Genes, neurons, and decisions: Using fixed circuits to generate flexible

behaviors.

Cornelia I. Bargmann, HHMI and The Rockefeller University, New York NY

The human brain is capable of astonishing things; it is the source of our thoughts,
memories, emotions, and behavior. But the human brain is also a biological organ,
and like other biological organs, it is built through the action of genes.

The importance of genetic factors in human behavior, and the limits of genetic
factors, can be illustrated using the example of psychiatric disease. There is
overwhelming evidence for genetic risks in psychiatric disorders; a person whose
identical twin has autism or schizophrenia has a much higher risk of developing the
same disorder than average, even if those twins are raised in different families. A
fraternal twin or a brother or sister also has an elevated risk, although less than an
identical twin. For some cases, we even know the exact genetic lesions that drive
that risk. But the risk, even for an identical twin with the same genes, is not 100%;
it is very roughly 50%. Genetic risk interacts with other factors to generate
psychiatric disorders. There are some factors that we understand, like stress to the
developing fetus and childhood trauma, but other factors are a mystery. Finally, the
natural history of psychiatric disorders shows that most of these disorders wax and
wane, so that individuals may be very severely affected at some points in life, but
unaffected or less affected at other times. The interaction between genes, context,
and individual experience is essential to these disorders, and to all aspects of
behavior.

Genes do not affect behavior directly. Genes, context, and experience must be
translated into the action of neurons and their connections in the brain to give rise
to behavior. The human brain has billions of neurons and trillions of connections,
and understanding this complexity in its entirety is daunting. One approach to
reducing this problem to a manageable level is to study the genes, brains, and
behaviors of simpler animals. The animal studied in my lab, the nematode worm
Caenorhabditis elegans, presents unique opportunities to make connections
between genes, brains, and behavior. Its nervous system consists of just 302
neurons in the adult hermaphrodite, each uniquely identifiable. Through the work
of John White and Sydney Brenner at the MRC in Cambridge, the anatomical
connections between these neurons have been completely described from serial-
section electron micrographs. With modern genetic techniques, each of the 20,000
genes of the worm can be manipulated, in the entire animal or in individual neurons
of interest. Because it is transparent, the worm is perfectly suited for genetically-
assisted visualization of neurons, proteins, and neuronal activity; it was the first
animal in which transgenic Green Fluorescent Protein and genetically-encoded
calcium indicators based on GFP were tested in vivo, by Martin Chalfie from
Columbia and by Roger Tsien and Bill Schaefer from UCSD, respectively.
Importantly, the 20,000 worm genes include homologs of most (but not all) genes
that are important in human brain development, function, and synaptic signaling,
and many (but not all) genes implicated in human psychiatric disorders.

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Homologous gene functions are generally conserved across the organisms that
share these genes, so by understanding how genes regulate behavior in this simple
nervous system, we may gain insights into how those genes function in more
complex brains, including our own.

Identifying the steps from gene, to nervous system, to behavior, begins with
knowing what behaviors an animal can generate. C. elegans lives in decaying matter
in gardens and orchards. These environments are rich in odors generated by the
metabolic activity of microorganisms, including the worms food, bacteria.
Accordingly, C. elegans is highly sensitive to chemical cues, and will demonstrate
that sensitivity by approaching bacterial metabolites through its senses of taste and
smell, as well as avoiding noxious compounds. Its olfactory capacity is impressive;
worms will show behavioral responses to about half of the random volatile organic
chemicals purchased from a chemical catalog, and discriminate between them based
on subtle chemical features. Smell and taste are the central means by which C.
elegans evaluates the world, and they regulate virtually every aspect of C. elegans
behavior and even its development and lifespan. By studying these cues that are
significant to the animal, we can gain insight into many elements of the animals
behavior.

