Hi,

I believe, sympathethic axons will have tyrosine hydroxylase, while the the motor will have choline acetyltransferase. Youcan also use calcitonin gene-related peptide for some sensory neurons. May I ask which system you are working in?

good luck! hope this helps.

I'd be interested to read the answers here in terms of potential antibody use.

Also, do not forget the contribution of sympathetic fibres. This paper does a good analysis in rat: http://www.ncbi.nlm.nih.gov/pubmed/3706794

Thanks, i'm afraid there's no way to discriminate motor-sensory-autonomic just with IHC, that's why i'm asking, but i might be wrong. Let's hope someone illuminates us

You can differentiate between different types of nerve fibers by immunolabeling their different neurotransmitters

Hi, I don't know if it helps but I think looking at papers studying motor neuron disease (e.g. Amyotrophic lateral sclerosis?) may be useful. One marker that may be helpful in identifying peripheral nerves is peripherin, a type III Intermediate filament (structural) protein, which identifies "small-sized [dorsal root] ganglion neurons, lower motor neurons, sensory and motor neurons of the cranial nerves, and autonomic neurons in ganglia and the enteric nervous system" (I admit I got this excerpt from wikipedia but I have read papers to back that up). I have used peripherin and can tell you that it is also expressed in some (not all) sympathetic fibres and I believe some researchers use it for identifying small sized sensory neurons in the dorsal root ganglion. It is best to use in adult rats because these marker is meant to be highly expressed during development. Peripherin is also upregulated in nerve injury. I recommend the Chemicon-MIllipore AB1530 rabbit-anti peripherin antibody. Also, to include or exclude the possibility of other nerves, I guess you would have to colabel with markers for other neurons you wish to identify (perhaps CGRP for peptidergic sensories?) or neurons you wish to exclude (like tyrosine hydroxylase for sympathetics). I hope this helps - good luck!

Hi,

depending on the model organism and the age (embryonal?), I would suggest trkA, B and C for sensory axons. DCC might work for motor axons...

Otherwise, maybe you could get the Hb9::eGFP marker line (mouse) for visualization of motor axons (in combination with Neurofilament), or the Brn3A reporter strain for sensory projections

depending on the model organism and age I would suggest reporter strains (mouse): Hb9::eGFP for somatic motor and Brn3a-Tau-LacZ for sensory.

Otherwise, Trk receptors A, B and C work for sensory axons

I've been wondering about something similar for a while. I'm not aware of any clear cut markers. I think it must theoretically be possible, to identify most of the types through a process of elimination, given enough reliable markers. Exactly which markers and whether they are expressed in the axons, is the primary stumbling block to me.

There are markers of autonomic fibres which will eliminate them (ACh etc).

So you are left (according to Ewan's reference) trying to separate a population of unmyelinated afferents, myelinated afferents and myelinated motor nerves.

There are markers such as myelin basic protein, which will label myelinated nerves. 200kDa neurofilament is also touted as a myelinated marker, though I am not 100% sure about it.

I would think you might need to separate the afferent from motor nerves with something like SubP, CGRP, NPY, NaV channel, P2X3, TRP markers, VGLUT1/2 etc. Though I am not sure about how universally reliable these antibodies are or which one you would ideally pick.

Would it be possible to inject tracers like fast blue at your site of interest and then do PCR/micro-array on the cell bodies labelled from there? That would give you a clue as to the expression patterns to look for on the nerves.

I'd be interseted to see what other people think.

This paper might be helpful:

Ruscheweyh, R., Forsthuber, L., Schoffnegger, D., & Sandkühler, J. (2007). Modification of classical neurochemical markers in identified primary afferent neurons with Abeta-, Adelta-, and C-fibers after chronic constriction injury in mice. The Journal of comparative neurology, 502(2), 325–36. doi:10.1002/cne.21311

For peripheral nervous system axons seems to be useful neurofilament proteins and peripherin as immunohistochemical markers, but I think, that exact function of peripherin is still unknown - maybe it plays an important role during nervous system development.

Hi,

I believe, sympathethic axons will have tyrosine hydroxylase, while the the motor will have choline acetyltransferase. Youcan also use calcitonin gene-related peptide for some sensory neurons. May I ask which system you are working in?

good luck! hope this helps.

Thank you all! You have provided me interesting markers to look at.

