animal models: "Illuminating Behaviors"

[Mary Finelli]

ILLUMINATING BEHAVIORS
The Scientist, Volume 17 | Supplement 1 | 18, Douglas Steinberg, Jun. 2, 
2003
http://www.the-scientist.com/yr2003/jun/feature6_030602.html
Courtesy of Genevieve Anderson


If not for Nobel laureates Thomas Hunt Morgan, Eric R. Kandel, and Sydney 
Brenner, the notion of a general behavioral model might seem odd. Behaviors, 
after all, are determined by an animal's evolutionary history and ecological 
niche. They are often idiosyncratic, shared in detail only by closely 
related species.

But, thanks to Morgan's research in the early 20th century, and Kandel's and 
Brenner's work over the past 35 years, the fly Drosophila melanogaster, the 
mollusk Aplysia californica, and the worm Caenorhabditis elegans have become 
general behavioral models. The newest member of the club is the mouse.

This quartet yields broadly applicable behavioral findings for two reasons: 
First, these animals are unusually amenable to cellular and molecular 
experimentation; second, such experimentation has turned up certain genes, 
proteins, and cells that underlie behavior across many species. Evolution 
did not "completely reinvent the wheel and come up with a new set of 
molecular rules for each phylum," notes Aplysia expert Thomas J. Carew, at 
the University of California, Irvine.

Learning from Sea Slugs
Lacking a cortex, Aplysia has just 20,000 neurons clustered into ganglia. As 
such, its nervous system appears incommensurable with those of higher 
organisms. But this hand-length, maroon sea slug has one quality trumping 
interspecies differences: huge neurons, many with cell bodies hundreds of 
microns across. Biologists can easily image and manipulate these neurons to 
determine their firing properties and responses to stimulation. Popular 
research areas include the mollusk's feeding behavior and its withdrawal 
reflexes when it is touched.

One important discovery, says Carew, is that facilitation--a phenomenon 
involving enhanced neurotransmitter release into the synapses separating 
neurons--underlies a simple form of learning known as sensitization. Other 
findings have elucidated the signal-transduction pathways triggered by 
learning. Cyclic AMP (cAMP) activates cytoplasmic kinases (e.g., protein 
kinase A), which translocate to the nucleus where they activate 
transcription factors (e.g., cAMP response element-binding protein). These 
factors then turn on genes whose protein products cause long-term changes in 
the neuron.

Many findings in Aplysia have been replicated in Drosophila and mice. 
Experiments on transgenic and knockout mice, for example, show that synaptic 
plasticity relies on several kinases and transcription factors first 
explored in Aplysia. Conversely, knowledge gleaned from higher organisms 
might apply to sea slugs. Using a paradigm tested in humans, Carew learned 
that training the mollusk induces long-term memory if sessions are separated 
in time but not if they are massed together.

Aplysia has two major limitations as a model for higher organisms: a modest 
behavioral repertoire, and a genome that has not been sequenced (unlike the 
genomes of the other three behavioral models). To manipulate this mollusk 
genetically, researchers inject mRNA directly into its neurons.


Lords of the Flies
Genetic plasticity is Drosophila's chief advantage. In 1915, the Columbia 
lab of fruit-fly pioneer Morgan conducted the first behavioral genetics 
study of any organism, recounts Brandeis University biologist Jeffrey C. 
Hall. Since then, scientists have discovered or created thousands of fly 
mutants. Tools for investigating Drosophila include heat-inducible and 
tetracycline-regulated transgenes, transposable P-elements, and the GAL4-UAS 
system, which allows precise spatial control of transgene expression.

Many fly researchers are examining circadian cycling between activity and 
inactivity, as well as learning and memory.1 Some are focusing on courtship, 
geotaxis, and reactions to odor and taste. At least 15 homologs of fly genes 
implicated in these behaviors have been found in other species, Hall says.

Drosophila learning occurs during natural behaviors--courtship, for example, 
is not totally hard-wired--and in conditioning experiments. Martin 
Heisenberg, at the University of Würzburg in Germany, has trained flies to 
avoid the heated side of a chamber and devised a complex flight simulator to 
test visual learning. One popular type of apparatus shocks flies as they 
sniff an odor. Some mutant or transgenic flies later forget to avoid the 
odor.

Neuroscientist Jerry C.P. Yin, at Cold Spring Harbor Laboratory, learned 
that one murine transgene actually enhances certain forms of Drosophila 
memory. It encodes a specific form of the signal-transduction enzyme protein 
kinase C. Based on this protein's functions in other cell types and species, 
Yin speculates that it allows a neuron to tag its recently active synapses.

Two other Cold Spring Harbor neuroscientists, Tim Tully and Josh Dubnau, 
used the odor/shock assay to uncover about 60 putative memory genes. 
Switching to DNA chip technology, Tully and Dubnau identified 42 genes that 
turn on or off during memory formation. Prominent among the genes implicated 
by both methods were staufen, whose protein product appears to be involved 
in transporting mRNAs to synapses, and pumilio, whose product seems to help 
repress translation during mRNA transport.


