olfactory plasticity in Caenorhabditis elegans: a separation of adaptation and habituation

NIRIT BERNHARD

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Anatomy and Ce11 Biology University of Toronto

O Copyright by Nirit Bernhard 1999

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OLFACTORY PLASTICITY IN CAENORHABDrrIS ELEGANS: A SEPARATION OF ADAPTATION AND HABITUATION

Master of Science 1999

NIRIT BERNHARD

Department of Anatomy and Ce11 Biology University of Toronto

Abstract

Continuous presentation of an oifactory stimulus causes a chemotaxis response

decrement in the nematode Cuenorhabditis eelegans, but the differences between the

leaming process of habituation (a reversible decrease in behavioural response) and other

olfactory plasticity such as adaptation (a decrement in response due to sensory/motor

fatigue, which c m o t be dishabituated) have not been addressed. Using the volatile

odorant diacetyl (DA) 1 assessed the distinct processes of olfactory adaptation and

habituation. Pre-exposing and testing worms to 100% DA caused a chemotaxis

decrement which was not reversible despite the presentation of potentially

dishabituating stimuli. This DA adaptation is abolished in odr-10 mutants but remains

intact in odr-1 mutants. Although DA pre-exposure to intermediate concentrations of DA

(0.01% and 25%) produced no chemotaxic response decrement, pre-exposure to low DA

concentrations (0.001%) produced either a dishabituable response decrement

(habituation) or sensitization. The distinct behavioural effects o b s e ~ e d after DA pre-

exposure highlight a concentrationdependent dissociation between adaptation and

habituation.

1 would to thank rny supervisor, Derek van der Kooy, for al1 his tirne, patience and advice over the past couple of years. From you 1 have gained a new appreciation for the critical scientific mind, leamed about the advantages of thinking laterdy and being BRIEF. Thank you for expanding my ability to use the words: shockhg, pathetic, pitiful and dandy. 1 would &O like to thank Mike Wiley for king an inspiring teacher and helpful graduate student cooridinator and the members of my supervisory committee, Marc Perry, h Shettleworth, and especiaily Marla Sokolowski for all the staüstics help.

Throughout these advenhirous two years in the windowless basement of MSB (a.k.a. the Zombie Zone) I have seen many people corne and go (some permanently). 1 thank al1 the lab rats who made every day memorable. Firstly, I would like to thank the excellent technical support that has been the backbone of the worm project: Sue - you the woman! Thanks for your wit and good humour, and for listering to me as I cut the parafilm. Rachel, Nilo and Jim, thanks for all yow help. I'd also like to thank Nadine Livaya and Inge Marge for their contributions to UUs project. nianks to Bill for the interesting worm discussions with a molecular twist. Glenn, thanks for putting up with all of my stupid questions - your help has been invaluable, and your patience inspirational. Of course, I am also indebted to: Brenda - for the smiles and stupid e- mails; Danka - for the smiles and the stories; Hance - for king so unorganized and teaching me the significance of the Benjamins; Catherine - for providing constant background stories; Steve - for being a source of cwiosity; Dave-Bob - for being out, about and in-my-face; Raewyn - for being yourself; Cindi - for bringing in the sunshine, for your advice and understanding about what a schlep life can be sometimes; Colleen - for being so sweet, understanding and a constant source of exercise and culinary knowledge and Vince - for everything. Cuesta bella persona - the wind at my back - every moment has k e n memorable. Thank you so much for the stimulating discussions, support, advice, incredible patience and understanding, and for being there.

Lastly, and most importantly, thank-you Mom, Dad, Roy, Eric, Igael, Beni and family, Bali Dora and all of the rest of my family, extended family and wonderfd friends (especially H.N., R.R. and A.B.) - you have been so supportive, understanding, caring and patient. Thankyou justdoesn'tsayenough, but it'sall Icancomeupwith. It's been a long ride, prepare for part II of the adventure!!

Abs tract

Acknowledgments

Figure Index

Introduction

Methods

Results

Discussion

Bibliography

TABLE OF CONTENTS

Page

iii

LIST OF FIGURES

Figure 1: Habituation/ Adap tation Apparatus

Figure 2: DA Dose Respowe Curve

Figure 3: tncreasing exposure time, but not volume, affects degree of approach decrement after pre-exposure to 100% DA.

Figure 4: Lack of dishabituation after pre-exposure to 100% DA.

Figure 5: No response decrement seen after exposure to intermediate DA concentrations (25%, 0.01 %)

Figure 6: Nonassociative learning occurs after pre-exposure to low concenhations of DA (0.001%)

Figure 7: Adaptation is odr-10 dependent, but odr-1 independent.

Figure 8: Adaptation, Habituation and Sensitization are separate forms of olfactory plasticity.

Page

13

17

20

INTRODUCTION

Introduction

Our understanding of the mechanisms underlying some forms of learning and

memory have been greatly facilitated by the use of invertebrate mode1 systems.

Extensive studies on associative and nonassociative learning have been camed out in

molluscs, such as Aplysia californica (Castelucci et al. 1970; Carew and Sahley 1986;

Walter et al. 1979, 1981) and Hennissenda crassicomiss (Rogers and Matzel, 1995), as

well as in other invertebrate species such as Drosophila melanogaster (Corfas and Dudai

1988; Qullui et al. 1974; Tully and Quinn 1985) and Apis melli@ra camica (Braun and

Bicker 1992; Hammer 1997; Hellerstern et al. 1998). More recently, the nematode

Caenorhabditis elegans has been identified as a useful organism for the study of

learning and memory (Hedgecock and Russell 1975; Morrison et al. 1999; Rankin et

al. 1990; Wen et al. 1997). C. elegans has a relatively simple nervous system,

composed of 302 neurow whose synapses have been fully mapped out at the electron

microscopic level (Hali and Russel 1991; White et al. 1986). Combined with its

detailed physical map and a nearly completely sequenced genome (C. elegans

Sequencing Consortium 1998; Sulston et al. 1992), C. elegans is well suited for

ident@ing specific molecular pathways and genes that play a role in learning,

memory and other forms of behavioural plasticity.

The search for molecular and genetic components of learning and memory in

any organisrn must first begin with a clear definition of the different types of

learning being investigated and distinguishing between leaming and other forms of

plasticity . Different temporal relationships between stimuli during training d o w s

for classification of two elementary types of leaniing: nonassociative and associative

3

learning (Byrne et al. 1987). Whereas associative learning involves a change in

behaviour due io specific temporal contingencies between stimuli or between a

stimulus and a behavioural response, nonassociative learning involves modification

of a behaviour due to presentation of a single cue (Brown 1998; Carew and Sahley

1986). Types of nonassociative learning include sensitization, habituation and

dishabituation. Sensitization refers to the enhancement of a behavioural response

caused by a strong and often noxious stimdus (Groves and Thompson 1970).

Habituation is the decrement in response (independent of sensory/motor fatigue or

adaptation) due to repeated or continuous presentations of a single stimulus (Groves

and Thompson 1970; Harris 1943; Thompson and Spencer 1966). On the other hand,

dishabituation is the restoration from an habituated respowe following presentation

of a novel or noxious sthnulus, which has been hypothesized to be a superimposition

of sewitization on the habituated response (Groves and Thompson 1970) or may

involve different processes from sensitization (Cohen et al. 1997). The re-

establishment of baseline response levels due to a dishabituating stimulus is distinct

from a spontaneous recovery of the habituated respowe, which occurs without a

dishabituating stimulus after a prolonged period of time (Thompson and Spencer

1966).

Harris (1943) and 'Thompson and Spencer (1966) suggest that continuous or

repeated stimulus presentation leading to sensory adaptation can be caused by

decreased receptor activity (receptor adaptation) or limitations of effector response

(efictor fatigue). Respowe decrements that can be accounted for by either of these

mechanisms are not referred to as habituation, since they indicate an inability to

respond to the stimulus as opposed to an active learning associated modulation in

4

behaviour. There are examples of newoadaptation within the central nervous system

(Ochoa et al. 1990), as well as in sensory systems such as the mechanosensory (Corfas

and Dudai 1990), visual (Dizhoor et al. 1991; Kawamura and Murakami 1991) auditory

(Rauschecker and Korte 1993) and olfactory systems (Chen and Yau 1994; Dawson et

al. 1993; Kramer and Siegelbaum 1992; Kurahashi and Menini 1997; Leinders-Zufall et

al. 1999). While the olfactory studies f o w mainly on the cellular changes that lead to

modulations in sensory transduction, they do not assess the behavioural processes

that underlie the changes in olfactory perception leading to olfactory behavioural

plas ticity .

