state-dependency in c. elegans

State-dependency in C. elegans

J. C. Bettinger and S. L. McIntire*

Programs in Biological Science and Neuroscience, Gallo Center

and Department of Neurology, University of California, San Francisco,

UCSF School of Medicine, Emeryville, CA, USA

*Corresponding author: S. L. McIntire, Programs in Biological

Science and Neuroscience, Gallo Center and Department of

Neurology, University of California, San Francisco, UCSF School

of Medicine, 5858 Horton St. Suite 200, Emeryville, CA 94608,

USA. E-mail: [email protected]

Memory and the expression of learned behaviors by an

organism are often triggered by contextual cues that

resemble those that were present when the initial learn-

ing occurred. In state-dependent learning, the cue elicit-

ing a learned behavior is a neuroactive drug; behaviors

initially learned during exposure to centrally acting com-

pounds such as ethanol are subsequently recalled better

if the drug stimulus is again present during testing.

Although state-dependent learning is well documented

in many vertebrate systems, the molecular mechanisms

underlying state-dependent learning and other forms

of contextual learning are not understood. Here we

demonstrate and present a genetic analysis of state-

dependent adaptation in Caenorhabditis elegans.

C. elegans normally exhibits adaptation, or reduced

behavioral response, to an olfactory stimulus after

prior exposure to the stimulus. If the adaptation to the

olfactory stimulus is acquired during ethanol adminis-

tration, the adaptation is subsequently displayed only if

the ethanol stimulus is again present. cat-1 and cat-2

mutant animals are defective in dopaminergic neuron

signaling and are impaired in state dependency, indicat-

ing that dopamine functions in state-dependent adapta-

tion in C. elegans.

Keywords: Dopamine, ethanol, olfactory adaptation, state-

dependent learning

Received 23 January 2004, revised 12 April 2004, accepted

for publication 19 April 2004

Caenorhabditis elegans is able to modulate its olfactory

responses based on experience. Worms can detect numerous

volatile and soluble odorants that act as attractants or

repellents in chemotaxis assays (Bargmann & Horvitz 1991;

Dusenbery 1974; Ward 1973). Chemotaxis of a population of

animals is determined in these assays by creating a gradient

of an odorant from a point source, observing accumulation of

animals at the point source over time and quantifying the

strength of the behavioral response by calculating a chemotaxis

index (CI, Fig. 1) (Bargmann & Horvitz 1991; Colbert &

Bargmann 1995). Previous exposure to an odorant can result

in a diminished behavioral response to the odorant, a process

termed olfactory adaptation (Fig. 1) (Colbert & Bargmann 1995).

These adaptive responses occur in an odorant-specific manner.

Olfactory discrimination and adaptation may be important

for C. elegans to identify food sources in a complex natural

environment (Troemel 1999). If an odorant is omnipresent in a

given environment, it may not be useful as a cue to a food

source. Odorant-specific adaptation would allow C.elegans to

ignore an uninformative odorant or modulate its olfactory

responses based on experience.

Chemotaxis Index (CI) =

total number animals in assay

Pre-exposure Chemotaxisassay

CI

NO ODORANT

*high

ODORANT

*low

(adapted)

*

(a)

(c)

(b)

number at odorant – number at diluent

- origin- diluent- odorant

Figure 1: Olfactory adaptation. (a) Worms pretreated with no

odorant move toward a spot of attractive odorant (filled circle)

when tested in a chemotaxis assay. (b) Worms pretreated with a

high concentration of an attractive odorant (represented as a

plate with a heavy outer border) demonstrate olfactory

adaptation and will not move toward a spot of the odorant

when tested in a chemotaxis assay. (c) The chemotaxis index is

calculated by counting the number of animals immobilized at the

spot of odorant – the number of animals immobilized at the

diluent spot (open circle)/the number of animals in the assay.

Localized sodium azide is used to anaesthetize animals that

reach either the odorant or diluent.

