CEPHALOPOD – OCTOPUS
They were still able to change colour and body patterns but in a seemingly random fashion. Anatomical evidence also shows that nerves in the lower brain connect directly to muscles surrounding the pigment sacs or chromatophores.
Like an artist spreading pigment on a pallet, activating the muscles pulls the sacs apart spreading the chromatophore pigments into thin discs of colour. But the octopus is not composing a picture. Hanlon’s experiments with cuttlefish show they are deploying one of three pre-existing patterns – uniform, mottled or disruptive – to achieve camouflage on diverse backgrounds.
As far as detailed brain circuitry goes, researchers have made little progress since the 1970s when legendary British neuroscientist J.Z Young worked out the gross anatomy of the distributed coleoid brain. Escaping Britain’s dismal winter for the Stazione Zoologica in balmy Naples, Young’s research was part of an American Air Force funded project to search for the theoretical memory circuit, the ‘engram’.
“They were ahead of their time,” says Hanlon, who experienced a stint with Young in Naples. Nevertheless they were limited by the paucity of brain-recording techniques that were suited to the octopus.
It’s a problem that has continued to hold back the understanding of how their brain circuits work. “Is it the same as the way mammals process information? We don’t know,” says Ragsdale.
UNWILLING SUBJECTS
It’s not for want of trying, as Kuba will tell you. In the 1990s, he joined the lab of neuroscientist Binyamin Hochner at the Hebrew University of Jerusalem. Hochner was a graduate of Eric Kandel’s lab, the Nobel laureate who pioneered studies on how the sea slug Aplysia learns.
All the action takes place in the gaps between individual neurons, the ‘synapse’. The synapse may look like an empty gap under the microscope but it’s a crowded place. It’s packed with over 1,000 proteins that assemble into a pinpoint-size microprocessor. If each neuron is like a wire, it’s up to this microprocessor to decide whether the signal crosses over from one wire to the next. When the sea slug learns a lesson, for instance withdrawing its gill in response to a tail shock, that’s because new computations at the synapse rerouted the connections.
Kuba, however, found an octopus to be far less obliging than a sea slug. Whatever electrical probe he stuck into its brain was rapidly removed thanks to all those opposable thumbs. Ragsdale also had his share of frustration. “We have a technical problem with sharp electrodes. For example, if you put an electrode into the optic lobe, the neurons will fire for about 10 to 20 minutes and then become silent.”
Kuba, who is now based at the Okinawa Institute of Science and Technology, hopes that a new kind of miniature brain logger that sits on the surface of the brain, hopefully out of reach of prying suckers, will kick-start the era of octopus brain-circuit mapping.
“There’s a lot of technical challenges, but they are surmountable,” agrees Ragsdale.
The irony is that the first insights into how the octopus brain sends signals came from a squid. In 1934 Young identified a giant squid nerve cell that controlled the massive contractions of its mantle, the bulbous muscular sac behind the eyes that both houses the organs and squeezes water through the siphon with such great effect!
Like mammalian neurons, the most distinctive feature of the squid cell was its wire-like axon, but with a diameter of around one millimetre, it was 1,000 times fatter than those of mammals. The colossal size allowed researchers to insert a metal electrode and measure the changing electrical voltage as a nerve impulse travelled along the axon.
All this foundational knowledge shed light on vertebrate brains, but the detailed circuitry of the squid brain was largely left in the dark.
BREAKING THE CENTRAL DOGMA
It was another frustrated neuroscientist who opened the latest front into the understanding of soft intelligence.
In the early 1990s, Josh Rosenthal, based at William Gilly’s lab at Stanford, was making use of the timehonoured giant squid motor axon. But with a new purpose. Rather than measure its electrical properties, Rosenthal wanted to isolate one of its key components: the ‘off’ switch. It is a protein called the potassium channel.
The squid neuron made this protein according to a recipe carried by its DNA blueprint, which is cached in the cell’s nucleus. To access the recipe, the cell makes a MRNA transcript, rather like transcribing a single recipe from a recipe book. Rosenthal wanted to isolate these transcripts and read the code sequence for the protein channels.
But he had a problem. Every time he read the sequence for the potassium channel, it was slightly different. Was it just an error? If so, it was highly consistent. The changes were not random. They always occurred at one or more precise positions in the code. And, invariably, the letter A was always changed to the letter G.
For instance, imagine a recipe for apple pie was supposed to read: Place the crust around the pie.
Instead it was being edited to: Place the crust ground the pie. Such a change might instruct the modern-day deconstructed apple pie rather than the traditional crusted version.
Unbeknownst to Rosenthal, Peter Seeburg at the University of Heidelberg was puzzling over a similar glitch in a recipe for a human brain protein, the glutamate receptor. When Seeburg’s paper was published in 1991, Rosenthal recalls, “everyone got excited”.
Clearly editing brain recipes was important for humans and squid. But why?
In the human (or mouse), editing the glutamate receptor changed how much calcium could flow into brain cells. In mice, failure to edit was lethal, as it allowed toxic levels of calcium to stream in. There’s also evidence that failure to edit the same receptor in humans is associated with the neurodegenerative disease Amyotrophic Lateral Sclerosis.
An enzyme called ADAR2 carried out these crucial edits to the RNA recipe. Just why evolution hasn’t gone ahead and ‘fixed’ the DNA source code of the glutamate receptor remains a mystery.
As for the squid potassium channel, Rosenthal had a hunch. After an electrical signal has passed through a neuron, it needs a ‘reset’ for the next signal. The potassium channel plays a crucial part. In cold temperatures, the reset might take longer, making the animal a bit sluggish. Could RNA editing be a way of fine tuning the system in response to temperature? Rosenthal tested his idea by spending several years collecting octopuses that live in either tropical, temperate or polar climates. It was indeed the polar octopuses that were the most avid editors of their potassium channels.