The ability of C. elegans to sense, approach and discriminate between volatile

odorants has been shown to be manipulable at the cellular and genetic levels

(Bargrnann et al. 1990; Bargmann et al. 1993; Colbert and Bargmann 1997). Laser

ablation studies have identified the AWA and AWC primary chemosensory neurons

as responsible for mediating the olfaction to specific odorants, while ethyl

methanesulfonate mutagenesis of wild-type Worms has identified novel genes

involved in olfaction (Bargmann et al. 1993; Colbert and Bargmann 1995; Sengupta et

al. 1996). The AWA neuron has been shown to mediate approach to pyrazine as weU

as lower concentrations of diacetyl (Bargmann et al. 1993), and the odr-10 gene which

encodes a seven trammembrane domain diacetyl receptor is expressed in AWA

(Sengupta et al. 1996; Troemel et al. 1997; Zhang et al. 1997). In addition, odr-3 (a Ga-

protein subunit proposed to be involved in dowwtream intracellular signaling) and

osm-9 (a putative ion channel with some homology to the Drosophila transient

receptor potential protein) have been identified and behaviourally characterized as

affecting AWA olfactory h c t i o n (Colbert et al. 1997; Roayaie et al. 1998). Laser and

5

genetic ablations of the AWC neuion indicate that this neuron mediates responses to

odorants such as benzaldehyde, butanone and isoamyl alcohol (Bargmann et al. 1993).

In addition to the high-affinity odr-IO diacetyl receptor on AWA, there appears to be a

putative low-affinity diacetyl receptor on AWC which mediates response to high

concentrations (100%) of diacetyl and 2,3-pentanedione (Chou et al. 1996). This low-

affinity DA receptor may signal to a downstream G-protein coupled receptor via the

candidate receptor guanylyl cyclase, odr-1 (N. L'Etoile and C. Bargmann 1997, penonal

communication) and lead to the opening of a cyclic nucleotide gated cation channel,

such as the tax-2/tax4 channels (Coburn and Bargmann 1996).

In C. elegans, habituation to tactile st imul i (Chalfie and Sulston 1981; Croll

1975; Rankin et al. 1990) and chemosensory stimuli (Wen et al. 1997) as well as

associative leaming (Wen et al. 1997; Morrison et al. 1999) and olfactory habituation

(Nuttley and van der Kooy submitted; Morrison et al. 1999) have been demonstrated.

In addition, Colbert and Bargmann (1995; 1997) have characterized olfactory

adaptation. While genetic components specifically underlying nonassociative

learning have yet te be identified, there has been extensive work at the cellular level

in idenhfying pathways in the tapwithdrawal response, a form of mechanosensory

habituation (Wicks and Rankin 1996,1997).

In the present study, I asked if both olfactory adaptation and habituation could

be obsemed in the same paradigm, and if these two processes could be differentiated.

After pre-exposure to very high concentrations (100%) of diacetyl @A), Worms

exhibited a chernotaxis decrement to a point source of DA which did not r e m to

baseline levels, with the presentation of various potentially dishabituating stimuli.

The adaptation of this response was dependent on proper odr-10 function (although

6

baseline chemotaxis to 100% DA did not), but odr-l was not required for DA

adaptation. h contrast to 1 0 % DA pre-exposure, after pre-exposure to low DA

concentrations (0.001%), the habituated response could be reinstated to naive

chemotaxis levels after presentation of a dishabituating stimulus (centrifugation at

250g). Moreover, at these low DA concentrations the baseline naive chemotaxic index

(CI) determined whether habituation or sensitization would occur such that High

initial Responders demowtrated habituation and Low initial Responders exhibikd

sensitization. Surprisingly, exposure to intermediate concentrations of DA (0.01 %

and 25%) did not c a w worms to exhibit a response decrement at all. Thus, there are

at least two distinct processes that underlie the observed behavioural response

decrement after continuous presentation of an olfactory stimulus, and these two

processes, adaptation and habituation, are disassociable in a concentration-dependent

METHODS

Materials and Methods

SmAINs

The Wild-type C. elegans Bristol strain (N2), the odr-10 (ky255) mutant s h a h

(Sengupta et al. 1996) and the odr-l (n 1936) mutant strain (Bargmann et al. 1993) were

utilized (mutant strains were provided by the Caenorhnbditis elegans Genetic Center).

The general culturing techniques w d are described by Sulston and Hodgkin (1988).

Adult worms were tested at approximately 4 days post hatching (3 day old from the

L1 stage). The population of worms was synchronized using the following method.

Populations of unsynchronized non-dauer plates containing many l a r d stage 1 and

2 worms were washed off a plate with distilled water (dHzO) into a 15 ml conical

centrifuge tube and spun down at 40g for 1 minute. This procedure causes the older

(heavier) worms to form a pellet, leaving the younger woms in the supernatant. The

supernatant was then transferred to a second 15 ml conical centrifuge tube and spun

down at lSOg for 1 minute. This technique of sequential centrifugation results in a

pellet of young larvae that could then be transferred to agar plates seeded with E. coli

(strain OP50) as a food source.

MATERLALS

Conditioning and testing were camed out using various different DA

concentrations, as specified. Lower concentration DA solutions (0.01 % and 0.001 %)

were prepared the day of the experiment from a stock solution of 1% diacetyl (in

ethanol) that was made the day @or. High concentration diacetyl and benzaldehyde

were aliquoted (100%) or diluted (1%, .1%, 25%) on the day of the experiment. All

attractants were obtained from Aldrich Chernicals and were always diluted in

9

anhydrous ethyl alcohol. Chemotaxic media (CD<) was prepared using an

autoclaved mixture of DIFCO Purified Agar with 100 ml/l of a 0.1 MOPS buffer (pH

7.2 with NhOH). Once the mixture was autoclaved and aliowed to cool, 2.5 ml/l

Tween 20 was added and test plates were made by pouring 6 ml of CTX into 10 ml

petri dishes (Fisher Scientific). Agar was evenly distributed throughout the plate and

aliowed to air dry for 20 minutes before capping with the iids; plates were generally

poured the day prior to the experiment. A plexiglass grid was used under the testing

plate to determine the location of placement of the odorant spots and the placement

origin of the worms (Fig. 1). A 1 pl spot of 1 M sodium azide OIJaN3) was placed on

test plates 15 minutes prior to testing in order to anesthetize animals as soon as they

chose to approach a specific attradant and to prevent the effects of odorant exposure

on leaming during the actual test period.

BEHAVIOURAL ASSAYS

Worms were placed in 15 ml conical centrifuge tubes, washed twice with dH20

and given two gentle 1 minute spins at 40g. The resulting peilet of adult worms was

carefully transferred to the conditioning CTX plate. Worms were gently dried with a

Kimwipe, and diacetyl was pipetted ont0 agar plugs piaced on the lids of the petri

dishes. The standard amount of diacetyl used was 5 4 distributed among 5 agar

plugs (variations from this amount are noted in a specific experiment). Control

animals were placed on CTX plates with no diacetyl added to the agar plugs. The

duration of odorant exposure ranged between 15 minutes for low concentration

experiments (0.001% and 0.01%) to 60 and 120 minutes for higher concentration

experiments (25%, 100%). The duration of pre-exposure was varied with different DA

10

concentrations since 15 minutes was sufficient to induce habituation at low DA

concentrations, whereas 60 to 120 minutes of pre-exposure were required to see a

similar approadi decrement at higher DA concentrations. After diacetyl pre-

exposure, dH20 was w d to wash habituated worms off of conditioning plates and

into a 15 ml conical centrifuge tube. Sufficient was added to the tubes to bring

the final volume up to 15 ml, and the worm suspension was allowed to settle for 5

minutes. The supernatant was removed and a 5 pl spot of Worms (containing as many

as two hundred worms) was aliquoted to the centre of the test plate at a point

equidistant from the aitractant and control spot using an Eppendorf micropipettor

with the tip cut short to prevent damage to the animals. The spot of worms was then

dned using a Kimwipe, and lpl spots of diacetyl or control solution (ethanol) were

placed on opposite sides of the plate. Plates were parafilmed, and left on the counter

for a 60 minute test duration, a standard tirne point (Bargrnaru et al. 1993). Each plate

consisted of 50-200 of worms and constitutes an n = 1.