Genes, Brain and Behavior (2004) 3: 266–272 Copyright # Blackwell Munksgaard 2004

266 doi: 10.1111/j.1601-183X.2004.00080.x

C. elegans is a good model organism for studying simple

learning behaviors and has been demonstrated to pair stimuli

in several different paradigms (Colbert & Bargmann 1995,

1997; Ishihara et al. 2002; Rankin 2000; Saeki et al. 2001;

Wen et al. 1997). We sought to determine whether or not

state-dependent modification of olfactory adaptation could

be observed after exposure to a drug. State-dependent

effects of ethanol are widely appreciated in vertebrate

systems (Goodwin et al. 1969; Lowe 1988; Overton 1966,

1972; Shulz et al. 2000), although the molecular mechanisms

underlying state-dependency are not understood. We

previously demonstrated a depressive effect of ethanol on

multiple behaviors of C. elegans (Davies et al. 2003). Similar

dose–response relationships were observed for the effects

of ethanol on locomotion and egg laying behaviors. The

intoxicating effects of ethanol occur at the same internal

tissue concentrations that cause intoxication in humans.

The similar depressive effects of ethanol on the behavior of

C. elegans and other organisms led us to ask if the simi-

larities in ethanol’s effects could generalize to its ability to

generate state-dependency.

State-dependent effects of ethanol on olfactory adaptation

were observed for multiple odorants. We analyzed state-

dependency in mutants that are defective in dopaminergic

signaling, and found that dopamine is required for the

generation of state-dependency in C. elegans. Neuroadaptive

processes in dopamine neurons have been linked to alcohol

dependence in vertebrate systems (reviewed in Weiss &

Porrino 2002).

Materials and methods

Nematodes were maintained as described (Brenner 1974).

Strains used were: Wildtype Bristol N2, cat-1(e1111),

cat-2(e1112), glr-1(n2461).

Chemotaxis and adaptation assays were performed as

described by Colbert and Bargmann (1995) with minor

modifications.

Chemotaxis assays

Chemotaxis assay plates were prepared as follows: 10 ml of

assay agar (2% agar, 5 mM KPO4 [pH 6], 1 mM CaCl2, 1 mM

MgSO4) were aliquoted into 10 cm Petri plates, and allowed to

dry overnight. The day of the experiment, all plates were dried

at 37 �C without lids for 1 h. If ethanol was to be used in the

experiment, the contents of one representative plate were

melted to determine volume, and 100% ethanol was added

to the desired final concentration (generally 200 mM). Adding

95% ethanol to the plates to the desired final concentration

generated similar results (data not shown). The plates were

sealed with parafilm, and ethanol was allowed to equilibrate

into the agar for 1.5–2 h. Animals were washed twice in S

Basal (0.1 M NaCl, 0.05 M KPO4 [pH 6], 5 mg/l cholesterol [in

100% ethanol]) and once in assay buffer (5 mM KPO4 [pH 6],

1 mM CaCl2, 1 mM MgSO4). A spot of diluted odorant (for

benzaldehyde, 1ml of 1:200 benzaldehyde:EtOH, for butanone

1ml of 1:1000 butanone:EtOH) was placed on one side of

the plate. Exactly opposite was placed a spot of diluent (1ml

EtOH). To each spot was added the anaesthetic sodium azide

(1ml of 1 M Na Azide) to immobilize the worms when they

reached a spot. Worms were considered to be at a spot

during counting if they were within 1 cm of the center of the

spot of odorant or diluent. Between 100 and 300 washed worms

were then placed onto a spot on the plate that was exactly

between the odorant and diluent spots and slightly off center

(see Fig. 1), and excess liquid was wicked off. After 1 h, worms

were counted, and a CI was calculated as follows: CI¼ (number

of worms at the odorant spot – number of worms at the diluent

spot)/number of worms in the assay. A high CI (close to 1)

indicates that the odorant acted as a strong attractant, a lower

CI indicates that the odorant was a less effective attractant or did

not act as an attractant.

