Contact: dkenney@mbl.edu; 508-685-3525

WOODS HOLE, Mass. -- The dialogue between neurons is of critical importance for all nervous system activities, from breathing to sensing, thinking to running. Yet neuronal communication is so fast, and at such small scale, that it is exceedingly difficult to explain precisely how it occurs. A preliminary observation in the Neurobiology course at the Marine Biological Laboratory (MBL), enabled by a custom imaging system, has led to a clear understanding of how neurons communicate with each other by modulating the “tone” of their signal, which previously had eluded the field. The report, led by Grant F. Kusick and Shigeki Watanabe of Johns Hopkins University School of Medicine, is published this week in Nature Neuroscience.

In 2016 Watanabe, then on the Neurobiology course faculty, introduced students to the debate over how many synaptic vesicles can fuse in response to one action potential (see this 2-minute video for a quick brush-up on neurotransmission). To probe this controversy, they used a “zap-and-freeze” imaging technology conceived by co-authors M. Wayne Davis, Watanabe and Erik Jorgensen, and built by Leica for testing in the Neurobiology course. They zapped a neuron with electricity to induce an action potential, then quickly froze the neuron and took an image. They saw multiple vesicles fusing at once at many synapses, the first novel finding of this Nature Neuroscience report.

Shigeki Watanabe, left, and Erik Jorgensen with the flash-and-freeze imaging system, a forerunner to zap-and-freeze. Both systems were developed in the Jorgensen lab and are descendants of the 1979 “freeze slammer” invented by John Heuser, Tom Reese and colleagues. Credit: Cveta Tomova

But there was more. Back at Johns Hopkins, Kusick and Watanabe decided to walk through the neurotransmission process with zap-and-freeze, taking images every 3 milliseconds after the action potential. That’s when they found an answer to an even larger question – how do neurons change the tone of their neurotransmission signal?

At any given time, only a few synaptic vesicles are in “docked” position, meaning loaded and ready to release neurotransmitter. Immediately after an action potential, the number of docked vesicles decreases by 40 percent, so after 2 to 3 action potentials, the docked vesicles would be depleted. (That is, their signal or “voice” would become weaker and weaker, as more action potentials are induced.) But they found that, within 14 milliseconds following an action potential, new vesicles are swiftly recruited to the docked pool that can fuse and release neurotransmitter, and this recruitment is transient such that neurotransmission can be strong or weak on a millisecond time scale. This is the first close-up look at neural communication that adds up from a temporal perspective.

“What this means is that we have identified a mechanism that neurons use to communicate through intonations,” Watanabe says. “Each docked vesicle is like a word that neurons can use for communication at any given moment. It has been known for decades that neurons can speak more than a few words at a time, and they can also change the tone of these words. The question was how. We’ve shown that neurons continuously bring in more words, but by simply changing the number of vesicles, they can raise or lower the voice. If you are asking a question, you will raise the intonation at the end of a sentence – neurons do so by changing the number of docked vesicles ready to go.”

Synapses before (left) and after stimulation, with arrows showing the moment of neurons talking to each other (fusion of synaptic vesicles). Credit: MBL Neurobiology course students Kristina Lippmann, Kadidia Adula, Thien Vu, and Eddie Hujber

The “zap and freeze” electron microscopy technology is  the 21^st-century version of the “freeze slammer” developed by John Heuser, Tom Reese et al., and used at MBL nearly 50 years ago to demonstrate how neurons communicate with each other.

The MBL Neurobiology course faculty and students co-authoring this paper include Jorgensen and Davis (University of Utah), Kristina Lippmann (University of Leipzg), Kandidia P. Adula (University of California, Los Angeles), and Edward J. Hujber and Thien Vu (University of Utah). Watanabe, who was a 2014 Grass Fellow at the MBL, returns nearly annually to teach in the Neurobiology course and as a Whitman Center scientist.

Left to right: Grant Kusick (Watanabe lab), MBL Neurobiology course students Christine Prater and Stephen Lee, and Shigeki Watanabe in Woods Hole in 2019.

Citation:

Grant F. Kusick, M. Chin, S. Raychaudhuri, K. Lippmann, K. P. Adula, E.J. Hujber, T. Vu, M.W. Davis, E.M. Jorgensen, S. Watanabe (2020) Synaptic vesicles transiently dock to refill release sites. Nature Neuroscience, DOI: 10.1038/s41593-020-00716-1

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The Marine Biological Laboratory (MBL) is dedicated to scientific discovery – exploring fundamental biology, understanding marine biodiversity and the environment, and informing the human condition through research and education. Founded in Woods Hole, Massachusetts in 1888, the MBL is a private, nonprofit institution and an affiliate of the University of Chicago.