The next component of this functional map was to characterize the neuronal basis of
olfactory behaviors. The simple C. elegans nervous system permits a precise
mapping of individual neurons to functions, a prospect that is still a dream in
complex nervous systems. In a series of experiments begun in Bob Horvitzs lab at
MIT, the C. elegans sensory neurons that detect odors, tastes, and pheromones were
mapped by killing individual neurons and examining the behavioral effects on the
operated animals. As first suggested by the anatomical reconstructions of the
nervous system, chemical are sensed by a cluster of neurons with ciliated endings
that are exposed to the external environment through specialized pores at the tip of
the nose. These neurons are so reliable in their morphology, position, synapses, and
patterns of gene expression that each has a three-letter name. The laser killing
experiments showed that each neuron detects a specific set of chemicals, and is
responsible for a specific, predictable set of behaviors. Thus, for example, the AWA
neurons are required for chemotaxis to diacetyl, but not benzaldehyde, whereas the
adjacent AWC neurons are required for chemotaxis to benzaldehybde but not
diacetyl, and a third pair of neurons, AWB, is required for avoidance of noxious
odors.

How does the animal detect and respond to odors? C. elegans has a generation time
of three or four days, and as a self-fertilizing hermaphrodite, it is ideal for
mutational genetics. Indeed, Sydney Brenner consciously developed this
experimental system for this purpose. Therefore, to find the genes for olfaction,
Piali Sengupta and others in the lab conducted behavioral screens for mutant
animals that lacked an interest in particular odors. The most specific of these was a
mutant called odr-10, the tenth gene we found with a defect in olfactory responses.
odr-10 mutants were active, healthy, and normal in all respects we could detect

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except that they were blind to diacetyl, a normally attractive odor. Piali cloned this
gene, which was a painful exercise at the time, and found that it encoded a seven
transmembrane domain receptor in the G protein-coupled receptor superfamily. At
the same time, the first large-scale genome sequencing of C. elegans had begun at
Washington University and what is now the Sanger Center. Scanning this sequence
showed us that the C. elegans genome had many additional genes that were similar
to odr-10, defining a gene family. In parallel studies around the same time, graduate
students in my lab found other gene families encoding G protein-coupled receptors
in the worm genome, and showed that most of these genes were expressed very
specifically in just one kind of olfactory neuron. When the dust had cleared a few
years later, we knew that the C. elegans genome had about 2000 genes in these
receptor families, most of them olfactory-specific in expression. Conservatively, a
single worm may have 1000 different functional olfactory receptor genes. An
animals genome is both a set of instructions, and a record of history: it tells us what
kinds of genes have been important in the evolution of that animal. The genome of
C. elegans tells us that it has been so important to the worm to detect environmental
chemicals that it has dedicated 10% of its genes to this one problem.

The discovery of odr-10 and the other C. elegans receptor gene families echoed prior
work by Linda Buck and Richard Axel at Columbia University, who had discovered a
similarly vast family of olfactory receptor genes in rodents a few years earlier
1000 G protein-coupled receptors that are each expressed in a few mammalian
olfactory neurons. With so many molecules, it is hard to assign receptors to specific
odors. Therefore, the odr-10 mutant, based on an unbiased behavioral screen,
formed the first connection linking a specific odor diacetyl -- to a specific receptor
gene and the resulting behavior of the animal. Subsequent studies in flies, rodents,
and even humans have reinforced the conclusion that each olfactory receptor
protein detects specific chemicals in the environment, and transmits that
information to the olfactory neurons and ultimately to the brain.

Having the functional receptor for a specific odor, in a genetically tractable animal,
provided tools to ask further questions about the logic of olfactory behavior. ODR-
10s activation by diacetyl explains how C. elegans can detect this chemical, but why
does C. elegans find diacetyl attractive rather than neutral or repulsive? TO explore
this question Emily Troemel took an odr-10 mutant that did not respond to diacetyl,
and restored odr-10 with targeted transgenes. Instead of expressing odr-10 in the
AWA neuron, its normal location, she expressed it in the AWB neuron, which detects
repulsive odors. The resulting animals detected diacetyl, but instead of approaching
it they avoided it, the behavior appropriate for the sensory neuron that now
expressed the receptor. The ability to rewire the connection between diacetyl and
behavior showed that individual sensory neurons are not neutral collectors of
sensory information: they can encode characteristic behavioral outcomes such as
attraction or repulsion. At the extreme, anything that activates AWA might drive
attraction, and anything that activates AWB might drive repulsion.