Adaptation assays

Adaptation plates were prepared as follows: 10 ml of adapta-

tion agar (3% agar, 5 mM KPO4 [pH 6], 1 mM CaCl2, 1 mM

MgSO4) or assay agar (2% agar, 5 mM KPO4 [pH 6], 1 mM

CaCl2, 1 mM MgSO4) was aliquoted into 10 cm Petri plates,

and allowed to dry overnight. The day of the experiment, all

plates were dried at 37 �C without lids for 1 h, and the con-

tents of a representative plate of each type was melted to

determine volume. Ethanol was added to appropriate plates

to the desired concentration, the plates were sealed with

parafilm and allowed to equilibrate for 1.5 h. Odorant was

aliquoted onto 5 agar plugs on the lid of adaptation plates (for

benzaldehyde adaptation, 2ml of 100% benzaldehyde was

used; for butanone, 5ml of 100% butanone was used). Typ-

ically, in our hands, butanone adaptation generated lower CIs

than we generally generated to benzaldehyde. This occurred

when animals were treated in the presence or absence of

ethanol, indicating that this feature was intrinsic to butanone

adaptation, and did not reflect a combination of effects of

butanone and ethanol. Animals were washed twice in S

Basal (0.1 M NaCl, 0.05 M KPO4 [pH 6], 5 mg/l cholesterol

[in 100% ethanol]) and once in assay buffer (5 mM KPO4

[pH 6], 1 mM CaCl2, 1 mM MgSO4), then placed on an adap-

tation plate, and the plate was sealed with parafilm. Animals

were incubated in all adaptation conditions for 90 min, then

washed twice with S Basal and once with assay buffer and

placed on chemotaxis plates. Chemotaxis was allowed to

proceed for 1 h, worms were counted, and a CI was calcu-

lated. Adaptation was observed as a decrease in chemotaxis

to an odorant after pretreatment with the odorant. Animals

were considered to have adapted if the CI (adapted with no

drug treatment, tested in the absence of drug) was less than

0.6. In all experiments in which animals were pretreated in

the same way but tested in different conditions, populations

were treated on a single pre-exposure plate, then washed

and the population was divided only when it was placed onto

different chemotaxis assay plates (Fig. 2).


Genes, Brain and Behavior (2004) 3: 266–272 267

Data analysis

The strength of olfactory adaptation in our experiments var-

ied considerably from day to day. When we used all data

from all experiments we found statistically significant state-

dependency (Tukey post hoc test: P< 0.05). However, we

found that the degree of olfactory adaptation strongly

impacted the expression of state dependency, such that

our olfactory adaptation assays naturally fell into two distinct

classes. We observed a natural demarkation between the

behavior of animals. On days when adaptation yielded very

low CIs (less than 0.35), we noted that state-dependency was

never total (see Table 1). On days when olfactory adaptation

was more modest (CIs between 0.4 and 0.6), expression of

state-dependency was more complete, such that there was

no evidence of adaptation when animals were tested in the

absence of ethanol (Fig. 2c). Thus, we chose to analyse the

data in two groups: All experiments that yielded CI (adapted

with no drug treatment, tested in the absence of drug)< 0.35

were considered to be ‘overadapted’, and were grouped

together for analysis. The results of these experiments are

shown in Table 1. All experiments that yielded CI (adapted

with no drug treatment, tested in the absence of drug)

0.35<CI< 0.6 were grouped together for analysis. The

results of these experiments are shown in Fig. 2. For all sub-

sequent analysis, we chose to use conditions that generated

complete state-dependency in the wild-type control groups.

Statistics

When only a pairwise comparison was made, we used two-

tailed t-tests. When more than two treatments were com-

pared, we used a one-way ANOVA with Tukey post hoc tests.

Results

We first characterized the acute effects of ethanol intoxica-

tion on chemotaxis. Although moderate slowing of locomo-

tion was observed at an exogenous dose of ethanol of

200 mM, chemotaxis to the volatile odorant benzaldehyde

Pre-exposure Chemotaxisassay

Chemotaxisindex

Adapted?

(0.80 ±0.04)Ethanol

–

(0.89 ±0.01) –(a)

Ethanol(0.72 ±0.08)

Ethanol –

(0.83 ±0.12) –(d)

EthanolBenzaldehyde

(0.44 ±0.04)Ethanol

+

(0.84 ±0.03) –(c)

Benzaldehyde

(0.55 ±0.05) +

(0.40 ±0.12)Ethanol +

(b)

