Any sensory experience in any animal species can originate from either an event in the outside world, such as a predator approaching, or from a stimulus generated by the animal itself. For example, if an animal emits a threatening growl, this sound will activate auditory pathways that could indicate the presence of an aggressive conspecific, but clearly no defensive action is initiated.
Our team of researchers from the Lichtman and Engert Labs, led by then-MCO student Iris Odstrcil, recently published an investigation into how zebrafish identify self-produced sensory signals. Responses to such self-induced sensory stimuli, often called reafferent stimuli, need to be very different from responses to signals that originate from the outside world, which are commonly known as exafferent stimuli. Since the biophysical nature of these two different versions of stimuli is identical at the level of the primary sensory receptors, the distinction between them needs to happen somewhere else within the nervous system. In the somatosensory modality, i.e. touch, this distinction between reafference and exafference is equally important. Tickling, for example, is for many people an intense sensation, if, and only if, performed by another individual.
We, Odstrcil and colleagues, tried to get to the bottom of this universal and important phenomenon by leveraging the simplicity and small size of the zebrafish larva. The results recently appeared in the journal Current Biology.
In the case of a baby fish, tickling is probably not the ideal assay, but these small animals will also self stimulate the touch-sensitive receptors on their skin simply by moving through the water and thereby inducing flow-induced sensations. The specialized organs in many aquatic organisms that detect water flow are the neuromasts of the lateral line. Each neuromast is populated with a few dozen hair cells which are equipped with sensitive cilia or filaments that stick out into the surrounding liquid and can detect even minute perturbations in the flow. These hair cells are the same ones that humans and all vertebrates use in their ears and vestibular systems to detect sound and acceleration.
Thus, swimming would elicit a reafferent stimulus to the neuromasts, whereas an exafferent stimulus could be caused, for example, by ripples in the water from an approaching predator.
When we looked at activity in the afferent sensory neurons that send information from the hair cells in the neuromast to the brain, we found that external water flow led to strong activity patterns. Activity in these afferent sensory neurons during swimming, however, was completely absent.
This finding is evidence for a strong inhibition of the hair cells themselves, which needs to be timed precisely to the occurrence of swimming-related tail flicks. We traced the source of this inhibition to a small population of cholinergic neurons in the hindbrain of the zebrafish.
We found that these hindbrain neurons are only active during locomotion, that their axons project to all the neuromast organs of the lateral line, and that each individual axon forms stochastic synaptic terminals directly onto a subset of the neuromast hair cells.
In most vertebrates, the sensory hair cells have cholinergic receptor subtypes that serve an inhibitory role, and such receptors were also found in the zebrafish neuromast.
We confirmed the specific role of these cholinergic receptors in reafferent cancellation by recruiting then Schier Lab postdoc Jamie Gagnon, who generated mutant fish that lacked this particular receptor. Notably, these mutant animals, unlike their wildtype siblings, exhibited strong activity patterns in the afferent sensory neurons while flicking their own tails.
Similarly, if the cholinergic neurons in the hindbrain were removed by targeted laser ablation, such sensory activity during swims was equally restored. These results reveal the detailed mechanisms by which larval zebrafish cancel out self-induced sensory stimulation and thereby avoid maladaptive responses, such as trying to escape when the stimulating motion is from their own tail.
In order to verify the inferred connectivity rules within the neuromast organ, where hair cells, afferent sensory neurites and efferent axons all come together in a dense and interconnected network, we generated — with the help of the experts from Jeff Lichtman’s team—a complete “microconnectome” of this critical region, which unveiled all ultrastructural details of this complex circuitry.
Some of the findings confirmed what was known from the literature, such as the existence of two types of hair cells–one that is tuned to water flow going from head to tail and another that is selectively excited by flow going from tail to head. Each afferent neurite is selectively targeting only one of these two hair cell types.
We also found that the cholinergic efferent axons innervate both types of hair cells indiscriminately, where any active cholinergic neuron will inhibit any hair cell it contacts.
But we also discovered features that were unexpected and novel. For example, we found a second class of efferent neurons that also innervate the neuromast in addition to the cholinergic inputs. These neurons are located in the dopaminergic efferent lateral line nucleus (DELL), and their role in hair cell modulation is unknown. A careful connectomics analysis of their efferent terminals revealed that they make synapses with afferent neurites that are leaving these hair cells, not the hair cells themselves. As such, these DELL neurons are poised to directly modulate the afferent sensitivity of this important somatosensory modality, and could, for example, sensitize the animal’s ability to detect exafferent perturbations in the water in environments where predators may be present.
Further investigations of these neurons’ activity will allow us to test specific hypotheses. For example, we can test whether these cells might modulate activation thresholds of these sensory input channels depending on the general arousal state of the animal, and we can extend our connectomics analysis to study specifically how these dopaminergic populations might be connected to modulatory centers in the zebrafish’s brain, such as the hypothalamus and the autonomic nervous system.