Brain’s Alertness Attributable to Ancestral Circuitry
In an NIH-funded study, scientists have now been able to identify the types of neurons supporting alertness using optogenetics technology and a molecular method called MultiMAP in transparent larval zebrafish.
“Vigilance is gone awry marks states such as mania and those seen in post-traumatic stress disorder and depression,” explained Joshua Gordon, M.D., Ph.D., director of the NIH’s National Institute of Mental Health (NIMH), which along with the National Institute on Drug Abuse, co-funded the study. “Gaining familiarity with the molecular players in a behavior – as this new tool promises – may someday lead to clinical interventions targeting dysfunctional brain states.”
Internal states of the brain profoundly influence behavior. Fluctuating states such as alertness can be governed by neuromodulation, but the underlying mechanisms and cell types involved are not fully understood.
Therefore, researchers have built a neural activity screening tool called MultiMAP, or Multiplexed-alignment of Molecular and Activity Phenotypes.
The researchers employed
this technology that allowed them to monitor vast numbers of nerve cells’ activity in the brain simultaneously and, afterward, to characterize the cells of interest in molecular detail. This new method lets scientists record the activity of any cell type, without needing to produce special, genetically modified animals for each new experiment.The MultiMAP, which is short for Multiplexed-alignment of Molecular and Activity Phenotypes, lets them track the activity of nearly every neuron in the zebrafish brain and then identify the cell type of every neuron of interest — the crucial step in determining which neuronal circuits are participating in the induction of a brain state such as alertness.
“We identified multiple monoaminergic, cholinergic, and peptidergic cell types linked to alertness and found that activity in these cell types was mutually correlated during heightened alertness,” wrote the article’s authors. “We next recorded from and controlled homologous neuromodulatory cells in mice; alertness-related cell-type dynamics exhibited striking evolutionary conservation and modulated behavior similarly.”
The researchers, Karl Deisseroth, M.D., Ph.D., Matthew Lovett-Barron, Ph.D., and colleagues—used the technique to screen activity of neurons visible through the transparent heads of genetically engineered larval zebrafish. They gauged vigilance by measuring how long it took the animals to swish their tails in response to a threatening stimulus.
The fish had been bioengineered so that calcium flux within a neuron — an excellent proxy for impulse conduction within that cell — triggered a fluorescent signal that could be picked up and recorded via high-powered microscopy.
To test alertness, the investigators presented the fish with a visual stimulus suggestive of an approaching predator. This evokes an instinctive tail-swishing response, as the fish attempts to veer away from impending danger. Each of 34 separate larval zebrafish was subjected to repeated exposures to the looming-predator stimulus. The time elapsed between each exposure and the consequent swishing of the fish’s tail — the animal’s reaction time- was measured. In each instance, fluorescence representing activity within tens of thousands of nerve cells just prior to the stimulus was captured and recorded.
After humane euthanasia and preserving the fish tissue in fixative, the team directed fluorescence-labeled molecular probes at the animals’ neurons and imaged the results. Using algorithms, they were able to align the neural-activity data from their experiments on live zebrafish with cell-identity data extracted afterward from those same fish.
Deisseroth and his colleagues identified multiple anatomically and biochemically distinct populations of neurons that were most active just before the fishes’ fastest responses to the looming-predator stimulus. One of these was the norepinephrine-secreting population originating in the locus coeruleus, already well-known to be involved in alertness.
Guided by their findings in zebrafish, the researchers then targeted the equivalent neuronal populations in the much more complex brains of mice. To test mice’s reaction times, they trained the mice to lick in response to a particular tonal cue; when the mice did so, they were rewarded with a drop of drinking water.
The scientists went one step further in the mice: They used optogenetics – a technology, developed in Deisseroth’s lab, that allows neurons to be excited or inhibited by a pulse of light at the flick of a switch — to prove, by variously activating or inhibiting neurons in one or another population, that only a subset of the circuits in question were individually capable of increasing alertness.
The new findings open the door to a whole new route of further exploration, Deisseroth said. “The more we understand the landscape of neurons that underlie a brain state like alertness, the more we understand the brain state concept itself — and we may even be able to help design brain-state targeted clinical interventions.“
