Zebrafish study links altered neuronal genes to hyperarousal states

The altered presence of tiny fragments of neuronal genes, called microexons, causes hyperarousal in zebrafish. This is the main conclusion of an international study led by the Pompeu Fabra University (UPF) and the Centre for Genomic Regulation (CRG). An abnormal pattern of neural microexon presence leads to a hyperarousal state characterized by heightened neural activity and insomnia, commonly associated with stress but also in neurodevelopmental disorders. Arousal regulation is highly conserved in evolution. Therefore, this finding could help understand the mechanism underlying some human neurodevelopmental disorders, such as autism and schizophrenia, conditions associated with microexon mutations.

To survive, animals need to be ready to react to external and internal stimuli. This activation of the central nervous system, arousal, is highly conserved throughout the animal kingdom. Proper regulation of arousal ensures that neural and thus behavioral responses maintain a balance between drowsiness or reduced responsiveness and insomnia and sensory hypersensitivity. Two states associated with stress and neurodevelopmental disorders.

To properly regulate arousal during development and adulthood, organisms require a broad range of diverse proteins that are achieved via alternative splicing. This is a process that can produce two functionally distinct proteins with similar but not identical amino acid sequence, in response to the presence or absence of one or more microexons.

The study published in Science Advances shows that, in zebrafish, an alteration in the presence of neural microexons leads to a state of hyperarousal. Abnormal fish larvae have an altered swim pattern and reduced sleep. "They sleep less frequently, for shorter durations and take longer to fall asleep" explains Tahnee Mackensen, first author of the study. Who adds, "It is fascinating to see how, by analysing the movement of this transparent larvae, you can recall fish internal states.".

In addition to the behavioral alterations, researchers found that mis-splicing alters cAMP levels –a signal produced within cells that regulates neuronal activity– making them more or less excitable. "Abnormal fish are permanently overexcited", clarifies Mackensen. They have increased activity in the forebrain and elevated cAMP signalling, responsible for the hyperactivity during the day. However, this hyperactivity can be normalized by manipulating cAMP pharmacologically.

According to the study, reducing cAMP with a chemical inhibitor lowers the activity of the mutated fish to a normal level, whereas maintaining elevated cAMP levels in normal fish using drugs-either by inducing its synthesis or reducing its degradation-imitates highly aroused behavior, confirming that cAMP is key to driving arousal behavior. Or in the words of the scientist, 'in neurons, cAMP acts as a thermostat for its activity'.

Study in zebrafish with a human angle

The constellation of behavioral and neuronal shifts observed in abnormal zebrafish had also been reported in flies in a previous study of the same group. "We do know that the alteration of these microexons causes sleep deprivation in fish and flies", explains Manuel Irimia, who has led the research. And adds, "this mechanism is likely to be conserved in mammals, including humans, but maybe not in the exactly very same way".

In humans, sleep disturbances and sensory hypersensitivity are frequent in neurological disorders like autism and schizophrenia, two disorders that are reported to have an altered microexon regulation.

Despite not being causative of the disease, we know that changes in protein production, can contribute to symptoms of the disorder, in this sense, it is plausible to study whether the treatment to restore the arousal state in fish also corrects or alleviates the symptoms in other species".

Manuel Irimia, leader of the Transcriptomics of Development and Evolution lab at UPF and CRG

This cAMP-regulated arousal pathway is also implicated in anxiety and depression. That is why Mackensen believes it is worth continuing to investigate because "this could be just the tip of the iceberg".