Potassium channels turned out to be just the tip of the iceberg. Rosenthal teamed up with computation geek Eli Eisenberg at Tel Aviv University to trawl through MRNA databases and find out just how much recipe tweaking was going on with squid genes. In humans, tweaking is rare – restricted to a handful of brain gene recipes. In the squid, the majority of brain recipes received this treatment. Many of them were related to proteins found at the synapses, the microprocessors for memory and learning.
Could this extemporising with brain protein recipes be important for soft intelligence? It’s a tantalising idea. “Coleoids show it. Nautilus – the stupid cousin does not, it’s like any other mollusc,” says Eisenberg.
“Coleoids are editing the same proteins that we
know are involved in learning and memory. By editing them or not, it’s not a stretch to hypothesise that they are adding flexibility and complexity to the system,” says Rosenthal.
CLUES FROM THE BLUEPRINT
Over in Chicago, Cliff Ragsdale, another frustrated octopus neuroscientist, was also turning his interest to octopus DNA.
In 2015, working with Daniel Rokhsar and Oleg Simakov of OIST, the Ragsdale laboratory managed to read the genome of the California two-spot octopus.
It turns out that the octopus has more genes that we do: 33,000 compared to our 21,000. But gene number per se doesn’t bear much relation to brain power: water fleas also have about 31,000. In fact most of the genes in the octopus catalogue were not all that different to those of its close relative – the limpet, a type of sea snail. But there were two gene families that stood out like a sore thumb. One was a family of genes called protocadherins. This family of ‘adhesion’ proteins are known to build brain circuits. Like labels on the tips of growing neurons, they allow the correct types of neurons to wire to each other — so neuron 370 connects up to neuron 471 at the right time and the right place. Limpets and oysters have between 17-25 types of protocadherins. Vertebrates have 70 types of protocadherins plus over 100 different types of related cadherins. These circuit builders have long been thought to be the key to vertebrate braininess.
So it was stunning to find that the octopus has a superfamily of 168 protocadherins. Ragsdale says the squid genome, also now being sequenced, shows it is similarly equipped with hundreds of circuit-building genes.
THE OTHER STAND- OUT
in the octopus genome was a family of genes called ‘zinc fingers’. They get their name because the encoded proteins have a chain structure that is cinched by zinc atoms into a series of fingers. These fingers poke into the coils of DNA to regulate the transcription of genes.
Limpets have about 413 of these zinc fingers. Humans have 764. Octopuses have 1,790! Perhaps this profusion of octopus zinc fingers is involved in regulating the network of brain genes?
So far, the octopus has revealed three big clues as to how it generates brain complexity: it has multiplied its set of circuit-building protocadherin genes and its network-regulating zinc fingers. It has also unleashed RNA editing to generate more complexity on the fly. There may also be a fourth mechanism at work. Genes are supposed to stay put. But ‘jumping genes’, which are closely related to viruses, have a tendency to up anchor and insert themselves into different sections of the DNA code. That can scramble or otherwise change its meaning. Imagine if the words ‘jumping gene’ just started appearing randomly in this text. Fred Gage’s group at the Salk Institute in San Diego has found that during the development of the nervous system in mice and humans, jumping genes start jumping.
What this means is that each individual brain cell ends up with slightly different versions of its DNA code. Gage speculates that this may be a way to generate diversity in the way neurons wire up. Perhaps it goes some way to explaining why twins, born with the same DNA, nevertheless end up with different behaviours.
“If you believe that theory,” says Ragsdale, “you’ll be struck by the fact that we also found a high number of jumping genes active in the brain tissues of the octopus.”
TESTING THE THEORY
Unravelling the details of how octopus and squid are using and abusing the genetic code is generating iconoclastic hypotheses about how they generate their complex brain circuitry.
And researchers are not blind to the problems of dogma-breaking. For one thing, playing fast and free with the genetic code creates an astronomical number of possible proteins, most of which would be toxic to the animal, says Eisenberg. “It’s very troubling; one hypothesis is that this may explain their short lifespan of one to three years.”
Troubling or not, Rosenthal and colleagues at Woods Hole are moving full speed ahead to test the role of RNA editing in the coleoids by bringing together researchers with different expertise. “There’s a lot of moving pieces,” says Rosenthal.
For starters, their Woods Hole team is cultivating four species of small squid and cuttlefish that reach sexual maturity in two to three months. The goal is to manipulate the squid’s genes using the genetic engineering tool, CRISPR. To see if they can get CRISPR working, they will try to ‘knock-out’ the pigment genes. If they’re successful they should see the result on the squid bodies. “It’s a beautiful in-built test,” says Rosenthal.
If that works, they will try the big experiment. Does impairing the ability to edit proteins at the synapse (by knocking out the ADAR2 gene responsible for RNA editing) tamper with learning and memory?
Meanwhile, collaborator Alex Schnell, a behavioural biologist based at the University of Cambridge in the UK, is developing rigorous tests for complex learning and memory in cuttlefish. In particular, she is testing their capacity for “episodic memory”, a detailed weaving together of memories once thought to be a strictly human attribute.
For instance, it’s thanks to your episodic memory that you recall exactly where you were and what you were doing on 11 September 2001. Since the late 1990s, we know that animals like great apes, crows and jays also have that capacity. Maybe cuttlefish do too. Schnell’s initial results show that cuttlefish can learn and memorise complex information about their favourite food, such as when and where it is likely to be found.
With other teams around the world pursuing similar strategies, it seems likely that after decades of awe and wonder, the mystery of soft intelligence may soon yield to hard science.