16 April 2013

Tuesday Crustie: Clarity

Last week, the science news world was all a-flutter about a new technique to clear brains described in the paper, “Structural and molecular interrogation of intact biological systems.” (Argh, what a title. Would you have guessed what they did from that title?)

We in the invertebrate neuroscience community have been clearing brains for decades. Here are some examples from my own work.

Assembled in the dying days of straight edges and Letraset and photographing photographs, here are leg motor neurons from spiny sand crabs (Blepharipoda occidentalis; Faulkes and Paul 1996). The nerve to the leg splits into two branches. A shows the neurons in the combined nerve, B shows the neurons just from the front branch of the nerve, and C shows the neurons in the back branch of the nerve.

Here are homologous neurons from the legs of slipper lobsters (Faulkes 2012), presented here in colour for the first time. There is the equivalent to part A in the composite above. This one is darker than some of the others because it has gone through a process called intensification.

Same species (Ibacus peronii) but this time we have neurons in the tail. These are abdominal fast flexor motor neurons that power the big tasty muscles that everyone likes to eat (Faulkes 2004).

Here are the homologous cells in a spiny lobster (Panulirus argus; Espinoza et al. 2006). This is a composite “stack” of images compiled with Helicon Focus. That’s why this one is prettier than the others; more of the neurons are in focus.

Now compare the two above to this one from crayfish (Procambarus clarkii; another Helicon Focus composite, previously seen in the 2011 J.B. Johnston Club calendar). Notice how there are seven in the pictures above but eight in the one below (two on the left are overlapping)? It’s because the species above lack a specialized giant motor neuron that crayfish have related to escape tailflips.

The technique create these images is called cobalt backfilling, developed in the 1970s (Tyrer and Altman 1974; Bacon and Altman 1977; Altman and Tyrer 1980). I think the clearing of neural tissue was developed at this time. All the water in the neural tissue is removed and replaced with absolute alchohol. The tissue is cleared in methyl salicylate.


Altman JS, Tyrer NM. 1980. Filling selected neurons with cobalt through cut axons. In: NJ Strausfeld, TA Miller (eds.), Neuroanatomical Techniques, pp. 373-402. Springer-Verlag: Berlin.

Bacon JP, Altman JS. 1977. A silver intensification method for cobalt filled neurons in wholemount preparations. Brain Research 138(2): 359-363. http://dx.doi.org/10.1016/0006-8993(77)90753-3

Chung K, Wallace J, Kim S-Y, Kalyanasundaram S, Andalman AS, Davidson TJ, Mirzabekov JJ, Zalocusky KA, Mattis J, Denisin AK, Pak S, Bernstein H, Ramakrishnan C, Grosenick L, Gradinaru V, Deisseroth K. 2013. Structural and molecular interrogation of intact biological systems. Nature: in press. http://dx.doi.org/10.1038/nature12107

Espinoza SY, Breen L, Varghese N, Faulkes Z. 2006. Loss of escape-related giant neurons in a spiny lobster, Panulirus argus. The Biological Bulletin 211(3): 223-231. Abstract and reprint

Faulkes Z. 2004. Loss of escape responses and giant neurons in the tailflipping circuits of slipper lobsters, Ibacus spp. (Decapoda, Palinura, Scyllaridae). Arthropod Structure & Development 33(2): 113-123. http://dx.doi.org/10.1016/j.asd.2003.12.003

Faulkes Z. 2012. The distal leg motor neurons of slipper lobsters, Ibacus spp. (Decapoda, Scyllaridae). NeuroDojo (blog): http://neurodojo.blogspot.com/2012/09/Ibacus.html

Faulkes Z, Paul DH. 1997. A map of the distal leg motor neurons in the thoracic ganglia of four decapod crustacean species. Brain, Behavior and Evolution 49(3): 162-178. http://dx.doi.org/10.1159/000112990

Tyrer NM, Altman JS. 1974. Motor and sensory flight neurones in a locust demonstrated using cobalt chloride. The Journal of Comparative Neurology 157(2): 117-138. http://dx.doi.org/10.1002/cne.901570203

Related posts

A virtual camera lucida
Tuesday Crustie: Neural

External links

See through brains clarify connections
Getting better views of brains by turning them invisible


Jason said...

I've done my share of leech ganglion clearing in methyl salicylate and I don't think it's quite fair to be smug about this.

For one, they've been able to clear big tissue like mouse brains for a long time too. Hell you can probably clear a whole animal if you wanted to. The cool thing with CLARITY is that they preserve a huge fraction of the proteins, and they can do repeated washes on and off of different antibodies.

Zen Faulkes said...

Jason: That's fair. The CLARITY work is a major step forward. I wasn't trying to suggest it isn't.

The CLARITY coverage I’d seen just seemed to imply that there had never been a way to clear neural tissue before. Regular readers will know that "most science is incremental" is a recurring theme here, so I wanted to point out that CLARITY isn’t the first time we’ve been able to look inside brains and see the 3-D structure of neurons in their place.