On the last day of Neuroscience 2017, I went to see this special lecture, by Dwight Bergles, from Johns Hopkins University School of Medicine.
Dr. Bergles’ team studies neuron-glia interactions in the nervous system, and in his lecture, Dr. Bergles explained how spontaneous activity on the auditory system during development can play the role of sensory input, allowing a correct arrangement of the circuitry, and preparing the system to be functional from the moment when the mouse starts hearing.
Developing sensory systems
The senses have to be present and functional very early in life. The developing sensory systems exhibit spontaneous activity that resembles the sensory input they get, and it is propagated from the periphery to the cortex, all along the sensory pathway.
Visual, olfactory, somatosensory and auditory systems show this spontaneous activity before sensation happens. For the results presented in this lecture, Dr. Bergles’ team focused on the spontaneous activity taking place during the development of the auditory system.
It is possible to extract the cochlea of newborn mice and study it in vitro. Rodents are born deaf, and they do not start hearing until two weeks after birth. Therefore, the cochlea of newborn mice offers a good opportunity for studying the development of the auditory system before the onset of hearing.
There are spontaneous action potentials that take place in the rodent’s auditory system in development. They emerge early after birth and they persist until the onset of hearing. They propagate along the whole auditory pathway, from the cochlea, to the thalamus, to the auditory cortex, and they are necessary for the maturation of the system.
The origin of this spontaneous action potentials is the cochlea. The application of tetrodotoxin to the round window membrane results in the elimination of this spontaneous neural activity throughout the auditory system.
Inner Supporting Cells
In the developing cochlea of mammals, there is a transient structure named Kölliker’s organ. Kölliker’s organ is formed by inner supporting cells, which resemble glial cells from brain cortex. However, they exhibit spontaneous activity –while glial cells exhibit very little electrical activity in the brain-. This activity may be a reflection of spontaneous activity on the inner hair cells -a consequence of their neuronal activity-, or it may be originated on the inner supporting cells -a cause of inner hair cells neuronal activity-.
This spontaneous activity is very robust. In patch-clamp of inner supporting cells, or when a field electrode is inserted in Kölliker’s organ, this activity is detected, and is characterized by slow kinetics and high frequency. Also, pharmacological tests showed that this spontaneous activity is sensitive to purinergic receptor antagonists, but not to adenosine receptor antagonists; therefore it is elicited by extracellular ATP.
If we look at DIC images of Kölliker’s organ, we see spontaneous shrinking events, in the developing cochlea. These events are ATP-dependent, and they take place near inner supporting cells.
The spontaneous activity on inner supporting cells from Kölliker’s organ, is correlated with the activity of inner hair cells, and they exhibit also a correlation with the activity of spiral ganglion neurons. This correlation seemed to indicate that ATP mediates the activity of these three types of cells, suggesting that the excitatory drive to inner hair cells would be the release of ATP from inner supporting cells in Kölliker’s organ. This would be the origin of the spontaneous activity in the auditory system.
How is spontaneous activity generated in inner supporting cells?
The extracellular ATP is probably released by the same inner supporting cells. These cells are capable of releasing ATP. Also, the extracellular ATP increases take place in Kölliker organ, on an area where they are almost the only cell type, and they express connexin 26; when a blocker of gap junctions was added to the cochlea, the release of ATP stopped.
Dr. Bergles’ team studied the relation between ATP release, the shrinking of inner supporting cells, and intracellular activity on these cells.
Inner supporting cells have ATP receptors, and the stimulation of these receptors increases intracellular calcium. This is sufficient to evoke the cells’ shrinking. Dr. Bergles’ team suggested that intracellular calcium increase may activate chloride channels. Chloride is taken out of the inner supporting cell, and as a consequence of this osmotic imbalance, the cell shrinks.
The increase in intracellular calcium caused by ATP receptor activation causes a depolarizing ionic current in inner supporting cells.
Having found the origin of activity in inner supporting cells, what is the link to the action potentials in inner hair cells? Is it ATP?