Dishabituated worms were pre-exposed to diacetyl using the same procedure

as above, but instead of allowing to settle for 5 minutes in a 15 ml conical centrifuge

tube, the animals were spun down once at 250g for 1 minute, the supernatant was

removed, and more dHQ was added to a final volume of 15 ml prior to centrifuging

for a second time at 250g for 2 minutes. The speed of centrifugation varied across the

different pre-exposure experiments, and the different speeds used are noted for those

instances. Worms were placed on the test plates in an identical manner as described

above for the habituated groups. Naive Worms were placed on blank CïX plates for

the duration of exposure time (i.e. 15 minutes for low concentrations, 60 minutes for

high concentrations), and given the same strong centrifuga1 spins as the dishabituated

11

groups before testing. There was no difference in chemotaxic approach between

unmanipulated naive animais and naive worms exposed only to the dishabituation

treatments (data not shown), but as a control Naive groups were always given the

dishabituating treatment without DA presxposure. The order of groups tested were

randomized within an experiment.

SCORING AND ANALYSE

After the 60 minute test period, plates were inverted and placed at 4OC for at

least 30 minutes prior to counting. Any worms within 20 mm of the spot were

considered to have chosen that odorant (Fig. 1). A chemotaxic index (CI) adapted

from Bargmann et al. (1993) was calculated based on the numbers of animals at the

attractant :

CI = # at test spot - # at control spot Totd on plate

The CI can Vary from + 1.0, indicating pure attraction, to -1.0 indicating pure

aversion. In general, less than 10% of the w o n s do not leave the origin, and since

this sniali aggregate of animals in the middle of the plate is likely due to possible

damage inmred during the handling procedures, such clumps of worms were not

included in the CI cakulation. The % Response was w d as a measure of change from

Naive baseline CI and was calculated as:

% Response = Mean of Pre-Exposed Treatments iOO% Mean Naive Treatments

where the Mean of each treatment represents the Mean for one experiment with n = 4

plates. Mean values, standard error of the mean (SEM), regression analysis, t-tests,

analyses of variance (ANOVAs) and post- hoc analyses (Neuman-Keuls) were

calculated using the Statistica software program (Macintosh StatSoft software version

Figure 1: Habituation/Adaptation Apparatus. Conditionhg agar plates were used for exposing worms to the various concentrations of diacetyl during training. Circles with hatched bars represent agar plugs saturated with 1 pl of diacetyl, and short wavy lines represent aggregates of worms. Following treatment, hundreds of worms were placed at the origin point on a test plate with a 1 y1 control spot (ethanol, E1OH) and test spot (DA) placed on either end. Plates were treated with 1 pl sodium azide (NaN3) at control and test spots to capture the animalsr initial respowes. After a 60 minute test period woms were placed at 4 ' ~ , and later counted to establish a chemotaxic index per plate. Each plate comisting of hundreds of worms was considered an n =l; each treatment group had an average of n = 4 per experiment.

Control spot Test spot of DA

Conditioning plate Test plate

CI = # at test m o t - # at control spot Total on plate

RESULTS

Results

C. ELEGANS APPROACHES DIFFERENT CONCENTRATIONS OF DIACETYL IN A DOSE-DEPENDENT MANNER

In order to assess behavioural plasticity in C. elegnns using diacetyl (DA) as an

olfactory stimulus, the naive baseline approach to a variety of different DA

concentrations was established. The animals approached DA in a dose dependent

manner, such that the highest chemotaxic response was elicited at the highest

concentrations and as the concentration of DA became more dilute, the CI decreased

(Fig. 2). This corresponds with previous data which show that DA is a significant

olfactory attractant over a broad range of concentrations (Bargmann and Horvitz,

1991). In addition, it was also noted that the variability was highest at the lowest

concentrations of DA.

PRE-EXPOÇURE TO HIGH CONCENTRATIONS OF DIACFPiIL (100%) LEADS TO A DECREMENT IN APPROACH THAT IS TIME-DEPJ3lDENT BUT V O L W - INDEPENDENT

Ln order to test whether worms are capable of showing nonassociative

learning at very high concentrations of diacetyl, populations of woms were pre-

exposed to 5 pl of 100% DA for varying exposure times and their CI was scored after

the standard 60 minute test period. Naive animals showed very high approaches

towards a test spot of 100% DA. A Naive Starved group that was starved for 2 hours

(there was no noted difference in naive DA approach for animals starved for 30

minutes, 1 hour or 2 hours, data not shown) was used as a control for effeds of

Figure 2: Dose Response Curve. C. elegans chemotax to a broad range of diacetyl concentrations. Approach was elicited to a variety of different concentrations of diacetyl, with the highest approach to diacetyl seen at the highest dilutions. n = 4 for all groups.

18

starvation during pre-exposure t h e , however there was no significant difference

between Naive or Naive starved groups (t(7) = 1.26, P > 0.05). Within 1 hour, animals

showed a signihcant decrease (Neuman-Keuls test, P < 0.05) in approach to DA, and

this approach decreased even further after a 2 hour exposure (Fig. 3A). A one-way

ANOVA comparing Naive or Naive Starved animals to Pre-exposed groups with

varying exposure times revealed a signihcant effect of pre-exposure time (F(5,18) =

26.0, P < 0.05). Neuman-Keuls post-hoc tests demowtrated that there was no

significant effect (P > 0.05) following a 30 minute pre-exposure, but the 1 hour and 2

hour pre-exposure treatments yielded Ch that were significantly different from

Naive starved levels and from each other (P < 0.05). A 3 hour pre-exposure time was

also tested, but despite the substantial decrement in diacetyl approach (data not

shown), this lack of response was likely due to damage to the animals as seen by

general tack of movement and gross clumping at the site of origin on the test plate.

Based on these results, the 2 hour exposure period was chosen to examine the effects

of varying the volume of DA on approach. A dose-independent decrease in diacetyl

approach was seen, such that regardless of the pre-exposure volume, the decrement in

CI remained constant compared to Naive levels (Fig. 38). A one-way ANOVA

comparing Naive treatment to Re-exposed treatments with vary hg exposure

volumes revealed a significant effect of pre-exposure (F(4,15) = 7.7, P < 0.05).

Neuman-Keuls post-hoc tests showed that followuig pre-exposure, ail groups were

significantly different from Naive (P < 0.05) and were not signihcantly different from

one another (P > 0.05). Thus, t h e of exposure but not exposure volume affects the

decrement in diacetyl approach.

Figure 3: Increasing exposure time, but not volume, affects degree of approach decrement after pre-exposure to 100% DA. (A) Worms pre-exposed to 5 pl of 100% diacetyl for 30 minutes to 2 hours had significantly lower mean diacetyl chemotawis. The unstarved naive group versus the naive group that was starved for 2 hours were not significantly different. After 1 hour of diacetyl exposure, worms showed a signihcant decrease in approach, but the effed was most pronounced after the 2 hour exposure. n = 4 for all groups. (B) A 2 hour pre-exposure time to diacetyl with vasring volumes of odorant shows that the approach decrement decreased to approximately 40% of Naive levels for all groups, regardless of pre-exposure volume. n = 4 for all groups.