By Raleigh McElvery

Hydra in control media before contraction (left) and during contraction (right). Credit: Wataru Yamamoto

WOODS HOLE, Mass. — Although it may seem counterintuitive, researchers are turning to an animal without a brain to crack the neural code underlying behavior.

Hydra vulgaris, a tiny, tentacled, freshwater organism, uses “nets” of neurons dispersed throughout its tube-like body to coordinate stretching, contracting, somersaulting, and feeding movements. This simple nervous system is one reason that Hydra is well suited for studying how electrical activity translates into motion.

In a study published in eNeuro, a duo from Columbia University and the Marine Biological Laboratory (MBL) has begun to crack the neural code behind Hydra’s simplest behavior, called  contraction bursts (when the torso shrinks down and expands outward, over and over again). The scientists found that the concentration of dissolved particles in the surrounding water — a property known as osmolarity — affects the activity of a neural circuit in one of Hydra’s nerve nets, which can trigger a specific set of muscle cells to contract the torso.

“One by one, we want to decipher the neural and muscular activity behind each of Hydra’s behaviors,” says senior author and MBL Whitman Center Fellow, Rafael Yuste of Columbia University. “This paper is the beginning of our journey.”

Yuste and first author Wataru Yamamoto conducted their experiments in Woods Hole during the summers of 2017, 2018 and 2019, in consultation with their MBL Hydra Lab research consortium. They used whole-body calcium imaging to visualize Hydra’s neurons and muscles, and tested whether tweaking various environmental conditions such as water temperature, body size, nutritional state, and osmolarity would affect contractions. They were surprised to find that just one of those parameters, osmolarity, had an impact.

Boosting the concentration of sugar particles in the water triggered fewer contractions and decreased activity in the “contraction burst” (CB) neural circuit as well as in one set of muscles. Lowering particle concentration had the opposite effect, increasing cellular activity and contractions. Although additional experiments are needed to confirm their theory, the researchers propose that CB neurons respond to changes in osmolarity by altering muscle activity, which in turn influences contraction frequency.

Five Hydra freely behaving in 50mM sucrose (high osmolarity) solution.
This video is sped up 300-fold. Credit: Wataru Yamamoto

Reacting to changes in particle concentration could mean the difference between life and death for Hydra — especially if salt is involved. As freshwater dwellers lacking advanced excretory systems, Hydra are not well-equipped to maintain the proper balance of salinity inside and outside their bodies. Water is constantly flowing in and out of their gastrovascular cavities, and too much internal salty solution causes them to balloon and literally explode. The researchers surmise that Hydra contract in response to high osmolarity to “wring” themselves out and expel excess water.

In addition to affecting contractions, osmolarity also influenced how often Hydra detached and repositioned its tube foot, likely preparing to move to a new location. Yamamoto plans to continue to investigate how and why this happens. He hopes his line of inquiry will eventually help decode a more complex behavior: somersaulting, when Hydra flips tentacles-over-tube foot, like a circus acrobat.

Hydra ectoderm muscle activity during contraction in control media.
The animal was allowed to move between coverslips in the mounted configuration.
This video is sped up 20-fold. Credit: Wataru Yamamoto

Homepage photo: Freely behaving Hydra exhibiting different motor behaviors (elongation, contraction, somersaulting). Credit: Wataru Yamamoto

Citation:

Wataru Yamamoto and Rafael Yuste (2020) Whole-Body Imaging of Neural and Muscle Activity during Behavior in Hydra vulgaris: Effect of Osmolarity on Contraction Bursts. eNeuro, DOI: 10.1523/ENEURO.0539-19.2020

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The Marine Biological Laboratory (MBL) is dedicated to scientific discovery – exploring fundamental biology, understanding marine biodiversity and the environment, and informing the human condition through research and education. Founded in Woods Hole, Massachusetts in 1888, the MBL is a private, nonprofit institution and an affiliate of the University of Chicago.

2019 Nikon Small World Virtual Exhibit

MBL Falmouth Forum Lecture Series 2020-21

Media Contacts:
Diana Kenney; dkenney@mbl.edu; 508-685-3525
Emily Greenhalgh; egreenhalgh@mbl.edu; 508-289-7119

WOODS HOLE, Mass. – The flashy Flamboyant Cuttlefish is among the most famous of the cephalopods (octopus, squid, and cuttlefish) – but it is widely misunderstood by its legions of fans.

A new paper from the Roger Hanlon laboratory at the Marine Biological Laboratory, Woods Hole, sets the record straight.

“This animal is well known in the Internet community, has been on TV many times, and is popular in public aquariums,” Hanlon says. “In almost all cases, [its skin] is showing this brilliantly colorful flamboyant display.”

