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4.04a0005 Molecular Mechanisms of Habituation in C. elegansM. P. Butterfield and C. H. Rankin, University of British Columbia, Vancouver, BC, Canada
ª 2008 Elsevier Ltd. All rights reserved.
4.04.1 Introduction to Habituation 1
4.04.2 Caenorhabditis elegans as a Model System 1
4.04.3 Olfactory Habituation 2
4.04.4 Mechanosensory Habituation 3
4.04.4.1 Mechanosensation in C. elegans 3
4.04.4.2 Habituation 3
4.04.4.3 Behavioral Analyses of Short-Term Habituation 3
4.04.5 Neural Circuit 5
4.04.5.1 Identifying Neurons Involved in Habituation 5
4.04.5.2 Roles of Identified Neurons in Habituation 5
4.04.5.3 Localizing the Site of Plasticity in the Neural Circuit 7
4.04.6 Genetic Dissection of Short-Term Habituation 7
4.04.6.1 Role of Genes Involved in Glutamate Neurotransmission 7
4.04.6.2 Other Identified Components of Habituation 8
4.04.7 Analyses of Long-Term Habituation 9
4.04.7.1 Dependence on Protocol 9
4.04.7.2 Molecular Correlates of Memory for Habituation Training 9
4.04.8 Summary 10
References 11
s0005 4.04.1 Introduction to Habituation
p0005 The most basic form of learning is nonassociativelearning, which involves alterations in response to asingle (sometimes repeated) stimulus. Habituation,dishabituation, and sensitization are the three mainforms of nonassociative learning. Habituation is thesimplest form of nonassociative learning and hasbeen defined as a decrease in response to repeated orlong-lasting stimulation (
b0085
Groves and Thompson,1970). This form of learning has been found in allorganisms studied, from protozoa to humans. If astrong, novel stimulus is presented after the organismhas been habituated to the initial stimuli, the organismwill immediately recover its habituated response. Thisphenomenon is known as dishabituation and has beenused to distinguish habituation from sensory adapta-tion or fatigue. Although a great deal is known aboutthe characteristics of habituation, very little is knownabout the molecular mechanisms that underlie it.
p0010 Habituation allows an organism to ignore irrele-vant stimuli; thus it is the basis for selective attention.If organisms did not habituate, then they would giveequal attention to all stimuli in the environment andcould not attend to stimuli important for survival.
Further, the behavioral rules that govern habituationdescribed by
b0085
Groves and Thompson (1970) are fol-lowed in all species and systems studied. For thesereasons, the mechanisms underlying this simple formof learning are likely to be highly conserved through-out evolution. To accurately study such a simpleform of learning, it becomes increasingly importantto reduce any other factors that could confound suchstudy. Because of this, organisms that exhibit verysimple behaviors and that can easily be studied at thecellular and genetic levels provide good model sys-tems to discover the molecular mechanisms involvedin habituation.
s00104.04.2 Caenorhabditis elegansas a Model System
p0015Caenorhabditis elegans is a very powerful and usefulmodel system in which to study molecular mechanismsof simple forms of learning and memory.
b0140
Rankin et al.(1990) first showed that C. elegans are capable of a varietyof simple behaviors including habituation, dis-habituation, and long-term memory lasting for at least24 h. This nematode has a small nervous system that is
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composed of only 302 neurons, which allows for theunique ability to map behaviors to specific identifiedneurons and sites of plasticity (Figure 1). The connec-tivity of the nervous system has been resolved at theelectron microscope level and has produced a completewiring diagram indicating 5000 chemical synapses and3000 electrical synapses (
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White et al., 1986). Further,the entire cell lineage has been investigated and deter-mined. Combining this knowledge, researchers havebeen able ablate single identified neurons and investi-gate the behavioral outcome (
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Chalfie et al., 1985). Also,since the cuticle of this nematode is transparent, the useof laser ablation of single, identified neurons has madecircuit analysis possible, and the use of genetic markerssuch as green fluorescent protein (GFP) has allowed theanalysis of changes in expression patterns of specificgene products in vivo (
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Chalfie et al., 1994). The worm’sgenome has been mapped and sequenced, which has ledto the identification of numerous genes and gene pro-ducts (
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Wood, 1988;b0155
Riddle et al., 1997). Also, thegenetics of C. elegans is easy to manipulate. The use ofboth forward and backward genetic screens and, morerecently, RNA interference techniques has led to theidentification of a large number of genes that play a rolein mediating various behaviors. Another major advan-tage of this model system is that it is relatively simple tomanipulate gene expression spatially or temporally,allowing for the investigation of the roles of specificgenes in targeted tissues and/or at specified time points.
s0015 4.04.3 Olfactory Habituation
p0020 The presentation of an olfactory stimulus can lead toa chemotaxic response, which is the migration towardor away from that stimulus. C. elegans will perform
both of these behaviors; it is attracted to various ions,amines, and some volatile substances (ketones, esters,etc.), and it is repelled by acidic pH, D-tryptophan,and various volatile substances (benzaldehyde, octa-nol, etc.;
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Bargmann and Mori, 1997). Continuous orrepeated presentation of such compounds can resultin a decrement of the chemotaxic response (
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Colbertand Bargmann, 1995).