- 1 E

0.9 PRE-EXPOSED

0.8 c 8 0.7 - 0-6 5 8 0'5 a g 0.4

$ 0.3 O O ri 0.2 O - 0.1 U

n V

Naive Naive 30 min Starved

PRE-EXPOSURE TIME

PRE-EXPOSURE VOLUME

EXPOSURE TO HIGH CONCENTRATIONS OF DIACETYL E L K E ADAPTATION, BUT NOT EIABITUATION

Worms demonstrate a very high CI to a test spot of 100% DA and can show a

decrement in approach foliowing various pre-exposure h e s and odorant volumes.

Is this decrement in response adaptation, cawd by receptor or effector fatigue, or is

it due to nonassociative leaming which is subject to reversal? By definition,

habituation requires a behavioural demonstration of dishabituation. In order to

determine whether the observed decrease in approach behaviour represented

adaptation or habituation, worms were pre-exposed to 3 pl of 100% DA for 2 hours,

and the dishabituating stimuli varied from centrifuga1 spins of lOOg to 1000g.

Regardless of the magnitude of the spin, the stimuli were not sufficient to induce

dishabituation of the observed response decrement after pre-exposure (Fig. 4A). A

one-way ANOVA comparing Naive, Re-exposed and Pre-exposed + Spin treatments

indicated a signihcant effect of treatment (F(5,17) = 8.2, P < 0.05). In a Neuman-Keuls

post-hoc analysis, the CIs of the Pre-exposed and Re-exposed + Spins groups were

significantly different from the Naive group (P c 0.05), but not from each other (P >

0.05). In a subsequent test 1 asked if a stronger dishabituating stimulus couid c a w

the response decrement to retm to naive levels. After pre-exposure to 5 pl of

diacetyl for 1 hou, two different groups of wonns were subjected to two different

dishabituating treatments: dishabituation group 1 was given two centrifuga1 spins at

2000g and 30 seconds of vortexing, and dishabituation group 2 was spun twice at lOOg

and Gven a 15 minute cold shock at 4 ' ~ (Fig. 4B). A one-way ANOVA revealed that

animals decreased their approach response after pre-exposure to 100% DA (F(5,52) =

229, P< 0.05). Neuman-Keuls post-hoc analysis showed that foiiowing

Figure 4: Lack of dishabituation after pre-exposure to 100% DA. (A) Pre-exposure to 3 pl of 100% diacetyl for 2 hows caused Worms to exhibit a response decrement to 100% DA which could not be reversed despite being presented with dishabituating stimuli ranging from lOOg to lOOOg spins. The Naive group was given lOOOg treatment without pre-exposure to DA. Ail groups given dishabituation stimuli were significantly different from the mean Naive, but not different from the pre-exposed group without dishabituation stimuli. 100g= a 1 minute spin at 100g followed by a 2 minute spin at 100g 300g= a 1 minute spin at 300g followed by a 2 minute spin at 300% 500g= a 1 minute spin at 500g followed by a 2 minute spin at 500g. 1000g= a 1 minute spin at lOOOg followed by a 2 minute spin at 1000g. n = 4 for all groups, except n = 3 for Naive. (B) Worms exposed to 5 y1 of 100% diacetyl for one hour do not show a dishabituated response to 100% DA following different dishabituating treatments. Pre-expose = pre-exposed without dishabituating treatments, Group 1 = pre-exposure + 2 x 2000g spins (3 minutes total) + 30 sec vortex, Group 2 = pre-exposure + cold shock treatment of 4°C for 15 minutes, followed by a 1 minute spin at 100g Spont. Recovery = Spontaneous recovery d e r 3 hours. n = 20,18, 4,8,4 respectively from left to right.

NAlVE

0 PRE-EXPOSED

El PRE-EXPOSED + DISHABITUATION TREATMENT

Naive Pre- lOOg 300g 500g lOOOg expose only

DISHABITUATION spins at various g

' ~ a i v e ' Pre- Group Group Spont. expose

only 1 2 Recovery

TREATMENTS

24

conditioning, the Pre-exposed group showed a deaeased CI that was approximately

50% of the naive CI (P c 0.05), and that regardless of attempts at dishabituation, this

decrement was not reinstated to naive levels. Moreover, the decrement in response

seen in the two groups given the dishabituating treatments was not caused by any

potentially damaging effects of the treatments, since the Naive groups (combined)

were given equivalent treatments without DA pre-exposure and these treatments did

not affect the high naive baseline CI. When left for 3 hours after an equivalent DA

pre-exposure to spontaneously recover prior to testing, worms exhibited chemotaxic

indices that were not significantly different from naive levels (Fig. 48, P > 0.05).

Thus pre-exposure to 100% DA results in adaptation, since dishabituation is not

revealed despite the potency of the dishabituating stimulus.

EXPOSURE TO INTERMEDIATE CONCENTRATIONS OF DIACETn (25% AND 0.01 %) LEADS TO NO RESPONSE DECREMENT IN DA APPROACH

Since exposure to 100% diacetyl leads to adaptation, might UA concentrations

lower than 100% elicit a respome decrement that is capable of dishabituation? Pre-

exposure to 25% DA was not suffident to cause a decrement in approach to the same

25% DA stimulus regardless of exposure time or amount of diacetyl present (Fig. 5A).

The dishabituating stimulus of two strong centrifuga1 spins at 500g did not affect

approach to diacetyl in either the Naive or Pre-exposed + Spin groups. A one-way

ANOVA examining a 90 minute pre-exposure to 2 ~ 1 of 25% DA in Naive, Pre-exposed

and Pre-exposed + Spin groups revealed no signihcant effect of treatments (F(2,8) =

2.3, P > 0.05). Similarly, a 2 hour pre-exposure to 3p1 of 25% DA yielded no significant

effect of treatment (F(2,9)=2.1, P > 0.05). Since approach to 25% DA was so high, the

effects of pre-exposure to a concentration of diacetyl whch was lower than 25% DA

by several orders of magnitude (0.01 % DA) was examined. Pre-exposure to 5 p l 0.01%

DA for 15 minutes was not sufficient induce a decrement in CI to 0.01% DA, A one-

way ANOVA comparing Naive, Pre-exposed and Pre-exposed + Spin groups revealed

no signihcant effects of treatment (F(2,15) = 0.2 P > 0.05) (Fig. 58). Pre-exposure to

the same concentration of diacetyl for a longer 60 minute duration of, however,

showed a trend toward a deaease in CI, but a one-way ANOVA revealed no

significant difference between these two groups (F(2,lI) = 2.63 P > 0.05). In

subsequent attempts to see if an even longer pre-exposure time of 90 minutes would

drive the CI down even further, there was no significant reduction from Naive CI

(F(2,12) = 1.97 P > 0.05, data not shown). Despite variations in length and volume of

pre-exposure to the odorant, intermediate concentrations of diacetyl (<IO098 and 2

0.01 %) do not produce decrementd respowes.

PR.-EXPOSURE TO LOW CONCENTRATIONS OF DIACETYL, (0.001%) FAVOURÇ NONASSOCIATIVE LEARNING

The effects of pre-exposure to low concentrations of DA (0.001%) were

observed and compared with pre-exposure to intermediate DA (25% and 0.01%)

concentrations to determine if there would be a similar lack of response decrement as

seen after 25% or 0.01% DA pre-exposure. At these low odorant concentrations, naive

response to 0.001% DA varied from 0.2 to 0.8 on any given day @ut was consistent

within days). A one-way ANOVA comparing Day x Naive CI demonstrated there

Figure 5: No response decrement seen &ter exposure to intermediate DA concentrations ( 2 5 O 0 , 0.01%) (A) Pre-exposures to 2 pl of 25% DA for 90 minutes or 3 pl of 25% DA for 2 hours were not sufficient to elicit a response decrement to 25% DA in wild-type animals, regardless of conditioning treatments. The dishabituating stimulus was a 1 minute centrifugation at 500g, followed by a 2 minute 500g spin. n = 4 for all, except n = 3 for Naive 2 pL/90 minute exposure group. (B) A 5 pl pre-exposure to 0.01% diacetyl did not cause a decreased chemotaxis approach to 0.01% diacetyl despite varying the time of exposure from 15 to 60 minutes. The dishabituating stimulus was a 1 minute centrifugation at 500g, followed by a 2 minute 500g spin. n = 4 for all 15 minute group, and n = 3 for au60 minute groups.