Withdrawal

Ethanol

(0.51 ±0.08) +EthanolBenzaldehyde

(e) 35’(0.76 ±0.04) –

NS

†

*

NS

NS

NS

*

*

*

*

Figure 2: State-dependent olfactory

adaptation. Animals were treated for

90 min as described and then the

population was divided and half were

tested in the absence (upper plate) and

half in the presence (lower plate, yellow) of

ethanol for chemotaxis to benzaldehyde

(blue dot). (a) Pretreatment with no odorant

or drug causes no effect on chemotaxis to

benzaldehyde. (b) Benzaldehyde pre-

treatment (outer blue border) causes

olfactory adaptation when tested in the

presence or absence of ethanol. (c)

Pretreatment with benzaldehyde and

ethanol causes olfactory adaptation to be

demonstrated only when animals are

tested on ethanol. This treatment does

not increase sensitivity to benzaldehyde

when animals are tested in the absence of

ethanol (not shown). (d) Ethanol

pretreatment does not significantly impair

chemotaxis to benzaldehyde (n¼8). (e)

Prolonged withdrawal from ethanol

between pretreatment and testing does

not eliminate state-dependency of

olfactory adaptation (n¼8). * Significantly

different as tested by one-way ANOVA with

Tukey post hoc tests. † Significantly

different as tested by t-test. NS, not

significantly different.

Bettinger and McIntire

268 Genes, Brain and Behavior (2004) 3: 266–272

was not inhibited by this treatment (Fig. 2a). As a result, we

could analyze the effect of ethanol on olfactory adaptation.

Adaptation to benzaldehyde is normally observed as a

decrease in the chemotaxis response of the animals to the

odorant after a 90-minute period of pre-exposure to a high

dose of this attractant (Fig. 2b) (Colbert & Bargmann 1995).

In order to determine whether or not ethanol is capable of

producing state-dependent effects, we treated animals with

ethanol during pre-exposure to benzaldehyde and subse-

quently tested them in the presence or absence of ethanol

(Fig. 2c). No benzaldehyde adaptation was observed when

these animals were tested in the absence of ethanol. How-

ever, if the animals were tested in the presence of ethanol,

normal benzaldehyde adaptation was evident, indicating that

ethanol exposure during pretreatment did not prevent the

animals from adapting to benzaldehyde. Rather, these data

suggest that the olfactory adaptation is state-dependent: if

ethanol is present during the adaptation phase then it must

be present during the chemotaxis phase for the learning to

be recalled. We noted that the state-dependency we

observed is asymmetric; ethanol treatment only during the

chemotaxis assay after adaptation had no significant effect

on the degree of adaptation demonstrated (Fig. 2b). Asym-

metry in state-dependency has also been observed in mam-

malian models of state-dependent learning (Overton 1987).

We sought to exclude other possible explanations for

these observations. A prolonged exposure to ethanol in the

absence of benzaldehyde did not itself result in an apparent

adaptation to benzaldehyde or a decrease in chemotaxis

(Fig. 2d). Sequential rather than simultaneous pre-exposures

to ethanol and benzaldehyde also failed to produce state-

dependency (for 20 minute ethanol exposure followed by

90 minute benzaldehyde exposure CI [benzaldehydeþethanol]¼ 0.35� 0.18, CI [benzaldehyde – ethanol]¼ 0.18

� 0.29 P > 0.05 [n¼ 3]; for 90 minute benzaldehyde expo-

sure followed by 20 minute ethanol exposure CI [benzald-

heydeþ ethanol]¼ 0.32� 0.07, CI [benzaldehyde – ethanol]

¼ 0.47� 0.09 P > 0.05 [n¼ 14]), suggesting that the olfactory

stimulus must be presented simultaneously with the drug in

order for the adaptive response to the odorant to become

state-dependent.

Benzaldehyde olfactory adaptation has been reported to be

disrupted following a strong centrifugation that appears to

act as a dishabituating stimulus (Nuttley et al. 2001). We

asked if the dependency of olfactory adaptation on continued

ethanol treatment could reflect a disruption of olfactory

adaptation where withdrawal from ethanol constitutes a

non-specific dishabituating physiological stress. In the course

of the state-dependent learning experiments, worms were

washed in the absence of ethanol between the adaptation

and chemotaxis steps for no longer than 15 min, so any

withdrawal shock must occur within this interval. We tested

for a withdrawal effect by allowing for a thorough 35 minute

withdrawal from ethanol between the pre-exposure period

and the subsequent chemotaxis assay (Fig. 2e). As before,

adaptation was not expressed unless the animals were

treated with ethanol during the subsequent chemotaxis

assay period. This result suggests that the dependence on

ethanol reflects an association of olfactory adaptation with

ethanol rather a shock response to withdrawal from the drug.

