Freshwater planarians are among the very few animals capable of fully regenerating a new functional brain from a tiny piece of their bodies. Despite being animals relatively simple from a morphological point of view, several studies have reported the complexity of their central nervous system (CNS). This complexity can be seen by the high degree of molecular compartmentalization based on the expression patterns of many neural-specific genes. In addition, a large number of distinct neuronal populations expressing different neurotransmitters and neuropeptides have been identified.
An important aspect of planarian CNS regeneration is that even though the available tools allow us to determine quite precisely when and where the different neuronal populations regenerate and how the new brain is formed again at the morphological level, much less is known about when this new complex CNS is fully functional again and how the animals recover their normal behaviours. The main reason for this is that few behavioural assays have been established in these animals. So, even in intact non-regenerating planarians there is no much information about which genes might be regulating different behaviours controlled by the planarian sensory system.
Now a recent paper from the laboratory of Kiyokazu Agata describes some genes related to thermosensory signalling in these animals and reports how thermotaxis is re-established during regeneration (http://www.ncbi.nlm.nih.gov/pubmed/25411498). First, they showed that planarians of the species Dugesia japonica displayed normal locomotor activity and morphological shape at a temperature range from 15ºC to 25ºC. At lower temperatures (5-10ºC) their bodies crumpled and lost their motility; above 30ºC they displayed hyperkinesia and became lethargic or even died just after 1 hour. Then, the authors developed a thermotaxis assay based on a method previously used in C. elegans. To put it simple, they created a radial thermal gradient from the centre of a Petri dish towards its edges: the Tª at the centre being 17ºC and at the edges, 25ºC. When the temperature was uniform all throughout the Petri dish planarians moved towards the edges. However, when a thermal gradient was created planarians tended to move towards the centre of the Petri dish and stay there, where the Tª was lower. Decapitated planarians put in a dish with a thermal gradient did not seem to recognize it and moved randomly towards the edges, as they were in a dish with a uniform Tª. In contrast, the small amputated head pieces were able to recognise the gradient and moved towards the centre of the dish, indicating that the head region was necessary and sufficient for thermotaxis in planarians. In all these experiments and assays all the animals displayed the same locomotor activity independently of being intact, headless or head pieces in Petri dishes with a uniform Tª or under a thermal gradient.
Next, the authors investigated when this thermotactic behaviour was recovered during head regeneration. During the first 4 days of regeneration no thermotactic response was seen even though at this stage planarians had already regenerated the eyes and a new small brain. However, a strong recovery of the thermotactic behaviour was observed at day 5 of regeneration. Previous studies had shown that negative phototaxis is also recovered after 5 days of regeneration.
To try to identify genes involved in planarian thermotaxis the authors focussed on the family of Transient Receptor Potential (TRP) ion channels as they have been involved in regulating a variety of sensory systems including thermosensation in different animals. They identified seven genes homologs to TRPs that displayed different expression patterns in planarians. One of them DjTRPMa was expressed in a scattered pattern throughout the body, although there were more cells in the head region that in the rest of the body. Remarkably, the silencing of DjTRPMa by RNAi resulted in a clear defect in thermotaxis. Thus, and contrary to controls, the animals in which DjTRPMa was silenced never rested in the coolest centre area of the Petri dish. Importantly, RNA treatment did not cause any locomotor defect in those animals. That TRP genes are also involved in thermotaxis in planarians was further supported by the results obtained after treating the planarians with AMTB that specifically antagonizes a thermosensitive TRPM family protein in mammals. These animals moved randomly in the Petri dish and did not tend to go the cold central area. Overall, these results suggested that DjTRPMa would be expressed in thermosensory neurons and might be required for thermotaxis.
During regeneration, DjTRPMa-expressing cells appeared de novo by day 2 whereas a normal thermotactic behaviour was not recovered until day 5, suggesting that thermotaxis would depend on something else other than these thermosensory neurons. Then, the authors analysed the effect on thermotaxis of silencing some genes related to different neurotransmitters expressed in specific neuronal populations. Interestingly, the silencing of DjTPH (a marker of serotonergic neurons) resulted in an abnormal thermotactic response (as DjTRPMa did), without disturbing other behaviours as negative phototaxis or proper locomotor activity. Double staining indicated that DjTRPMa and DjTPH were not coexpressed in the same cells. However, neural projections from serotonergic neurons extended towards DjTRPMa-positive cells, pointing out the possibility that serotonergic neurons could somehow transduce to the brain the temperature signals received by DjTRPMa neurons.
In summary, the authors have characterized for the first time a gene that regulates thermotaxis in planarians and analysed how this behaviour is recovered during regeneration. Further studies should help to better characterize the neural circuit that transduces those external signals to the brain and trigger the proper behaviour.