A Exposure to 25% Diacetyl

Naive Pre-

2 pl exposure for 90 3p1 exposure for 2 hr min

B Exposure to 0.01% Diacehrl

F Pre-

Expose ' ~ r e - ~ x ~ ;se

+ Spin

5p1 exposure for 15 min

5p1 exposure for 60 min

28

was a significant effect of Day (F(2S173) = 3.0, P < 0.05). In an analysis of ail

experiments (including ail ranges of Naive CI), Pre-exposure treatment (habituation)

was compared to Pre-exposure + Spin treatment (dishabituation). Worms exhibited a

25% decrease in approach to 0.001% DA after 0.001% DA pre-exposure, and after the

Dishabituation treatment their CI retumed to Naive levels (Fig. 6A). If left to recover

for two hours after exposure, animals spontaneously recovered back to naive DA

approach levels . A one-way ANOVA corn paring Naive, Habitua ted, Dishabituated

and Spontaneous Recovery treatments showed that there was a significant effect of

treatment (F(2,282) = 10.0 P < 0.05) and post-hoc tests revealed that the Habituation

treatment was significantly different h m the Naive, Dishabituation, and

Spontaneous Recovery treatments (P < 0.05), which were not significantly different

from each other (P > 0.05).

Investigating the day to day variability, 1 noticed that when the Naive baseline

CI was higher, the habituation tended to be greater than the average 25% response

decrement. 1 therefore hypothesized that as the Naive CI increased, the % Response

after habituation treatment would decrease. A regression analysis of the % Response

to the Mean Naive CI (for each day's experiment) demonstrated a correlation of r = - 0.59 (F(1,26) = 13.6, P < 0.05) (Fig. 68). There were two noted outliers ( > 2 s.d. away

from mean CI and mean % Response). When each of these points were excluded from

the analysis, the correlation stiîl remained significant (F(1,24) = 6.1, P < 0.05, r = -0.45).

Based on this regression analysis, the experiments were divided into two groups: the

Sensitized Group, which exhibited an increase in % Response after 0.001 % DA pre-

exposure, and the Habituated Group, which demonstrated a decrease in % Response

after 0.001% DA pre-exposure. A closer analysis of the Çewitized group revealed that

29

the mean Naive CI was 0.34 f 0.02 with a mean % response of 51.9% f 7.7% (n = 33)

after DA pre-exposure, while the mean Naive CI of all scores in the Habituated group

was 0.48 î 0.01 with a mean % response of -51.5% I 4.3% (n = 67) after DA pre-

exposure. A one-way ANOVA comparing Group to Naive CI and % Response

revealed a main effect of Group x Naive CI (F(1,98) = 32.9, P < 0.05) and % Respowe

(F(1,98) = 162.3, P < 0.05). Neuman-Keuls post-hoc analyses demonstrated that both

Naive CI and % Response were significantly different between the Habituated and

the Çensitized groups (P < 0.05).

Based these results and previous expeninents which suggest that Low initial

Responders (in this case, those with low Naive CI) tend to demonstrate sensitization

and High initial Responders (those with a High Naive CI) demonstrate habituation

(Eisenstein et al. 1991; Eisenstein 1997), all the experiments were divided into two

groups: the Habituated group with % Response less than zero as the High initial

Responders (naive CI values > O.%), and the Sensitized group with % Response

greater than zero as the Low initial Responders (naive CI < 0.34). An analysis of Low

initial Responders and High initial Responders in a two-way ANOVA comparing

Group x Treatment demonstrated a signihcant effect of Group alone (F(1,279) = 33.7, P

< O.OS), and a signihcant interaction between Group and Treatment (F(2,279) = 17.9, P

< 0.05). Worms in the High Responder group (naive CI > 0.34) that were pre-exposed

to 5 111 DA for 15 minutes showed a 40% decrement in approach to the odorant in the

standard chernotaxis test (Fig. 6C). In addition, this decrement could be dishabituated

upon presentation of a strong stimulus of two centrifugai spins of 250g. A one-way

ANOVA comparing Naive, Habituated, Dishabituated and Spontaneous Recovery

treatments in the High initial Responder group revealed a signihcant effect of

30

sigruficant effect of treatment (F(2,214) = 27.4, P < 0.05). Post-hoc tests indicated that

following the habituation treatment, the CI to 0.001% DA was significantly different

h m naive and dishabituatecl leveis (P < 0.05). Following the dishabituation

treatment, the CIs increased to a level that was significantly different from

Habituated (P < 0.05), but not statistically different from naive levels (P > 0.05).

Interestingly, under the exact same Pre-exposure and Pre-exposure + Spin

treatments, pre-exposure in the Low initial Responder group (naive CI < 0.34)

showed sensitization, as seen by a 25% increase above baseline levels (Fig. 6D). A

one-way ANOVA comparing Naive, Pre-exposed and Preexposed + Spin

(dishabituation treatment) groups demonstrated a significant effect of treatment

(F(2,89) = 3.6, P < 0.05) and post-hoc analysis indicated that the pre-exposed group was

significantly different from Naive (P < O.OS), while the Pre-exposed + Spin group was

not significantly different from either Naive or Pre-exposed groups. Thus, when

baseline approach to DA is high (> O%), wild type Worms exhibit habituation and

when baseline CI is low (< 0.34) they display sewitization.

Figure 6: Nonassociative leamhg occurs after pre-exposure to low concentrations of DA (O.OOlO/o) (A) Pre-exposure to 0.001% DA leads to habituation. Worms pre-exposed for 15 minutes to 5 pl of 0.001% DA showed a 25% response decrement when tested to 0.001% DA compared to naive values. Dishabituated groups centrifuged once at 250g for 1 minute and again at 250g for 2 minutes exhibited a recovery of chemotkwic approach to 0.001% DA that was not significantly different from the Naive or Spontaneous Recovery groups. Al1 naive wonns were given the dishabituahg treatment without diacetyl pre-exposure. n = 100,106, 103,10, respectively from leh to right. (B) Linear regression of % Response versus Mean Naive CI. Analysis revealed a correlation of r = -0.59 (represented by the slope of the diagonal line) between % Response (% difference between Mean Naive CI and Mean Pre-Exposed CI) and Mean Naive CI. Mean Naive CI is calculated as the mean of 4 plates within one day's experiment. n = 28 individual experiments. The dashed horizontal line represents no change in behavioural response (0% Response) after pre-exposure treatment; all points above the line reflect the experiments where sewitization was obsewed (Sewitized group) and those below the 0% Response represent the Habituated group. (C) High initial Responders to 0.001% DA show habituation after 0.001% DA pre-exposure. Naive animals with baseline CIs of greater thm 0.34 were classified as the High initial Responder group. Worms exposed for 15 minutes to 5 pl of 0.001% DA showed a 40% response decrement to 0.001% DA compared to naive values. Dishabituated groups exhibited a recovery of chemotaxic approach to DA that was not significantly different from the Naive or Spontaneous recovery groups. 11 = 69, 77/71, respectively from left to right. (D) Low Naive Baseline CI to 0.001% DA favours sensitization. in Low initial Responders where wiid-type naive CIs are less than or equal to 0.34, pre-exposure to 5 pl 0.001% DA caused a 25% increase above baseline levels. n = 31,29,32, respectively from left to right.

