledad,

fluorescein-labeled β-estradiol-6-(O-carboxymethyl)oxime-bovine serum albumin

Sigma Aldrich E6507-1MG euro 433,00

LP

Hey Mauro,

check this out:

Efficient isolation yields 20-30 million hepatocytes/liver after Percoll gradient.

Typical seeding would be 0.25 million cells/mL. That would be 0.5 million / well in a 6-well plate. 

I think its absolutely possible to combine antipsyhotic and dopamine agonists. But for such cases PRL levels are not  best criterias to assess current tumour control.

Thank you mam

I definitely will study this paper and expecting to be benifited with this. Thanks for your helpful cooperation.

Thank you very much

Well, it depends on the type of cancer. In most of the cancers such as pancreatic cancer, head and neck cancer and breast cancer, inhibition of notch pathway has been suggested to be effective. On the other hand, valporic acid, a well known drug for epilepsy treatment, is known to increase activation of notch with its anti-tumor effect on carcinoid tumors. 

You're correct that various investigators use different SPE cleanup methods for steroids and oxytocin, and indeed it doesn't matter which method you use as long as you verify the results yourself. There are two main things you need to do to check out for a particular method: (1) determine recovery from the SPE column (using known amounts of pure standards, spiking samples with known amounts of a standard, or using radiolabeled materials), and (2) determine whether the clean-up procedure works to give you reliable results. If you have access to a mass spectrometry facility, you can directly check the purity of the column eluate and how well the EIA values compare to values from LC-MS/MS measurement. More commonly, accuracy of an antibody-based assay (e.g., EIA) is assessed by preparing serial dilutions of your unknowns (purified and non-purified) and determining whether the readout from the set of serially diluted samples parallels the readout from your standard curve. One final note is that in my lab we have been happy with using the Phenomenex StrataX cleanup method for salivary oxytocin measurement. Good luck!

first of all, their are many studies regarding PCS in relation to experimental pain sensetivity.

second, HPA axis disruption will be likely be only in a disease state, but it can only influence pain sensitivity and not PCS because catastrophization is a trait and it cant be changed by hormonal changes.

Compare serial dilutions of goat serum with two standard different curves, one using purified human LH, FSH and prolactin and one using goat LH, FSH and prolactin (if available). If the goat standards are not available, test parallelism between the standard curve and the dilutions of goat serum. If the slopes in the linear portions of the curve are parallel, then you can use the human standards. If not, you  may need to develop an adjustment factor to correct for non-parallelism. Make sure your samples fall in the linear portion of the curve. If they are not, increase or decrease the volume to make sure the values fall within the linear portion. Testosterone is the same in both species, so it should work either way.

Dear Megan, you have three ways. 

1) Find a E2 ELISA kit which needs 10 ul sample,

   in this way you can use Rat E2 ELISA kit or human E2 ELISA kit, because E2 has the same structure in human and Rat/Mouse.  Human kits for animal case control or before after study is suitable.

2) Dilute sample with suitable diluent such as zero standard solution of the kit. 

   in this way, the sample estradiol may be low and with dilution, it may be fall below the assay sensitivity.

3) add standard of kit as diluent, e.g add 1:1 ratio of sample and standard (e.g 10pg/ml). if the final result be 10 pg/ml, so the sample result was 10 pg/ml. if the final result be 15 pg/ml, the sample amount be 20 pg/ml, and so on.

because you have  final concentration=(C1+C2) / 2 

in this way you will have enough sample volume and escape from falling below the assay sensitivity amount.

best

mehdi

thanx

The PDE5 inhibitors were first tested in the clinic for hypertension.  It was good observational (behavioral)  science that led to the current indication for erectile dysfunction. The prescribing information for the PDE5 inhibitors is similar.  For example, there is  a contraindication for use with nitrates because, together, the two types of drugs can lead to rapid vasodilatation and serious episodes of hypotension.  There are also warnings and precautions because patients with hypotension or uncontrolled hypertension may be at risk for pronounced and rapid changes in blood pressure. And again, in the label there are warnings about taking PDE5 inhibitors with other drugs such as alpha-adrenergic-blockers or alcohol, because of the possibility of serious decreases in bloood pressure.

Most of what I read about OXT is now oriented toward its mind/social effects when used at high dose to affect the brain. Traditionally it's known for birth related effects. My interest is in its most 'basic' physiological effects, on the water metabolism as governed by the hypothalamus.