I need to identify motor axons to check if serum from multifocal motor neuropathy patients, that are known to have antibodies against gangliosides, binds to motor axons specifically. We know that serum binds to nerves (nodal staining) but it looks like it binds to all fibers (and motor ones seem to be around 6% of all). However, patients only have motor involvement so that strikes me a little bit. Is controversial whether those antibodies are pathogenic so, showing that they bind specifically to motor axons could help in that without making an animal model (immunizing with gangliosides is rather hard).

I wanted to check if there's any specific marker for (myelinated) motor neurons that could allow me to make co-localization experiments. I can make a negative selection process but that would be more difficult than just using a marker to detect motor neuron axons with a specific marker.

So, let me, re-formulate the question... do you think is there any specific histochemical marker to detect motor neuron axons (not a marker in endplates or NMJ)?

I'm checking your references and literature on the markers you suggest anyway because i might use the negative selection approach. But, it would be much easier to do it in a positive way, if possible.

Thank you very much sincerely.

Well, motor axons are myelinated, have you tried using myelin proteins (MBP or PLP) to distinguish them?

Maybe a double labelled immunohistochemical experiment with either MBP or PLP, and ChAT, will show you myelinated motor axons. ChAT should be present in the axons, since it made in the cell soma then transported to the axon terminal.

(I would not recommend using the myelin proteins alone in your experiments because certain sensory fibers, such as the acute pain fibers, are also myelinated.)

Hope this helps a little.

That helps, of course! Since the serum antibodies recognize nodal components i already was pretty confident that the stained axons were all myelinated. My doubt was if they were myelinated sensory or motor. Using ChAT is a good idea...

i'm going to search for some literature on that and try to see if i'm able to use it in my experiments.

Thanks again!

Great! Good luck.

Here a method to staining sensory nervous fibers in the skin "Arch Neurol. 1998 Dec;55(12):1513-20.

Epidermal nerve fiber density: normative reference range and diagnostic efficiency.

McArthur JC, Stocks EA, Hauer P, Cornblath DR, Griffin JW.

Source

Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA. [email protected]

Abstract

BACKGROUND:

The sensitivity of neuron-specific antibodies permit the identification of the small unmyelinated nerve fibers within the skin.

OBJECTIVES:

To develop a reference range of epidermal nerve fiber density in humans, and to evaluate their diagnostic efficiency for sensory neuropathies.

METHODS:

Ninety-eight normal controls (age range, 13-82 years) were examined with both directed neurologic examinations and quantitative sensory testing. The diagnostic utility was examined in 20 patients with sensory neuropathies. Each subject had 2 punch biopsies performed at each site in the thigh and distal part of the leg (total of 392 biopsies). After formalin fixation, 50-microm-thick free-floating sections were stained with a polyclonal antibody to neuron-specific ubiquitin hydrolase, anti-protein gene product 9.5. We enumerated intraepidermal nerve fibers per millimeter to derive a "linear density." The linear density technique was validated against a stereological technique that used the fractionator to measure the total length of intraepidermal nerve fibers per 3-mm punch.

RESULTS:

The biopsy technique was well tolerated, with no notable complications. The linear density quantitation was rapid and had high intraobserver and interobserver reliability. We determined that the density of intraepidermal fibers in normal controls was 21.1+/-10.4 per millimeter (mean +/- SD) in the thigh (fifth percentile, 5.2 per millimeter), and was 13.8+/-6.7 per millimeter at the distal part of the leg (fifth percentile, 3.8 per millimeter). Significantly higher intraepidermal fiber densities were seen in the youngest group (P = .004), and we observed no significant effect of race, sex, height, or weight. The density at the thigh was significantly correlated with that at the distal part of the leg (P = .01) and was consistently higher by about 60%, a reflection of the normal proximal-distal gradient. The results obtained with stereology and the linear density correlated significantly (P=.001), providing internal validation for the technique. Epidermal nerve fiber density was significantly reduced (P = .001) in patients with sensory neuropathies. With a cutoff derived from the fifth percentile of the normative range for the distal part of the leg, the technique had a positive predictive value of 75%, a negative predictive value of 90%, and a diagnostic efficiency of 88%.