Inner supporting cells express TMEM16A channels, which are calcium-activated chloride channels. TMEM16A are necessary for supporting the depolarizing calcium-activated currents in these cells. In the presence of inhibitors, both depolarizing currents and shrinking are greatly reduced, and the same happened in a conditional TMEM16A KO mouse model.
Chloride efflux in other epithelial tissues is usually accompanied by the efflux of water and potassium. Considering this fact gave a great idea to Dr. Bergles’ team: could it be possible that action potentials in inner hair cells were not mediated by the release of ATP, but by extracellular potassium?
To test this hypothesis, they evoked a depolarization of inner supporting cells by loading them with caged calcium and stimulating with UV light, in the presence of ATP-receptor inhibitors. On these conditions, inner hair cells did not show spontaneous inward currents before the photorelease of calcium, but, as expected, they could induce a depolarization in these cells when stimulating inner supporting cells with UV light. This depolarization was abolished with chloride channel antagonists. To verify the implication of potassium, they introduced TEA in the intracellular liquid on patch clamp. TEA blocks most, but not all potassium channels, and its introduction reduced the amplitude of spontaneous inward currents on inner hair cells by 66%. These results indicate that potassium was the main responsible of inner hair cells activation, and its release depends on the release of chloride.
However… This seems a quite complicated approach to regulate the depolarization of inner hair cells. The question is: why not just using ATP? In his talk, Dr. Bergles interpreted this as the result of the way evolution acts: “Evolution works in unpredictable ways, and it uses the toolbox it has, as it can, to do what it needs”. It seems that sometimes evolution does not find the easiest pathway to do something (at least according to our own, human interpretation…).
To conclude with his talk, Dr. Bergles mentioned some of the future directions his team will take on the study of spontaneous activity on the developing auditory system. This includes the study of the transduction of spontaneous neuronal activity along the auditory pathway, towards the inferior colliculus, the effect of some mutations in the gap junctions that are related to deafness, and a possible role of this spontaneous activity in tinnitus after traumatic injuries.
All of this is work in progress, so you will not have to wait for long to see it published!
Bergles DE, Jabs R, Steinhäuser C (2010). Neuron-glia synapses in the brain. Brain Res Rev 63(1-2):130-137.
Dayaratne MW, Vlajkovic SM, Lipski J, Thorne PR (2014) Kölliker’s organ and the development of spontaneous activity in the auditory system: implications for hearing dysfunction. Biomed Res Int. 2014:367939.
Tritsch NX, Yi E, Gale JE, Glowatzki E, Bergles DE (2007). The origin of spontaneous activity in the developing auditory system. Nature 450(7166):50-5.
Tritsch NX, Bergles DE (2010). Developmental regulation of spontaneous activity in the Mammalian cochlea. J Neurosci 30(4):1539-1550.
Tritsch NX, Rodríguez-Contreras A, Crins TT, Wang HC, Borst JG, Bergles DE (2010). Calcium action potentials in hair cells pattern auditory neuron activity before hearing onset. Nat Neurosci 13(9):1050-1052.
Tritsch NX, Zhang YX, Ellis-Davies G, Bergles DE (2010). ATP-induced morphological changes in supporting cells of the developing cochlea. Purinergic Signal 6(2):155-166.
Wang HC, Bergles DE (2015). Spontaneous activity in the developing auditory system. Cell Tissue Res 361(1):65-75.
Wang HC, Lin CC, Cheung R, Zhang-Hooks Y, Agarwal A, Ellis-Davies G, Rock J, Bergles DE (2015). Spontaneous Activity of Cochlear Hair Cells Triggered by Fluid Secretion Mechanism in Adjacent Support Cells. Cell 163(6):1348-1359.
Video tutorial for cochlear dissection: https://www.masseyeandear.org/research/otolaryngology/investigators/laboratories/eaton-peabody-laboratories/epl-histology-resources/video-tutorial-for-cochlear-dissection (consulted on 12/01/17)
Aix-Marseille Université, France
Universitat Autònoma de Barcelona, Spain
ICN PhD Program in Neuroscience: http://neuro-marseille.org/en/phd-program-en/le-phd-program/