Does the ethanol dependency of olfactory adaptation

reflect the intoxicating actions of ethanol or other stimulus

properties of the compound? Ethanol itself can act as a weak

attractant in chemotaxis assays (Bargmann & Horvitz 1991).

Table 1: State-dependency in wild-type and mutant animals, and effects of overtraining on state-dependency in wild-type animals

Chemotaxis Indices (CIs) (�SEM)

Strain N2 N2 N2 glr-1 AMPA

receptor

cat-2 TH cat-1 VMAT N2

Odorant Benz. Benz. But. Benz. Benz. Benz. Benz., overadapted

n¼8 n¼ 3 n¼ 11 n¼3 n¼7 n¼ 5 n¼ 48

EtOH pre-exposure

concentration (mM)

200 50 200 200 200 200 0 200

CI off ethanol 0.84 0.39 0.71 0.83 0.44 0.23 0.13 0.37

(� 0.03) (� 0.03) (� 0.06) (� 0.02) (� 0.06) (� 0.07) (� 0.03) (� 0.03)

CI on ethanol* 0.44 0.46 0.29 0.55 0.37 0.23 � 0.02 0.18

(� 0.04) (�0.02) (� 0.05) (� 0.02) (� 0.07) (�0.06) (�0.03) (�0.04)

Benz., benzaldehyde; But., butanone; n¼ number of experiments.

All values for chemotaxis tested on and off ethanol for animals pre-exposed to an odorant are significantly different from each other (P< 0.05)

except for N2 pre-exposed to benzaldehyde and 50 mM ethanol, and cat-1 and cat-2 animals pre-exposed to benzaldehyde and 200 mM ethanol.

All numbers except overadapted were tested by t-test. Overadapted numbers were tested by ANOVA with Tukey post hoc tests (see Materials

and methods).

Values for overadapted animals are significantly different for animals adapted on ethanol and off ethanol tested off ethanol, and for animals

adapted on ethanol and off ethanol tested on ethanol as tested by ANOVA with Tukey post hoc tests (P<0.05).

* Concentration of ethanol was the same for pre-exposure and for chemotaxis assay when ethanol was present during the chemotaxis assay.



We tested an exogenous dose of ethanol (50 mM) that is

insufficient to induce intoxication in C. elegans but acts as a

volatile odorant (Davies et al. 2003). No effect on olfactory

adaptation was observed (Table 1), suggesting that the rele-

vant ethanol stimulus is its ability to change intoxication

state, not its odorant properties. Doses of ethanol higher

than 200 mM were also tested and shown to generate

state-dependency (400 mM, data not shown), although

these doses also produced greater impairment of locomotion

which made the experimental results more difficult to

interpret. The doses of ethanol required for generating

state-dependent effects correlated with doses required for

producing the neurodepressive effects, but not with lower

doses that act only as olfactory stimuli.

State-dependency should result in a change in response to

olfactory cues that are specifically paired with ethanol during

the pre-exposure period rather than a generalized change in

olfactory response. The volatile odorant butanone is sensed

by the two AWC neurons, the same chemosensory neurons

that sense benzaldehyde (Bargmann & Horvitz 1991). We

demonstrated that state-dependency is not unique to benzal-

dehyde adaptation by showing state-dependent effects of

ethanol on butanone adaptation (Table 1). Although benzalde-

hyde and butanone are sensed by the same neurons, adap-

tation to these odorants is odorant-specific; that is,

adaptation to benzaldehyde does not result in a change in

response to butanone. State-dependency also appears to be

odorant-specific. Animals pretreated with benzaldehyde and

ethanol do not show significant adaptation to butanone, with

or without ethanol treatment during the chemotaxis assay

(after adaptation to butanoneþ ethanol, CI [butanoneþethanol]¼ 0.29� 0.05 [n¼ 11]; after adaptation to benzalde-

hydeþ ethanol, CI [butanoneþ ethanol]¼ 0.59� 0.03;

P< 0.001 [n¼ 7]). While these results do not rule out

the possibility that there is some small general effect of

combined odorant and ethanol treatment on AWC function,

they do preclude the possibility that state-dependency is due

to overall AWC dysfunction.