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These results indicate that the decision about which behavior to generate is encoded
by the first C. elegans neuron that detects an odor, and imply that there is a sensory
prepattern for innate preferences. Subsequent results from Charles Zuker and Nick
Rybas labs at UCSD and the NIH suggest that a similar prepattern underlies
mammalian taste preferences. They showed that different taste cells in the
mammalian tongue sense sweet and bitter tastes, and then showed that expressing a
foreign receptor transgene in a sweet taste cell causes a mouse drink the foreign
ligand, and expressing the same receptor in a bitter taste cell cause the mouse to
reject the foreign ligand. Thus mouse taste preferences can be specified by the
pattern of transgene expression, just like worm odor preferences. An innate
prepattern allows a nave animal to accept nutritious sugar and avoid bitter toxins
the first time they are encountered, a suite of innate behaviors that are useful to all
animals.

If sensory neurons encode different behavioral outcomes, then different sensory

neurons must have distinct connections with other neurons in the brain. In C.
elegans, the existence of Whites complete synaptic wiring diagram allows us to
trace the connections from sensory neurons to their target neurons, and ultimately
to the motor output. Using quantitative behavioral assays, Jesse Gray defined a
complete pathway for food search behavior from the sensory neuron that detects
attractive odors, to interneurons that integrate sensory information and issue motor
commands, to the motor neurons that control specific turns and reorientation
maneuvers in chemotaxis. The path from a sensory input to a motor neuron is
usually indirect, with one or two intermediate layers of processing. Although
shallow, the path is not simple; after information leaves the sensory neurons, it is
distributed across a broader set of processing networks, not segregated into a single
sensory-to-motor stream. As a result, one sensory cue can regulate multiple actions,
each of which can promote the neurons overall connection to attraction or
repulsion.

The functional connections between the sensory neurons and their multiple targets
were elucidated by Sreekanth Chalasani, using genetically encoded calcium
indicators and microfluidic imaging chambers developed by Nikos Chronis.
Sreekanth observed the activity of the olfactory neurons and interneurons in real
time, and mapped the molecules that act at excitatory and inhibitory synapses in the
circuit. In addition to characterizing the basic properties of the neurons, these
experiments showed directly what we had begun to infer from indirect behavioral
experiments: these functional properties could change based on experience.
Indeed, the flow of olfactory information could be regulated as early as the primary
olfactory neuron, and also at later steps. This conclusion extends rather than
contradicting the idea of an innate prepattern; it shows that the nave prepattern
can be a substrate for learning, allowing an animal to tune its behaviors to its own
experience. The fact that learning and plasticity occur in a tiny worm with a lifespan
of a few days shows how fundamental these processes are to the nervous system.
Learning and plasticity are not secondary features of complex nervous systems; they

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are primary processes that every animal uses to predict the future based on the
past.

Variation within and between individuals is a hallmark of behavior, and every

student of animal or human behavior is struck by its variability. Learning is one
source of variability, and a second source is genetic differences between individuals.
As genome sequencing has accelerated, it has revealed just how much sequence
variation exists within any one species. Estimates of human variation and C. elegans
variation are very similar; two individuals within each species have a genetic
difference, on average, about once in 1000 bases. Many of these sequence changes
are functionally silent, but some sequence changes lead to changes in outward
appearance, metabolism, or behavior. For example, different wild-type strains of C.
elegans show behavioral variation in an interesting aggregation behavior first
noticed by Randy Cassada in Freiburg. Whereas the standard laboratory strain, the
British strain N2, is largely solitary when feeding on a bacterial lawn, the German
strain RC301 and other wild strains aggregate into large feeding groups at the lawn
border. Aggregation is perhaps the simplest social behavior; it is observed in many
animals and even in unicellular organisms. It can provide advantages for
cooperative predation, defense, migration, or foraging, but it comes with costs like
food-sharing. In keeping with these potential tradeoffs, aggregation behavior is
regulated by factors like sex, season, or feeding state, and it is genetically plastic as
well closely related species vary greatly in their preference for solitary versus
collective behaviors.