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Bernhard and Van der Kooy(2000) showed that this decrease in behavioralresponse could be mediated by two forms of olfactoryplasticity: adaptation and habituation. Althoughadaptation can be considered a form of plasticity, itis not considered to be a form of learning because theresponse decrement is mediated by sensory fatigue,and the response will return to baseline levels onlyafter sufficient time is allowed for the sensory systemto recover. On the other hand, habituation to olfac-tory stimuli is considered a learning process because,although a similar response decrement occurs as withadaptation, when a novel or noxious stimulus isadministered, dishabituation will occur.
p0025Using solubilized Naþ (an attractant ion),b0195
Wen et al.(1997) showed that olfactory habituation can occur inC. elegans. They showed that, when exposed to anattractant (75 mmol�1 NaCH3COO) for a prolongedtime, the chemotaxic response to migrate toward thatattractant diminished. But when worms were exposedto a much higher concentration of NaCH3COO(300 mmol�1) for a brief period of time and thengiven the same behavioral assay, the habituatedresponse returned to near baseline levels, indicatingthat dishabituation had occurred and that this beha-vioral decrement was habituation and not adaptation.
p0030To investigate the differences between the pro-cesses of habituation and adaptation,
b0025
Bernhardand Van der Kooy (2000) varied preexposure
Head sensory receptors Ring ganglia
Nerve ring
Retrovesicularganglion
Motor neuron cell bodies in theventral cord
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Motor neuroncommissures Dorsal cord Dorsorectal ganglion (internal)
Lumbar ganglion (lateral)Preanal ganglion
DA7DB7
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f0005 Figure 1 The nervous system of C. elegans. Top: C. elegans nervous system labeled with a pan-neuronal GFP (photo
courtesy of W. Materi and D. Pilgrim). Worm is 1 mm in length. Bottom: Anatomical drawing of the nervous system of
C. elegans. Drawing courtesy of Richard Durbin.
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concentrations of the volatile odorant diacetyl (DA)within a single paradigm. They found that preexpos-ing and testing worms in high concentrations of DAinduced a nonreversible decrement in chemotaxicresponse despite the introduction of a strong, novelstimulus. When preexposed to an intermediate con-centration of DA, no decrement of response wasobserved. Interestingly, at very low concentrationsof preexposure and testing, worms exhibited a decre-ment in response that could be dishabituated. Takentogether, these data suggest that the processes ofolfactory plasticity can be dissociated from habitua-tion by the concentration of DA used, withadaptation requiring high concentrations of DA andhabituation requiring low concentrations of DA.
p0035 Although a number of genes important for adap-tation have been identified, little is known about thegenetics of olfactory habituation.
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Wen et al. (1997)showed that mutant worms, lrn-1 and lrn-2, had def-icits in classical conditioning associative learningparadigms but showed no deficits in nonassociativehabituation. However, more recently,
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Morrison andvan der Kooy (2001) showed that a mutation in analpha-amino-3-hydroxyl-5-methyl-4-isoxazolepro-pionate (AMPA)-type glutamate receptor subunit,glr-1, impaired both olfactory associative learningand habituation. These data suggest that the mechan-isms involved in associative learning and habituationmay be dissociable at the level of the lrn-1 and lrn-2
genes but share a common pathway that involves theglr-1 gene.
s0020 4.04.4 Mechanosensory Habituation
s0025 4.04.4.1 Mechanosensation in C. elegans
p0040 Mechanosensation, which is the transduction ofmechanical force into intracellular signals, allowsorganisms to sense touch, vibration, and other tactilestimuli. In C. elegans, mechanosensation has been stu-died in two major ways: first, touch to the body fromgentle stimulation with a small hair and, second,vibration felt through the surrounding environmentresulting from the administration of a mechanical tapdelivered to the side of a Petri dish (
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Garcia-Anoverosand Corey, 1997). Most of the research on habitua-tion has been done using the tap stimulus. Theadvantage of using the tap stimulus as opposed tothe body touch when studying mechanosensorybehavior is that the strength of stimulation can becontrolled using a machine, whereas there isuncontrollable variation when delivering touch
using a handheld device. In response to a tapstimulus, a worm will respond by swimming back-ward (a reversal), which has been termed thetap-withdrawal response.