See the reference given in response to 1 question I asked: What is the most basic role of oxytocin in the body's physiology? @ https://www.researchgate.net/post/What_is_the_most_basic_role_of_oxytocin_in_the_bodys_physiology

See references in 1 case report I wrote related to the role in water metabolism: https://www.researchgate.net/publication/236215979_Low-dose_oxytocin_stops_unexplained_%27burning%27_pain_in_fibromyalgia_A_case_report?ev=prf_pub

 https://www.researchgate.net/publication/236216001_Additional_file_1_Oxytocin_in_the_hypothalamic_osmostat_%28a_physiologic_strain_system_viewed_as_%27the_first_stress_system%27%29?ev=prf_pub

I'd be interested, in return in your references concerning OXTR found in amygdala. thanks

Hi Andre,

We buy the gelatin from SIGMA, and we prepare it as a 10% gelatin-10% sucrose in PBS solution. Also, when I cryoprotect my tissue, I only go up to 10% sucrose, not 30% like you'd do when using OCT. I changed to the gelatin because i found it's much simpler to cut the gelatin than the oct (OCT rolls up too much for my taste).

The brain molds are like these:

They are very useful if you don't want to put the whole brain in the cryostat but have issues cutting it freehand.

If you want the complete gelatin freezing protocol let me know, but you basically need to embed the tissue in the gelatin, using little molds like you'd use with the OCT, and then rapid-freeze at -50 to -60ºC using methylbutane and dry ice in a cold bath.

Our secondary antibodies are all invitrogen goat anti rabbit and goat anti mouse, now that I think about it. I do have NDS for a donkey anti goat secondary antibody. Like Aaron said, those are the two typical serums you'd need to perform most IFs.

Let me know if you have any other question and best of lucks!

-Daniela

Thank you! I am investigating the connection with early dysregulation symptoms in infants (colic) and later development of Sensory Processing Disorder in children. I am looking at the microbiome's possible influence in this neurodevelopmental disorder. As you may know, rodent studies are finding a disturbed HPA axis in GF mice with a critical window of development for a healthy stress response. I wonder if this is also happening in a sub-set of infants ( those who do not regulate at 3 months per Wessel's criteria).

Thank you so much for your response and support!! My daughter has SPD and I need to find answers to help our family and others like us.

Another great area to look into would be the role of Leptin inducing puberty.

Also, stress effects on leydig cell spermatogenesis.

Talking to your mentor is the first step and a lot of research depends on the capabilities of the lab.

hi rosie, I have not seen this in a paper. usually people use serum gonadotropins and/or vaginal smears, after all these could be done by someone else or someone else could give the samples a different coded name so you still don't know until decoded which samples go with which brains.

but assuming you are asking because you have the brains but do not have these samples, I wonder if you could look at the levels of GnRH and GnRH receptor messenger or better yet a ratio of the two. (see Schirman-Hildesheim TD, Bar T, Ben-Aroya N, Koch Y. Endocrinology. 2005 Aug;146(8):3401-8.)

unless someone knows if this has been successfully done before, it would need to be validated, so you would need those samples anyway but I wonder if anyone reading has an opinion/experience?

note, probably the best, most sensitive test is behaviour with a sexually experienced male rat

I'm doing some right now. One of the major issues we're having is to get the timing down correctly. Our research is using salivary cortisol assays to measure specific stress response to the Trier Social Stress Test. The hight of cortisol concentration seems to be 30 minutes prior but all of our filler stuff doesn't last that long. Currently we're just writing down the times and hope to statistically control for it. Most are between 15 and 30 minutes for us.

The timing differs if you aren't using saliva.

McRae, A., Saladin, M., Brady,K., Upadhyaya,H., Back,S., and Timmerman,M.(2006) Stress reactivity: biological and subjective responses to the cold pressor and Trier Social stressors. Human Psychopharmacology. 21, 377-385, DOI 10.1002/hup.778

goes over it a little bit.

The KNDy neurons, probably through either their connections with the AVPV or with the GnRH terminals, can rapidly regulate GnRH pulses in response to exteroceptive stimuli as would occur with the first instance. The reason there is no current answer is that few studies have explored the inputs to the KNDy neurons. We have begun to explore this question and find a population of neurons in the midbrain/ventral thalamus that make TIP39 and innervate the KNDy neurons. Those neurons are stimulated by either mating or suckling and likely other stimuli as well. They work by augmenting glutamate transmission but the sources of glutamate innervating KNDy neurons has yet to be defined. For the nutritional aspects, the time delay could involve resetting of the clock (and that has been demonstrated for feeding at non-feeding times, and or connections of the KNDY cells with systems that receive and process those peripheral signals like leptin or insulin. Again those connections are not well studied, but some of us are working on those as we 'speak'