Culturing TG neurons

We culture DRG neurons and have become fairly consistent at it but are continuously looking to increase the yield of neurons we get. Would adding trigeminal ganglion neurons into the mix be one way of doing this? From what I could find in the literature, neurons from both the DRG and TG are remarkably similar, with a few key differences in certain genes (according to this paper from Stephen McMahon group…"The Molecular Fingerprint of Dorsal Root and Trigeminal Ganglion Neurons")

Anybody done this or have experiences with it?

Depends on what your goals are. But I imagine reviewers would object to combining them, since they may be similar at transcriptome level, doesn’t mean they are at protein level or functional. For example, see this from a paper from Fan Wang’s group:

Our previous studies and other work suggested that parvalbumin may be another good candidate for labeling subtypes of touch receptor neurons (Hasegawa and Wang, 2008, Ichikawa and Sugimoto, 2003). To examine this possibility, we crossed the Pv::Cre knockin mice (Hippenmeyer et al., 2005) with several Cre-dependent reporter lines (Rosa-loxP-STOP-loxP-PLAP [RΦAP], or Rosa-loxP-STOP-loxP-XFP [RΦGFP] or [RΦtomato] mice) (Arenkiel et al., 2011, da Silva et al., 2011, Que et al., 2008). All reporter lines were generated in our lab using the same strategy and gave essentially similar labeling (for details, see Extended Experimental Procedures). Using the PLAP reporter (RΦAP), we found that in the adult mouse, Pv::Cre selectively labeled only two types of vibrissa neurons: SA Merkel-ending neurons, and a small number of RA longitudinal lanceolate-ending neurons (Figure 1C). This is different from what has been known for DRG sensory neurons, of which most Pv+ cells are muscle spindle or Golgi tendon innervating proprioceptive neurons (Arber et al., 2000, Ernfors et al., 1994). This difference between TG and DRG is further evidenced by the expression of neurotrophin receptors. The majority of Pv+ DRG neurons coexpress TrkC (Arber et al., 2000). By contrast, of the TG neurons labeled by Pv::Cre, 62% expresses TrkC, 17% coexpresses TrkA, whereas 26% colocalizes with Ret (Figure 1E; the total number exceeds 100%, implying that some neurons coexpress two of the receptors). Although the Pv gene itself is known to express in second-order neurons in the brainstem TG nuclei (Bennett-Clarke et al., 1992), the reporter expression in the brainstem was only observable beginning at around postnatal day 7 (P7). This provides us a window of opportunity to examine the axonal projection patterns from Pv-expressing TG neurons at P7–P8 without the confounding of the neuronal processes from central neurons. At this neonatal age, more than 90% of Merkel cells in smaller vibrissae are innervated by axons labeled with Pv::Cre (Figure 1F, upper panels); whereas in the larger vibrissae (those that are located more caudally), many fewer Merkel cells are innervated by Pv::Cre-positive axons (Figure 1F, lower panels).

Personally, I wouldn’t mix them. You add another unknown which is sure to be met with resistance. I would just pool more animals.

@tberta @liz @gcorder @esypek @ShanTan @runDRG @CandlerPaige @sshiers @RPSeal @thicunha
What do you think?

Pooling more animals is probably the best way to go, just reluctant to give it the resources that will require. Thanks for the advice.

Ways to make the process better:

  1. Practice your technique and just get better and faster
  2. Hire helpers to do it for you or with you (undergrads, post-bacs, techs)
  3. Try different protocols to see if you can get better yield

I think reviewers would have a meltdown if you combined DRG and TG neurons. Our lab does both, but always separately - and we assume that they have completely different functions. How many neurons are you looking for? If we’re looking for extremely dense cultures we find one mouse/dish works.

1 Like

I also cross posted on Twitter with a poll. Overwhelming answer is don’t do it.
Thanks so much for starting the convo @DaraBree
Getting collective wisdom from other scientists in real time is why this site exists. Hope that helps.

The people have spoken! Thanks @achamess this was very helpful

1 Like