During the course of these experiments, we noted that the

degree of state-dependency we saw was strongly affected

by the degree of olfactory adaptation we induced. In experi-

ments in which animals responded extremely strongly to the

pre-exposure stimulus such that they lost all attractive

response to the odorant (CI< 0.35), state-dependency was

significant but less robust (Table 1). In these cases, animals

still showed significantly stronger adaptation when tested in

the presence of ethanol, but adaptation was also observed in

the absence of the drug. These results indicate that olfactory

adaptation is not entirely state-dependent under these con-

ditions, and that the animal can completely modify its

response to an odorant state-dependently only if the odorant

cue is not overwhelmingly large. This characteristic of state-

dependency is shared by classic state-dependent learning

paradigms; overtraining of the learned response appears to

abolish state-dependent learning in mammals (Overton 1987).

In other systems, changes in dopaminergic function have

been implicated in drug dependent behavioral changes and

cue-conditioned responses (Berke & Hyman 2000). We ana-

lyzed mutants with known abnormalities in dopaminergic

function to determine if dopamine is required for state-

dependency. cat-1 mutants are defective in the monoamine

vesicular transporter that packages dopamine and serotonin

into synaptic vesicles (Duerr et al. 1999). cat-2 animals are

deficient in the synthetic enzyme for dopamine, tyrosine

hydroxylase (Lints & Emmons 1999). Norepinephrine and

epinephrine, both of which are enzymatically derived from

dopamine, have not been identified as neurotransmitters in

C. elegans (Riddle et al. 1997). cat-1 and cat-2 mutant ani-

mals exhibit wild-type sensitivity to ethanol and show normal

chemotaxis and adaptation to benzaldehyde (for cat-2: CI

[benzaldehyde]¼ 0.95� 0.03 [n¼ 4], after adaptation to ben-

zaldehyde CI [benzaldehyde]¼ 0.48� 0.07 [n¼ 4]; for cat-1:

CI [benzaldehyde]¼ 0.88� 0.04 [n¼ 5], after adaptation to

benzaldehyde CI [benzaldehyde]¼ 0.23� 0.07 [n¼ 4]). Intri-

guingly, both cat-1 and cat-2 animals are deficient in the

state-dependent effect. Treatment of these mutant animals

with ethanol during the benzaldehyde pre-exposure period

had no significant effect on subsequent adapted responses

to benzaldehyde observed in the absence of ethanol

(Table 1). Abnormalities in state-dependency were not

observed in animals deficient in glr-1, an AMPA type gluta-

mate receptor subunit, that is known to play a role in another

nematode learning paradigm, habituation to tap (Hart et al.

1995; Maricq et al. 1995; Rose et al. 2003) (Table 1). The

abnormalities in cat-1 and cat-2 animals indicate a role for

dopamine in the generation of state-dependency in C. elegans.

Discussion

The finding of state-dependent effects on olfactory adapta-

tion indicates that C. elegans is able to modify its response to

an olfactory stimulus in the presence of a drug stimulus. Pre-

exposure to an odorant and ethanol results in an ethanol-

dependent change in the olfactory response to the odorant.

The recall of the adaptation, or the expression of the changed

response to the odorant, requires the same ethanol stimulus

that was present when the adaptation to the odorant

occurred. If the odorant is presented in the absence of the

associated ethanol stimulus, the animal exhibits no decrease

in response to the odorant, and acts as if it had not encoun-

tered the odorant previously.

The development of state-dependency is distinct from pre-

viously established associative learning phenomena in worms.

Nuttley et al. (2002) showed that worms can form an associ-

ation between olfactory adaptation and the absence of food.

Rather than a simple association between olfactory adaptation

and the presence of ethanol, we have observed an association

between olfactory adaptation and an internal state of intoxica-

tion. The presence of a subintoxicating dose of ethanol is insuf-

ficient to generate this association. The state-altering properties



of ethanol act as the stimulus recognized by the worm, and this

change in internal state is what is being used as a cue in the

development of the association with olfactory adaptation.