0.55

0.5

E: W 0.45 rn +l 0.4 4 O S

; 03 0.25 s Sensitizcd Cmup s" 0.2

$ 0.15 Hahiiuatcd Gmup CI t; 0.1

0.05

O 9C Naive Habituatcd Dishabituateci Sporitcncous O .1 .2 .3 -4 .5 .6 .7 Rt?Covcry

Habituafed Group - % Rrsponsc < O Sensitizd Group - % Response > O

High Initial Respondcrs: Mean Naive CI>0.34

Low [ni tial Responders: Mean Naivc. CIc0.34

33

ADAPTATION AND NONASSOCIATIVE LEARNING ARE DISTINCT PROCESSES

THAT ARE SEPARABLE IN A CONCENTRATION-DEPENDENT MANIER

Re-exposure to both 100% DA and O.ûûl% DA elicited a response decrement,

however the decreased response after pre-exposure to 0.001% DA (in High

Responders) could be dishabituated whereas the decreased response after 100% DA

pre-exposure could not. In addition, pre-exposure to intermediate concentratiow of

DA did not elicit any signihcant response decrement. In order to test if there is a

signihcant difference between the various concentrations (100%, 2596, 0.01% and

0.001 % DA) and treatments (Naive, Pre-exposed/ Habituation, and Pre-exposed +

Spin/ Dishabituation), a two-way ANOVA was carried out. This showed a signihcant

interaction of Concentration x Treatment (F(6,432) = 5.27, P < 0.05), demonstrating

that the effects of the treatments are different at differing DA concentrations.

DIA- ADAPTATION REQUIRES odr-10, BUT NOT odr-l

Wild-type N2 worms demonstrate adaptation after being pre-exposed to 100%

DA, however the receptor which mediates th& response is unknown. Approaches to

high concentrations of diacetyl are likely mediated by a low-affinity DA receptor on

the AWC neuron, since animals lacking the odr-20 gene or function of the AWA

neuron s till approach high DA concentrations (Chou et al. 1997; Sengupta et al. 1996),

but animals with deficiencies in AWC function show impaired chernotaxis to high

DA concentrations (Bargmann et al. 1993). Thus, mutations that affect the hinction of

the two putative DA sensing primary chernosensory neurons (odr-IO which is the

34

high-affinity receptor in AWA (Sengupta et al. 1996) and odr-2 which affects AWC

function (Bargmann et al. 1993)) were tested for their adaptation respowes to 100%

DA. Wild-type, odr-10 and odr-l strains were pre-exposed for 60 minutes to 5 pl of

100% DA, and tested for their responses to 100% DA, 0.1% DA and 1% benzaldehyde

(BZ) (Fig. 7). As expeded, Md-type worms pre-exposed and tested to 100% DA

demonstrated an -50% decrease in CI compared to wild-type Naive. Wild-type

worms pre-exposed to 100% DA and tested to 0.1% showed an even larger decrement

in approach (-75% decrease). Pre-exposed wild-type anirnals tested to either 100%

DA or 0.01% DA did not display any signs of dishabituation after being spun for three

minutes at 500g (Pre-exposure + Spin treatment). Approach to BZ was high and while

there did appear to be a small yet signihcant decrease after DA pre-exposure

(Neuman-Keuls post hoc tests, P < 0.05), this slight decrease was not different from

Pre-exposed + Spin CI levelç which were not significantly different from Naive (P >

0.05). A two-way ANOVA revealed a signihcant interaction of treatments with

olfactory stimuli (F(4,125) = 9.84, P < 0.05) for wild-type and post-hoc analyses

confirm that 100% DA pre-exposure caused a significant reduction in DA approach to

both 100% DA and 0.1% DA @<0.05) that could not be dishabituated.

Worms of the odr-20 strain exhibited high naive chernotaxis to 100% DA and

1% BZ, with approach to 0.1% DA almost completely eliminated (shown by Sengupta

et al. 1996). Intereshgly, testing to 100% DA revealed that unlike wild-type, odr-10

worms did not dernonstrate any signihcant decrement in response to DA after a 60

minute pre-exposure to 100% DA. A three-way ANOVA of odr-20 and wild-type

worms comparing Strain x Treatment x Olfactory Stimulus showed signihcant

interactions (F(4,197)=7.69 p<0.05) and a closer cornparison of Strain x Treatment in

35

response to 100% DA using a two-way ANOVA showed a significant interaction

(F(2,68) = 5.87 pd.05). Post-hoc cornpansons of wild-type and odr-10 groups pre-

exposed and tested to 100% DA yielded signihcant differences between wild-type

Naive and Pre-exposed or Re-exposed + Spin groups (P < 0.05) (with no difference

between Pre-exposed and Presxposed + spin (P 1 0.05)), while no significant

differences between odr-20 Naive and Pre-exposed or Pre-exposed + Spin groups (P >

0.05) were observed. Thus, unlike wild-type, odr-20 worms did not adapt.

The odr-1 worms displayed a compromised naive CI to 100% and 0.1% DA

@<0.05, significantly different from the comparable wild-type values). Despite this

lower baseline, however, after 100% DA pre-exposure the odr-1 worms still

demonstrated a -50% and -80% decreased CI when tested to 100% DA, and 0.1% DA,

respectively. This decrement in the odr-1 worms could not be dishabituated with a

diçhabituating stimulus (3 minute sp.h at 500g). An almost complete lack of BZ

approach in Naive odr-l worms confirms the prior classification of the odr-2 stralli as

particularly deficient in AWC function (Bargmann et al. 1993). A three-way ANOVA

comparing Strain x Treatment x Olfactory Stimulus interactions between wild-type

and odr-2 strains revealed signihcant interactions (F(4,200) = 4.03, P < 0.05). Within

the wild-type and odr-1 strains, a simple cornparison of response to 0.1% DA (the

concentration where wild-type and odr-1 woms had naive CI values that were most

similar) in a two-way ANOVA investigating Strain x Treatment interactions showed

a signihcant interaction (F(2,78) = 5.20, P < 0.05). Excluding the previously noted

differences in BZ approach, the interactions in the above cornparisons could be

accounted for entirely by the differences in Naive CI between the wild-type and odr-1

strains, since the odr-1 Naive CI to both 100% DA and 0.1% DA were significantly

36

different from wild-type Naive CI (Neuman-Keuis, P < 0.05). Both straiw

demonstrate a signihcant decrease in CI after 100% DA pre-exposure tested to either

100% DA and 0.1% DA (P < 0.05). Neither the wild-type Pre-exposed nor the odr-1

Pre-exposed groups differed significantly from each other, although unlike wild-type

woms, there appeared to be a trend towards dishabituation in the odr-l Pre-exposed

+ Spin group. Closer analyses of this trend using a two-way ANOVA to investigate

interactions of Strain x Pre-exposure or Pre-exposure + Spin treatments to 100% DA

and testing to 0.01% DA, revealed no significant interaction between Strain or

Treatment (F(1,53) = 3.7, P > 0.05) and that the differences between odr-1 Pre-exposed

or Pre-exposed + Spin treatments were not signihicantly different from each other or

control values (P > 0.05). The overall low baseline approach to DA and BZ in odr-1

may suggest a broad inability to sense volatile odorants or to chemotax normally.

To test this possibility, 1 camed out an additional experiment to ask if the odr-1 strain

had any generalized motor or sensory deficits by obsewing the chernotaxis approach

to the AWA sensed odorant, pyrazine (Bargmann et al. 1991). Baseline (naive)

approach to 2 mg/ml of pyrazine was tested and 1 observed that odr-2 naive CI did

not differ signüicantly from wild-type naive CI (wild-type Naive CI = 0.47 f 0.05, odr-

1 Naive CI = 0.61 f 0.12, n = 4, 4, respectively; P > 0.05). Overall, these experiments

demonstrate that wonns are stili capable of exhibiting normal adaptation to DA

(through an odr-2-independent pathway), and that lack of odr-10 prevents adaptation.