This flexibility and plasticity of aggregation behavior made it interesting as a subject

of further characterization. Aggregating mutants could be isolated after a
mutagenesis of the laboratory N2 strain, and when Mario de Bono was in the lab he
cloned one such mutant, npr-1. npr-1 encodes a G protein-coupled neuropeptide
receptor related to mammalian neuropeptide Y receptors and fly neuropeptide F
receptors; knocking it out stimulates aggregation, indicating that it normally
promotes solitary behavior. This mutant and the affected gene then led to an
understanding of the behavior of the wild social strains. The npr-1 gene in wild
social strains differed from npr-1 in the solitary strain at a single amino acid residue.
The npr-1 gene in social strains had lower activity in biological assays, indicating
that the sequence change was functionally relevant, and that npr-1 was a gene that
generated variability in social behavior.

Understanding social behavior and the role of npr-1 took a decade after this initial
discovery, leading along the way to a set of unexpected discoveries about the
sensory biology and the natural history of C. elegans. Leon Avery at UTSW and
David Tobin and Mario de Bono in my lab realized that social behavior was
enhanced under stressful conditions, and promoted by neurons that sense stress. A
set of convergent experiments from Mario de Bonos now-independent lab at the
MRC, and Jesse Gray in my lab and David Karow and Michael Marletta at Berkeley,
showed that the major stressor driving aggregation was atmospheric oxygen
which is much higher in the lab than in the decaying material that C. elegans

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normally favors. Patrick McGrath, studying another regulator of oxygen responses,
showed that the solitary npr-1 allele in our wild-type N2 strain actually arose in
the laboratory after the worms domestication, likely as an adjustment to the high
oxygen levels in the lab. Together, the results helped explain the environmental
context for aggregation, but left the circuit logic unsolved.

The circuit logic fell to Evan Macosko, who found the neurons that connected the
npr-1 gene to behavior. Carefully mapping the site of npr-1 action without
misexpressing it (a key detail), he identified the RMG neuron as the site that was
both necessary and sufficient for npr-1 to suppress aggregation. Moreover, killing
RMG with a laser suppressed aggregation in social strains, underlining the
importance of the neuron. RMG has a striking and unusual wiring pattern, with
relatively few chemical synapses, but an abundance of gap junctions (which in C.
elegans, as in humans, represent about 10% of all anatomical synapses). RMGs gap
junction partners include multiple sensory neurons, including all of the known
neurons that promote aggregation, and additional neurons that detect pheromones.
This wiring motif was sufficient to explain the elemental properties of aggregation
the coupling of stress-sensing neurons (such as the oxygen-sensing neurons) to the
detection of other animals, in this case their pheromones. Environmental regulation
of aggregation was neatly encapsulated in this RMG-hub-and-sensory-spoke wiring
motif, and RMG was also the site of genetic regulation of aggregation by npr-1 gene
variation. Genetic variation and environmental regulation converged on a common
neural circuit.