CONCLUSIONS:

We have established a reference range for intraepidermal nerve fiber density in normal humans by means of a simple quantitation method based on enumeration of individual intraepidermal nerve fibers on vertical sections of punch skin biopsy specimens stained with the sensitive panaxonal marker anti-protein gene product 9.5. The utility of the density measurement was confirmed for sensory neuropathy with a diagnostic efficiency of 88%. Skin biopsies may be useful to assess the spatial distribution of involvement in peripheral nerve disease and the response to neurotrophic and other restorative therapies.

PGP9.5 is more or less pan-neuronal, not likely to be that useful for Luis.

Aminata do you know of a good ChAT antibody?

Luis am I right in thinking that you are using human tissue? If so, I would caution you to check the antibody specificity very carefully. My opinion is that very few of the neuronal marker antibodies cross over well from rodent to peripheral human tissues.

Anti-PGP9.5 and anti-200kDa neurofilament work well, I have had some success for CGRP and have seen results from Substance P although I think these markers are much more sparse in humans than in rodents.

I spoke to someone only last week at a conference who was trying IHC in human sciatic nerve and had not had much luck so I'd be very interested in how you get on.

Hi Peter,

ChAT antibody from Millipore, has worked well for us (for both IHC and westerns).

you can try Parvalbumin antiboby for the sensory afferents- know that it is working for labeling sensory proprioceptive afferents in spinal cord so you can try it for the peripheral sensory fibers

PGP 9.5 is an antibody for peripheral sensory fibers

Hi again,

I'm amazed of the quality of your responses, thank you very much. Peter, what I work with both human and murine nerves. But, for the purpose of this question I'm using human serum over rat nerve, so the marker I wold need should work well for rat sciatic nerve. I've been digging a little bit more and I definitely think that ChAT is probably the marker I was looking for. It fits with what I want. In case in does not work I would try then a sensor marker.

Whatever the outcome I will let you know if I have anything interesting.

And, again, thank you very much to all. I would have never believed to have such a nice feedback on such an specific topic. Amazing.

Adriana,

I have posted below a few un-published pictures of autonomic innervation of mucosal blood vessels, bronchial smooth muscle and mucosal glands labelled with PGP9.5 (green) and smooth muscle actin (red). Functional studies show that these are mostly cholinergic parasympathetic fibres which control processes such as mucous production and brochodilation. They are most certainly not sensory fibres, which have a very different morphology.

http://i669.photobucket.com/albums/vv52/biotechpete/PGPSMA_zps6fd2391a.jpg

In most tissues in all species that I know of, PGP9.5 will label almost all nerves. As best I can tell from lung tissue, ironically, the nerves it does not label are a sub-group of sensory fibres. I hope that this might convince you that PGP9.5 is not a reliable marker to discriminate peripheral sensory fibres.

Hi Peter the PGP 9.5 is used to stain the sensory nerve fibers in skin

PLoS Negl Trop Dis. 2012;6(12):e1975. doi: 10.1371/journal.pntd.0001975. Epub 2012 Dec 13.

Degree of skin denervation and its correlation to objective thermal sensory test in leprosy patients.

Rodrigues Júnior IA, Silva IC, Gresta LT, Lyon S, Villarroel Mde F, Arantes RM.

Source

Departamento de Patologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

Abstract

BACKGROUND:

Leprosy is an infectious disease affecting skin and peripheral nerves resulting in increased morbidity and physical deformities. Early diagnosis provides opportune treatment and reduces its complications, relying fundamentally on the demonstration of impaired sensation in suggestive cutaneous lesions. The loss of tactile sensitivity in the lesions is preceded by the loss of thermal sensitivity, stressing the importance of the thermal test in the suspicious lesions approach. The gold-standard method for the assessment of thermal sensitivity is the quantitative sensory test (QST). Morphological study may be an alternative approach to access the thin nerve fibers responsible for thermal sensitivity transduction. The few studies reported in leprosy patients pointed out a rarefaction of thin dermo-epidermal fibers in lesions, but used semi-quantitative evaluation methods.

METHODOLOGY/PRINCIPAL FINDINGS:

This work aimed to study the correlation between the degree of thermal sensitivity impairment measured by QST and the degree of denervation in leprosy skin lesions, evaluated by immunohistochemistry anti-PGP 9.5 and morphometry. Twenty-two patients were included. There were significant differences in skin thermal thresholds among lesions and contralateral skin (cold, warm, cold induced pain and heat induced pain). The mean reduction in the density of intraepidermal and subepidermal fibers in lesions was 79.5% (SD = 19.6) and 80.8% (SD = 24.9), respectively.