Figure 7: Adaptation is odr-10 dependent, but odr-2 independent. Wild-type, odr-IO and odr-2 worms were pre-exposed to 5 pl of 100% DA for 60 minutes and tested to 100 % DA, 0.1% DA or 1% BZ. Wild-type worms displayed a 50 % decrease in CI to 100 % DA and a 75% decrease in CI to 0.1 % DA (compared to their respective Naive values), but the CI to BZ did not show a similar decrement after 100% DA pre- exposure. Pre-exposed group = pre-exposure with a gentle wash and settled in a tube; Pre-exposed + Spin = presxposed and spun at 500g for 1 minute followed by another 500g spin for 2 minutes. Within each strain, there was no significant difference behveen Pre-exposed or Pre-exposed + Spin treatments. odr-IO animals, which showed a nearly zero response to 0.1% DA, did not demonstrate DA adaptation after 100% DA pre-exposure and testing, and did not show any difference in BZ approach compared to wild-type. odr-1 animals diçplayed lower baseline CIs to 100% DA and 0.1 % DA and no response to BZ, yet still demonstrated DA adaptation to 100% and 0.1% DA after 100% DA pre-exposure. Respectively from left to right: for Wild-type 100% DA, n = 17,16,15; Wild-type 0.1% DA, n = 17,18,15; 1% BZ, n = 12,11,14. For odr-10 100% DA, n = 9,9,8; 0.1% DA, n = 9,9,10: 1% BZ n = 9 for all groups. For odr-1 100% DA,n=7,6,8,O.l% DA,n = I l , 12,12andl% B Z n =11,9,8.

DISCUSSION

DISCUSSION

The decrement in chemotaxic approach subsequent to continuous presentation

of the volatile attractant diacetyl c m be accounted for by hvo distinct foms of

olfactory plasticity depending on the odorant concentration. After pre-exposure to

high DA concentrations (100%), the obsewed chemotaxis decrement could not be

reversed to naive approach levels despite strong dishabituating stimuli, and thus the

cause of such a decrement is likely due to some form of sensory or motor fatigue

yielding behavioural adaptation of the response. M e r pre-exposure to intermediate

DA concentrations (0.01% to 25%) there was no observable decrease in DA

chemotaxis. After pre-exposure to low concentrations of DA (0.001%) the behavioural

plasticity observed was due to nonassociative learning: the decreased response was a

result of habituation in High initial Responders (naive CP.34) since a strong stimulus

was sufficient to induce dishabituation, and an increased CL above baseline levels in

tow initial Responders was indicative of sensitization. On a mechanistic level, lack

of the ODR-10 high-affinity DA receptor on the AWA neuron prevented DA

adaptation without affecting high baseline naive CI to 100% DA, and knocking out

AWC primary chemosensory function by eliminating the odr-2 gene reduced baseline

naive CI, but did not prevent normal DA adaptation.

In order to account for the concentration specific effects of DA pre-exposure the

following three processes are postulated to underlie the observed behavioural

responses: adaptation (receptor fatigue), sensitization and habituation (Fig. 8).

Although 100% DA is sensed by both low-affinity and high-affinity receptors, naive

approach to 100% DA requires the low-affinity DA receptor through odr-1 signahg

while odr-10 may account for only half of 100% DA approach in the absence of odr-1

41

function (Fig. BA, and Fig. 7). After pre-exposure to 100% DA, excessive activation of

the high-affinity ODR-IO DA receptor causes ODR-10 downstream targets to interact

with the AWC low-affinity DA receptor pathway leading to its down-regulation and

causing adaptation of the approach to 100% DA. This interaction between the two

primary chemosensory neurons may be through common downstream targets such

as the AIY or A U interneurons. Evidence of such cross-talk between AWA and AWC

is supported by studies which demonstrate that butanone (an AWC-sensed odorant)

sensitivity is slightly increased when odr-10 is knocked out (Sengupta et al. 1996).

After pre-exposure to 100% DA, approach to this concentration of odorant can then

only be reinstated to naive levels after sufficient time for upregulûtion of the AWC-

mediated approach pathway. Despite the strong intewity of the stimulus,

sensitization is not favoured to occur at this concentration since cellular fatigue

prevents any modulation due to nonassociative learning from occurring.

At intermediate DA concentrations (0.01% to 25%) there is less activation of the

AWC low-affinity receptor as well as strong activation of the high-affinity ODR-10

receptor (Fig. 8B). While the stimulation of the ODR-10 receptor is still strong, it is

not as great as with 100% DA pre-exposure so that adaptation is less and /or

sensitization is greater. Thus, there is less cellular fatigue/ adaptation and now both

sensitization and adaptation are equally favoured, which leads to a cornpetition

between the two processes. While sensitization is favoured due to the strength of the

stimulus (high stimulus intensity), adaptation is also occurring. Any potential

decrement in response that would be caused by adaptation is opposed by the

sensitization which is attempting to facilitate the respowe. Hence, the overau

obsewed behavioural response afkr DA preexposure to such intermediate

42

concentratiow is a failure to decrease the chernotaxis approach. It muçt be noted,

however, that only two different concentrations of DA that were tested within the

intermediate DA concentratiow. It is therefore possible that the ideal conditions to

observe a net behavioural response lies somewhere in between the tested

concentrations. While this remains a possibility, it may be uniikely since the

assessed concentrations (25% and 0.01% DA) covered several orders of magnitude

without any obsewable difference of treatments.

At the lowest DA concentrations (0.001%) only the ODR-IO receptor is

stimulated without activation of the AWC low-affinity DA receptor, which elhinates

any possible adaptation and d o w s the nonassociative learning processes of

sensitization or habituation to occur (Fig. SC). At low stimulus intensities, Groves

and Thompson (1970) would predict that habituation should occur; this is observed in

the High Responders (but not the Low Responders, see below) which demonstrate an

habituated response after DA pre-exposure that can be dishabituated with a

sufficiently strong stimulus. The data here suggest that on a day when the animal

does not find the diacetyl particularly appetitive, it will not habituate as well as on a

day when the cue is particularly salient. What then accounts for sensitization seen in

Low Responders? This trend of habituation in High initial Responders and

sewitization in Low initial Responders has been reported previously in the ciliate

protozoa, Spirostomurn and in the galvanic skin response of humans after being given

a shock stimulus (Eisenstein et al. 1991; Eisenstein 1997). However, the dual process

theory (Groves and Thompson 1970) would predict that a weaker stimulus is more

conducive to eliciting an habituated response while a stronger stimulus would elicit

Figure 8: Adaptation, Habituation and Sensitization are separate processes -

which underlie the changes in behavioural response after pre-exposure to diacetyl. (A) Under baseline conditions the putative low-affinity DA receptor on AWC mediates a portion of the naive approach to 100% DA via an odr-1-dependent pathway. Although the ODR-IO receptor on AWA is not necessary for naive approach to DA, it can account for half of the response to 100% DA in the absence of AWC hinction. Pre-exposure to 100% DA causes excessive activation of the high-affinity odr-10 receptor on AWA which leads to a down regulation of the AWC mediated approach pathway causing adaptation. Thiç interaction between AWC and AWA may be via direct connections between the two primary chemosensory neurons or tluough a common downstream target. Lack of the putative receptor guanylyl cyclase in AWC encoded by odr-l partially eliminates baseline approach to 100% DA, but does not prevent the odr-20 mediated adaptation. Any sensitization that may be occurring in AWA is overshadowed by the adaptation. (8) Pre-exposwe to intermediate DA concentrations causes less stimulation of the low-affinity DA receptor on AWC and strong stimulation of the ODR-IO DA receptor @ut less stimulation than with 100% DA so that adaptation is less and/or sensitization is greater). This leads to the processes of adaptation and sensitization to be equivalently favowed and the cornpetition between these two opposing processes results in no net behavioural decrement of DA response. (C) Pre-exposure to low DA concentrations causes stimulation of only the odr-10 receptor (no activation of the low-affinity DA receptor), eliminating any possible adaptation. This results in either of two nonassoaative learning processes: habituation or sensitization, depending on the baseline initial response to the odorant. Gray = inactive neurons; Blue = activated AWA neuron; Red = activated AWC neuron.

3 A High DA Concentrations , B intermediate DA Concentrations I C Low DA Concentrations -

(1 W/O) I BASELINE RESPONSE I

ADAPTATION > SENSITIZATION

DECREMENT IN D A RWPONSE, NO RETURN TO BASELINE WLTH DISHABïIWG STIMULI

ADAPTATION NO DECREICIENT IN DA RESPONSE

NON-ASSOCIATIVE LEARNING

45

sensiti~ation~ therefore questionhg why there should be any sensitization in Low

Responder animals exposed to a weak DA stimulus (0.001%). in this case, the

demonstration of sensitization in responders with low initial CI to DA is likely due

to the fact that once the animals' baseline response has reached su& a low output

response, the probability of the response to be driven down any hrrther is less likely

and with a less prominent habituation, the cornpethg process of sensitization is

unmasked.