But Hanlon’s field studies in Indonesia, reported here, tell a different and richer story.  “It turns out in nature, flamboyant cuttlefish are camouflaged nearly all of the time. They are nearly impossible to find,” he says. In the blink of an eye, they can switch from some of “best camouflage known in the cephalopods” to their dazzling flamboyant display. But they only use this display on certain occasions: For elaborate male courtship rituals; or when males are fighting over a female; or to flash briefly at a threatening object when it approaches too close, presumably to scare it away.

“The flamboyant display is common when a diver approaches close enough to photograph, which is why the public may think this species always looks so colorful,” Hanlon says. “But it is rare to see this species in flamboyant display in the wild.”

Video by Emily Greenhalgh, MBL

A Flamboyant Courtship

The courtship displays by male flamboyant cuttlefish (Metasepia pfefferi)  are among the most elaborate of all cephalopods! This study reveals new observations about the sex life of the flamboyant cuttlefish -- from courtship to mating to egg laying – gleaned from hours of video taken during many SCUBA dives in Indonesia with teams of volunteers.

Males, which tend to be significantly smaller than females, approach and court a camouflaged female with flamboyant displays and elaborate rituals, which include “waves” (rapidly waving three pairs of arms while displaying “passing cloud”) and “kisses” (male darts forward and briefly, gently touches his arms to hers).

Females generally ignore males while they are courting; they stay camouflaged and motionless or just keep on foraging and hunting. Male courtship goes on non-stop for prolonged periods (6 to 52 minutes observed in this study).

In three observations, two males competed simultaneously for a female. Males can display flamboyant courtship signaling on one side of the body while flashing white (signaling aggression) on the other side toward the rival male.

In one case, male competition ended abruptly when one of the males, while facing the female and waving and kissing, backed into a camouflaged scorpionfish and was eaten! “Sex can have a real cost,” Hanlon notes.

Females were choosy and often rejected courting males. Female receptivity was obvious when she widely spread her first three pairs of arms (while standing on the fourth pair of arms). The male would then swim within the arm crown and quickly deposit spermatophores in the buccal region where the seminal receptacle is located. Average duration of mating was only 2.89 seconds.

After fertilization, the successful male guarded the female for a while but not, curiously, up to egg laying, as is common with other cuttlefish. When another male was present, mate guarding was aggressive.

The female lays her eggs while camouflaged and staying still. She then pushes her eggs under a coconut shell and affixes them to the inside of the shell. When the hatchlings exit the egg case and jet away, they are fully formed and capable of camouflage and signaling.
 

Flamboyant cuttlefish (Metasepia pfefferi) in flamboyant display. Courtesy Roger Hanlon

Camouflaged flamboyant cuttlefish (Metasepia pfefferi).
Credit: F. Bavendam

 
Flamboyance for Secondary Defense

The primary mode of defense for both male and female Metasepia pfefferi is camouflage, and they remain camouflaged almost all the time. If a predator or threatening object (such as a diver) comes too close, though, the cuttlefish will flash the flamboyant display – switching from camouflaged to flamboyant in 700 milliseconds!

Elaborate Dynamic Signaling Rivaling That of Any Vertebrate

The vibrant colors (white, yellow, red and brown) of the flamboyant display are combined with apparent “waves” of dark brown color that produce a dazzling and dizzying kaleidoscope of motion, color, and patterning. The fast neural control of many thousands of chromatophore organs in the skin enable this unique signaling capability – all turned on or off in less than a second, and changed depending on the behavioral context of the courtship, or in the case of defense, the fish predators that discover them.

“Birds are renowned for highly evolved visual displays that depend partly on dramatic postural changes (with wings of different color and pattern, in particular), yet this invertebrate cuttlefish species has evolved equally dramatic and complex displays mainly with its skin coloration,” Hanlon says.

Hanlon is a senior scientist in the MBL's Eugene Bell Center for Regenerative Biology and Tissue Engineering.

More video resources are here: https://www.youtube.com/playlist?list=PLZYs-G4nf1Mb0srHYeZPeIGsuIVVyxxz8

Citation:

Roger T. Hanlon and Gwendolyn McManus (2020)  Flamboyant cuttlefish behavior: Camouflage tactics and complex colorful reproductive behavior assessed during field studies at Lembeh Strait, Indonesia. J. Exp. Marine Biology and Ecol., DOI: 10.1016/j.jembe.2020.151397

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The Marine Biological Laboratory (MBL) is dedicated to scientific discovery – exploring fundamental biology, understanding marine biodiversity and the environment, and informing the human condition through research and education. Founded in Woods Hole, Massachusetts in 1888, the MBL is a private, nonprofit institution and an affiliate of the University of Chicago.