Tammy Irvine,
Rear View Illustration


Hall specializes in the fruitless gene, which encodes a transcription factor 
critical to male courtship. He regards fruitless as the best example of a 
single gene specifying a set of behaviors. His lab found that fruitless is 
expressed throughout the fly's nervous system--a discovery, he notes, that 
is consistent with the gene's broad effects.

Genetic malleability, Drosophila's greatest strength, also can be a 
weakness. In rapidly breeding mutant populations, further mutations often 
cause loss of phenotype, which is difficult to reverse. (Labs cannot 
preserve the original phenotype because there are no reliable methods to 
freeze and thaw fly embryos.) For neurobiologists, the fly's main drawback 
is that its 150,000 or so neurons are too small to manipulate in situ, 
except at the neuromuscular junction.


Nothing to Hide
The nematode C. elegans joined the behavioral-model menagerie basically 
because it has nothing to hide. The tiny transparent worm's nervous system 
has been completely plotted, revealing 302 neurons and 5000 synapses. With 
this knowledge in hand, researchers employ various cell-ablation and 
gene-manipulation techniques to link behaviors to specific cells and genes. 
Investigators also can record from nematode neurons and recently began 
culturing them.

Worms have a limited behavioral repertoire. Besides life- and 
species-preserving activities such as feeding, mating, and egg-laying, 
nematodes "can't do much--swim forward, swim backward, stop, start, curl in 
a circle," observes psychology professor Catherine H. Rankin, at the 
University of British Columbia.2 Nevertheless, their exquisite sensitivities 
to temperature and ambient chemicals facilitate behavioral conditioning 
experiments. Homologs of Drosophila learning-associated genes are known, but 
their effects on C. elegans learning are unclear, says Rankin. Altering 
these signal-transduction genes, she explains, often results in an 
uncoordinated worm, a common and unrevealing mutant phenotype.

Even when a mutation's impact seems unambiguous, the full story is probably 
far from simple. Five years ago, neuroscientists Mario de Bono, now at the 
Medical Research Council's Laboratory of Molecular Biology in Cambridge, 
England, and Cornelia I. Bargmann at UC-San Francisco, discovered a 
loss-of-function mutation in a receptor gene that switched C. elegans from a 
solitary food-forager to a "social" one that gathers with its mates on their 
common source of nourishment, a lawn of bacteria.

Follow-up work suggests the existence of "multiple layers of antagonistic 
stimuli that are regulating whether you are social or solitary," says de 
Bono. "There's often this assumption that because a single gene flips 
behavior from one form to another, it is the critical gene." But, he demurs: 
"You can still have that effect when you have a gene that's only one player 
in a larger number of players."



Tammy Irvine,
Rear View Illustration


Knockouts and Their Discontents
Rats were long the rodent behavioral model of choice because of their 
intelligence and large brains. Over the past decade, however, transgenic 
mice have increasingly hogged the spotlight. (Transgenic rats are relatively 
rare, because foreign DNA does not incorporate readily into the genomic DNA 
of rat oocytes, explains Markus Heilig, at the Karolinska Institute, 
Sweden.) Jacqueline N. Crawley, chief of the National Institute of Mental 
Health's behavioral genomics section, notes that transgenic mice have become 
invaluable in experiments involving learning and memory, motor disorders 
such as Parkinson disease, and obesity.3

Mice lacking certain genes can display complex behavioral phenotypes, such 
as social deficits (knockout of the hormone oxytocin); less huddling and 
nest-building (knockout of the intracellular signaling molecule 
dishevelled-1); and male aggression (knockout of neuronal nitric oxide 
synthase). But Crawley cautions that different murine strains with an 
identical genetic alteration might each exhibit a unique phenotype. The 
likely reason: Each strain harbors a different set of polymorphisms that 
temper the genetic alteration's effect.

Transgenic studies involve other complexities and pitfalls. Crawley 
observes, for example, that some murine wild-type strains are already so 
aggressive that an aggression-causing mutation might be undetectable. She 
also warns of "a lot of variability in behavior that requires larger numbers 
of animals and more rigorous statistics than molecular geneticists are used 
to." When these requirements are not met, she adds, investigators often 
overinterpret results, causing the field of behavioral neuroscience to lose 
some credibility.

Crawley's mantra is that "behavior is not so simple." But that's a challenge 
that Drosophila expert Hall relishes. He insists that biologists "should 
want the phenomenon [they study] to be complicated, because life is 
complicated."

Douglas Steinberg (dougste at attglobal.net) is a freelance writer in New York 
City.

References
1. M.B. Sokolowski, "Drosophila: Genetics meets behaviour," Nat Rev Genet, 
2:879-90, 2001.
2. C.H. Rankin, "From gene to identified neuron to behaviour in 
Caenorhabditis elegans," Nat Rev Genet, 3:622-30, 2002.
3. J.N. Crawley, What's Wrong With My Mouse? Behavioral Phenotyping of 
Transgenic and Knockout Mice, New York: John Wiley & Sons, 2000.

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