This does not address the curious phenornenon of why there is such great

variability in the approach to low concentrations of diacetyl on different

experimental days. Environmental factors, such as temperature and relative

humidity are known to affect the response of C. elegans in a variety of behavioural

paradigms (Hedgecock and Russell, 1975; Gannon and Rankin, 1995). 1 attempted to

maintain these variables as consistent as possible hom day to day, however, formal

assessmentç on the degree to which these factors affect nematode leaming are still

lacking. In addition to these environmental signals, food availability and density of

worms on growth plates change dauer signals and have been shown to affect

chernotaxis and benzaidehyde adaptation (Colbert and Bargmann, 1997). Although

ali woms are prepared in the same way, it is possible that variation in the amount of

E. coli present on the NGM plates from one experiment to another may lead to

differences in nematode development that a f k t the worms abiiity to withstand the

effects of starvation at a later stage, such as during the odorant exposure period.

Another developmental consideration might be the absolute age of the worm; the

stage at which the worms are tested is on the cusp of adulthood, straddling the

reproductive peak. If it is the case that age plays such an important role, then a

46

simple experiment comparing naive chemotaxic approach to low concentratiow of

diacetyl in different aged Worms should address this issue. Attempts at tlus have

yielded inconclusive results, since woms that are younger or older in age than those

presently used have movement deficits which affect their chemotaxis to volatile

odorants (unpublished obseruntions). The multitude of environmental factors which

may affect the sensitive response to low DA concentratiow do not appear to have

such a great influence on more intense stimuli such as the higher DA concentrations,

since cues such as 100% DA are so strong and may be potentially noxious so that it

would be evolutionarily disadvantageous for the animal to have a variable response

to such a salient cue.

Other experiments in drosophila have demonstrated that the mutants rutabaga

(rut) and dunce (dm) can show normal adaptation despite their inability to habituate

normally (Corfas and Dudai, 1990). Within studies investigaüng C. rlegans olfactory

plasticity, however, there has been a lack of interest in systematically categorizing

these independent processes. For example, Colbert and Bargmann (1995) have

demonstrated benzaldehyde 'adaptation' by showing that following a 90 minute pre-

exposure to benzaldehyde (sensed by the AWC neuron), worms exhibit a diminished

chemotaxis towards a test spot of this odorant. This decrement in approach was not

reversed after washing and centrifugation three times in a buffer, and animals were

only able to restore their initial attraction to the odorant after a waiting period of

three hours (spontaneous recovery). The animals' ability to restore their olfaction to

the odorant atiests that there was no permanent damage done to the worms.

However, a similar experiment (Nuttley and van der Kooy, subrnitted) has

demomtrated that dishabituation can be achieved after BZ pre-exposue if worms are

47

washed in dH20 and given a stronger centrifuga1 spin. Thus, a phenomenon

previously identified as olfactory adaptation has all the hallmark characteristics of

nonassociative learning, suggesting this type of odorant pre-exposure termed

adaptation may be a misclassifica tion and shouid be considered habituation. Here, 1

demonstrate that a decrement in DA chernotaxis after pre-exposure to DA can be

caused by both of these processes, and it is the ability of the worms to exhibit a return

to naive approach subsequent to a dishabituating stimulus that defines whether

adaptation or habituation is occurring. Although habituation has been traditionaily

defined as a decrement in response after repeated presentation of a single stimulus

(Harris, 1943; Thompson and Spencer, 1966), 1 consider this equivalent to a continuous

presentation of a stimulus for a given period of tirne, and my controls demonstrate

that the criteria for both nonassociative learning and adaptation c m be h M e d by

changing only the odorant concentrations (stimulus intensities) and the tirne of

exposure. The longer duration (60 minutes) necessary for animals to display a 50%

response decrement after pre-exposure to 100% DA (as opposed to the 15 minutes

needed to see similar habituation after 0.001 % DA pre-exposure) is likely a reflection

of the differences in molecular timing of the two distinct processes. Whereas

olfactory habituation may involve readily reversible processes such as receptor

phoshphory lation or similar manipulations in downstream secondary messenger

pathways, the longer the-course of adaptation may involve changes in gene

transcription leading to downregulation of olfactory receptors or upregulation of

other unidentified proteins which may act to inhibit chernotaxis via an adaptation-

dependent process. Such differences in timing also serve to explain why habituation

can be easily reversed with a dishabituating stimulus, whereaç adaptation requites a

48

two to three hour spontaneous recovery period before animals can demonstrate a

non-decremented respowe.

Experirnents with the odr-10 and odr-l mutants have allowed genetic dissection

of some of the molecular pathways involved in adaptation. Initial baseline approach

can be dissociated from adaptation, since odr-20 animals exhibit high naive CIs to

100% DA but do not adapt to this odorant, while odr-1 animals have a compromised

baseline CI to higher concentrations of DA (100% and 0.1%) but still demonstrate

adaptation. 1 conclude that the interactions of the hgh-affinity ODR-10 receptor

pathway in AWA with some downstream targets of the proposed low-affinity

receptor on AWC, either within AWC via a direct connection or through a common

downstream interneuron, such as AIZ, are essential for adaptation and that despite

the importance of odr-1 for mediating naive approach to 100% DA, it is not involved

in DA adaptation. In addition, the ODR-10 receptor alone also mediates responses

that lead to olfactory habituation and sensitization, however, the current lack of

knowledge regarding many downstream targets in the odr-10 pathway combined

with the fact that odr-10 animais do not approach low DA concentrations presents a

challenge to investigations of the role of the odr-10 gene in nonassociative learning.

The low naive approach to DA that was observed in odr-1 mutants is not

surprising given the postulated presence of a low-affinity receptor on AWC. It is

possible that this AWC receptor is signaling via the ODR-1 guanylyl cyclase and loss

of odr-1 leads to loss of the signaling such that only ODR-10 activity in AWA can

account for the residual approach to DA seen in odr-1 mutants. Although previously

published data suggest that approach to 0.1% DA is not irnpaired in odr-l mutants

(Bargmann et al. 1993), 1 find that the CI to 0.1% DA in odr-2 Worms is - 20% lower

49

than wüd-type. This deficit, however, was specific to DA because their approach to

another AWA-sewed odorant, pyrazine, was the same as wild-type controls.

Interesüngly, the observation that a compromised baseline approach to DA in odr-2

mutants did not prevent a 50% response decrement after 100% DA presxposure

highlights yet another difference between adaptation and habituation, since it was

noted that an habituation-related response decrement is less likely to occur when the

baseline approach to low DA concentrations is diminished. However, the trend of

habituation and dishabituation seen in odr-l animals pre-exposed to 1 0 % DA and

tested to 0.01% DA suggests that as the baseline to DA decreases and the effect of the

low-affinity receptor as part of a stimulus for adaptation is diminished,

nonassociative learning processes may be revealed.

These findings allow distinction of three separate behavioural effects of

diacetyl pre-exposure. The first type of behavioural plasticity caused by odorant pre-

exposure is habituation, as characterized by a decrement in diacetyl approach which

can be dishabituated back to naive levels. The second process underlying a

behavioural decrement in response is adaptation, which is likely caused by receptor

down-regdation or fatigue since the baseline response can return over tirne, but c m

not be dishabituated. The third form of plasticity, which has not been demonstrated

before in C. ekgans olfaction, is sensitization. This form of plasticity facilitates the

behavioural response causing an increase above baseline DA approach, or competing

with adaptation to prevent a decrement in DA approach. The development of these

paradigms in C. elegans have allowed the exploration of behavioural adaptation at a

genetic level. 1 propose that DA adaptation may rely on interactions between an

AWC-dependent (but odr-1-independent) process that relies on ODR-10 receptor

50

function in AWA. Further analyses at the molecular level will help elucidate the

underlying relationship between nonassociative learning and other forms of

olfactory plasticity by idenhfying both common and distinct genetic and cellular

pathways.

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52

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