The association of olfactory adaptation with ethanol

appears to require dopaminergic function. Dopamine is

found in only eight cells in C. elegans. No direct synapses

exist between the chemosensory neurons (in which olfactory

adaptation is presumed to occur) and the dopaminergic neu-

rons of C. elegans (Ward et al. 1975). Thus, the role for

dopaminergic input suggests that state-dependency is not

due solely to a direct effect of ethanol on the AWC neurons,

but requires activation of a neural circuit.

Dopamine may play a role in learning in other systems.

Midbrain dopaminergic neurons respond to natural rewards

such as food and liquid as well as to conditioned reward-

predicting stimuli (Schultz 1998). In vertebrate learning para-

digms, dopaminergic neurons are active during the early

learning phase of conditioned reinforcement, but cease to

respond as the predicted stimuli and reward are repeated

(Hollerman & Schultz 1998). These experiments have led to

the hypothesis that dopamine could act during learning by

signaling errors in the prediction of reward. A selective

increase in dopamine release has also been demonstrated

in the nucleus accumbens when an association is formed

between two stimuli, neither of which independently affects

dopamine release (Spanagel & Weiss 1999; Young et al.

1998). Hence, dopamine may more generally modulate learn-

ing that does not involve reinforcement. These experiments

indicate that dopaminergic neuron activity correlates with

learned behavioral changes. Our results now reveal that

genetic elimination of dopaminergic function disrupts a

simple form of learning in C. elegans.

Worms are able to modify their responses to stimuli by

developing contextual associations in other paradigms.

Rankin has shown that the habituation to tap response can

be modified by the context in which the habituation regimen

occurs (Rankin 2000). Worms will respond to a tap by moving

away from the stimulus, but this response habituates in

response to repeated tapping. If the habituation occurs in

an environment with a contextual cue (an odorant), the habitu-

ation is strengthened if tested in the presence of the cue.

This is somewhat reminicent of the development of

state-dependency to olfactory adaptation, and may reflect a

commonality in C. elegans between these two behaviors.

Worms may have to signal salience of particular previously-

encountered stimuli in their complex natural environment,

and this could be accomplished by development of associa-

tions, represented, in these cases, as the development of

associations between stimuli and external (contextual) cues

or internal (state) cues. The mechanisms of the development

of these associations may not overlap at the molecular level

because these two behaviors do differ in their requirements

for neurotransmitters. The glutamate receptor glr-1 is

required for long-term habituation to the tap response

(although it was not required for short-term habituation to

the tap response) (Rose et al. 2003) but not for olfactory

adaptation. Furthermore, dopamine is required for state-

dependency and has not been reported to affect the contextual

conditioning paradigm.

The effect of ethanol on olfactory adaptation in C. elegans

has provided an opportunity to study the mechanisms under-

lying state-dependency in this genetically manipulable model.

Previous studies of state-dependent learning have not eluci-

dated the molecular mechanisms that are responsible for the

generation of state-dependency. State-dependency may be

important in addictive behavior. Chronic drug use and alco-

holism are frequently linked to neuroadaptive processes

within the dopaminergic system (Berke & Hyman 2000;

Koob & Le Moal 1997; Phillips et al. 1998; Rubinstein et al.

1997; Spanagel & Weiss 1999). Ethanol alters the levels of

dopamine in specific brain regions in mammals, and alterations

in dopamine levels are thought to be important for the addictive

properties of ethanol (reviewed in Weiss & Porrino 2002).

In both vertebrates and C. elegans, intoxicating doses of

ethanol are required for state-dependent effects. State-

dependent learning provides a mechanism whereby behav-

ioral changes evolving during drug exposure become at

least partially dependent on the drug for continued expres-

sion. Such a mechanism could contribute to the addictive

properties of ethanol and other drugs of abuse.

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Acknowledgments

We thank members of the McIntire and Bargmann laboratories

for helpful discussions and comments on the manuscript, and

Catharine Eastman for technical assistance. Strains used were

provided by the Caenorhabditis Genetics Center (funded by the

NIH National Center for Research Support). Support was provided

by the State of California for medical research on alcohol and

substance abuse through UCSF, and by the NIAAA, NIH (J.C.B).



state-dependency in c. elegans

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