CONCLUSIONS/SIGNIFICANCE:

We observed a good correlation between intraepidermal and subepidermal fibers deficit, but no correlation between these variables and those accounting for the degree of impairment in thermal thresholds, since the thin fibers rarefaction was homogeneously intense in all patients, regardless of the degree of sensory deficit. We believe that the homogeneously intense denervation in leprosy lesions should be objective of further investigations focused on its diagnostic applicability, particularly in selected cases with only discrete sensory impairment, patients unable to perform the sensory test and especially those with nonspecific histopathological finds.

dear luis querol,

cholinesterase if you are looking at neuromuscular junction. somehow i did conventional silver cholinesterase staining for motor endplate and motor fibers, not by immuno.

Neurons of dorsal root ganglia can be stained by Akt and ERK1/2 (if you are looking at peripheral nerve regeneration).

just my humble suggestion...

Thank you very much! That helps!

There are some nice results for ChAT here:

http://link.springer.com/article/10.1007/s00418-013-1078-9/fulltext.html

What do you think of Substance P, Neuropeptide Y for sensory fibers and thyroxine hidroxilase for autonomic fibers.

Roberto, there are a number of papers which show that Sub P and NPY are not universally expressed in sensory neurons. Conversely tyrosine hydroxylase is also expressed in subsets of sensory fibres.

http://link.springer.com/article/10.1007%2FBF00297551

All these techniques have been used previously. I guess there's no gold standard, so just try and choose the one that works. 

- Motor axons: you can use cholinesterase (Karnovsky's staining, histochemical), agrin (IHC) or ChAT (IHC). 

- Sensory axons: carbonic anhidrase (Hansson's staining, histochemical) or annexin V (IHC). 

- Simpathetic axons: tyrosine hidroxylase (IHC). 

- Panaxonal: antineurofilament (IHC) or PGP9.5 (IHC). 

I hope that helps. Please let me know if you need the references. 

Outstanding... thank youj very much. I'm awaiting other experiments to do this one...

Hi Victor,

That's an interesting idea about carbonic anhydrase, I found this staining quite difficult to do and not had much success in peripheral tissues as it also stains some tissue cell. This paper also suggests it labels some motor nerves: http://www.sciencedirect.com/science/article/pii/0306452284902690#

Can AnnexinV been used in mixed tissues? I have only ever used it to label phosphatidyl serine in activated immune cells.

Hi Maria,

I have just published the staining of peripheral nerves in human lung tissue in the current issue of AJRCCM (http://www.atsjournals.org/doi/abs/10.1164/rccm.201412-2293OC#.VbII8vnF-ig) and there is  a similar great publication by Scott, G, et al in AJRCMB. Greg has produced  some exceptional whole mount lung staining in mice. There are some good publications by Canning, Mazzone and Lamb & Sparrow too. It is difficult, but the key is fresh tissue. This is easier in animals than humans but it is clear to both Greg and I that the nerves decay rapidly ex-vivo.

My advice would be don't bother with sections or archival FFPE tissue, but I can share with you some images I have of human lung sections if you wish. Unfortunately, I think the PGP antibody I used is unavailable outside the UK but one available from Serotec works well in FFPE. Feel free to email me (details  are on the paper I linked to).

All motor axons are myelinated and all are cholinergic i.e. express choline acetyltransferase (ChAt) (for protocol see Exp Neurol. 2010 ;223(1):207-20) and probably also vesicular acetylchoine transporter (VAChT) (Neurochem Int. 1998;32(5-6):457-67).  Antibodies to ChAt need to be tested on your tissue - use spinal ventral horn or sciatic nerve.

The only other cholinergic axons in peripheral nerve trunks can be a few sympathetic vasodilator axons (also express vasoactive intestinal polypeptide (VIP)) and some unmyelinated afferent axons that also express calcitonin gene-related peptide (CGRP).  However these are easy to distinguish morphologically as they are unmyelinated and of much smaller diameter.

It is clear, not only from the comments above, that distinct subpopulations of the much more numerous afferent axons express a variety of peptidergic and other markers, but there is not one for all afferent axons selectively.  The paper by Ruscheweyh et al is helpful regarding this.

Brilliant and very useful discussion

I thank all of you for the information you provided

Victor, could you give me please the references?

thanks