These genetic experiments and functional imaging of the circuit began to explain
how npr-1 prevented aggregation behavior. High npr-1 activity functionally
uncoupled the RMG circuit, so that the stress-sensing neurons were no longer
cooperating with pheromone-sensing neurons. Further analysis by Heeun Jang, in
collaboration with Kyuhyung Kim and Piali Sengupta at Brandeis, added an
interesting element to the circuit mechanism. Solitary animals do not just ignore
pheromones from other animals; they are actively repelled. A sensory neuron called
ADL mediates this repulsion, through chemical synapses. However, ADL is also an
element of the hub-and-spoke circuit for aggregation, and is itself a neuron that
promotes aggregation in social animals with low npr-1 activity. Thus ADL is coupled
to two antagonistic behaviors, repulsion from pheromones driven by its chemical
synapses, and aggregation through the gap junction circuit. Its functional
ambivalence is resolved into aggregation in the presence of environmental cues like
oxygen that drive hub-and-spoke neurons, and can also be resolved into repulsion
by the activity level of npr-1. Seen from the perspective of ADL rather than RMG,
npr-1 shapes the flow of information downstream of a sensory neuron, resolving
antagonistic behavioral outcomes.

The larger implication of this result is that it is impossible to read the C. elegans
wiring diagram and know the flow of information through its circuits without
knowing the status of neuromodulatory systems like npr-1. The wiring diagram is
important; the chemical and electrical synapses defined by electron microscopy are

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essential for rapid coordinated behaviors. Anatomic connections that are visible in
electron micrographs act locally and reliably. They can function within
milliseconds, leading to immediate depolarization and hyperpolarization.
Neuropeptides and the biogenic amine neuromodulators such as serotonin and
dopamine are less easily studied by neurophysiology and neuroanatomy than
classical synapses. They typically act slowly over seconds or minutes, the
biochemical timescales of their G protein-coupled receptors. They do not usually
depolarize or hyperpolarize cells strongly, but instead alter neuronal excitability or
cause neurons to release more or less transmitter when excited. They are released
extrasynaptically, so their functional connections are not visible by electron
microscopy. Only by knowing the expression patterns of the neuropeptide
receptors and the availability of the relevant neuropeptide can functional
connections be uncovered.

Without the neuropeptides, however, the anatomical wiring diagram is incomplete.

These slow molecules that act at a distance can reshape the functional connectivity
between neurons to sculpt the anatomical wiring diagram. In the case of ADL, the
functional changes can reverse a behavioral outcome. Thus the anatomical wiring
diagram is ambiguous, containing within itself the potential for different behaviors
based on the neuromodulatory state. This design creates flexibility in behavior,
allowing rapid remodeling based on stimulus history, internal cues, the activity of
distant circuits, and all of the other features that can regulate neuropeptide release.
However, the same design means that there is not one C. elegans wiring diagram, but
a number of overlapping wiring diagrams, of which a subset may be functionally
active at a given time.

The regulation of neuronal wiring diagrams by neuropeptides and other

neuromodulators is not a new observation. In the stomatogastric ganglion of crabs
and lobsters, neuromodulators can fundamentally change neuronal dynamics and
circuit composition, leading to changes in digestive rhythms. In the vertebrate
retina, neuromodulators help reorganize the switch between the overlapping neural
circuits that are active in dim light (rod-driven) and bright light (cone-driven). The
functional connections of motor circuits in the vertebrate basal ganglia and spinal
cord are modified by neuromodulators. These few examples may be only the
beginnings of behavioral regulation by neuromodulators that shape fast circuits.
Mammalian neuropeptides regulate social behaviors, hunger, thirst, sleep, stress
responses, pain, and other critical behavioral processes. Their patterns of
distribution in the brain and the signals for their release are only partly described,
and their effects on target neurons are even less well-characterized. If many brain
circuits are overlapping, flexible, and regulated by neuromodulators, like C. elegans
circuits, we have a large job ahead of us in mapping them. But at the same time, the
idea of flexible, reversible rewiring presents opportunities for understanding the
brain and behavior, and perhaps even for intervention in brain disorders. Partly
disabled pathways might be strengthened and overactive pathways suppressed by
targeting neuromodulation. While these ideas emerge from genetics, they are the

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opposite of the determinism that some fear in a genetic description of behavior;
instead, they emphasize the diversity and flexibility latent in all nervous systems.

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