s00304.04.4.2 Habituation
p0045Using the tap-withdrawal response,b0140
Rankin et al.(1990) were the first to show that C. elegans is capableof nonassociative learning. When they administered atap stimulus to the side of the Petri plate holding theworm, they observed that the distance that the wormsreversed in response to the tap decreased when thestimulus was repeated at regular intervals. Theresponse returned to baseline levels (spontaneousrecovery) a few minutes following the last tap stimu-lus. To ensure that this response decrement washabituation and not sensory fatigue, they followedhabituation training with a brief electrical shock inorder to dishabituate the worms. Following shock,their response to tap increased significantly abovethe habituated level, indicating that the electricalshock had induced dishabituation, and the originalresponse decrement observed was habituation andnot adaptation or fatigue.
s00354.04.4.3 Behavioral Analyses of Short-Term Habituation
p0050b0185
Thompson and Spencer (1966) andb0085
Groves andThompson (1970) laid out the behavioral character-istics of habituation. These same criteria are usedtoday to define habituation, and so far, all speciesstudied show these same characteristics. Having abehavior well characterized is an asset when tryingto determine underlying cellular mechanisms. It isimportant for researchers to make constant compar-isons between behavior and the hypothesized cellularmechanisms in order to develop a greater under-standing of the factors that govern habituation.
p0055In their descriptions of habituation,b0185
Thompsonand Spencer (1966) and
b0085
Groves and Thompson(1970) missed one aspect of habituation that iscommon in all systems studied and can be used tofind clues about possible molecular mechanisms ofhabituation. These early papers stated that habitua-tion is sensitive to frequency, with high-frequencystimuli producing more rapid habituation than low-frequency stimuli, and that habituation recoversspontaneously. Both of these are correct; however,in all species studied, frequency also affects the rateof spontaneous recovery, with high-frequency
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stimulation leading to more rapid spontaneousrecovery than low-frequency stimulation (
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Rankinand Broster, 1992; Figure 2(a)).
b0145
Rankin and Broster(1992) showed that in C. elegans this relationship ofspontaneous recovery to frequency of stimulationheld, regardless of the number of stimuli delivered(as long as decrement had reached asymptotic levels)and regardless of the level of habituation reached(when levels of habituation were matched betweenworms habituated with high and low frequencies, rateof recovery was still dependent on frequency of thehabituation). This is important for two reasons. Thefirst is that this difference is the opposite of whatwould be predicted by fatigue or adaptation in that,
in both of those cases, the more complete the decre-ment the longer the recovery. With high frequencythe decrement is rapid and often complete, butrecovery is very rapid, while with low frequencythe decrement is not complete and yet recoverytakes much longer than for high-frequency stimula-tion. Thus, the sensitivity of spontaneous recovery tothe frequency of stimulation is a second way, inaddition to dishabituation, to distinguish whether abehavioral decrement is the result of habituation, orthe result of sensory adaptation or motor fatigue. Thesecond reason that the sensitivity of recovery tofrequency of stimulation is important is the deduc-tions one can draw from this about molecular
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f0010 Figure 2 (a) Reversal responses of wild-type worms shown as the mean percent initial response across 30 tap stimuli with
three recovery taps at 30 s, 5 min, and 10 min after habituation training. The 10-s interstimulus interval (ISI) group shows more
rapid habituation and a lower asymptotic level when compared to the 60-s ISI group. The 10-s ISI group also shows faster and
more rapid recovery following habituation training than the 60-s ISI group. (b) Mean percentage of control responsemagnitude for long-term memory for five test stimuli 24 h following habituation training given at a 60-s ISI. The significantly
lower level of responding in the trained group compared to the control group indicates the retention of memory for habituation
training. (c) Mean percentage of control response magnitude for long-term memory for five test taps 24 h following habituation
training given at a 10-s ISI. The lack of memory observed when training is given at a 10-s ISI suggests either that 60-s ISItraining selectively recruits molecular mechanisms needed to induce the formation of long-term memory for habituation
training, or that 10-s ISI training recruits molecular mechanisms that block the formation of long-term memory (Butterfield and
Rankin, unpublished results, 2006).
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mechanisms of habituation. If an animal that has habi-tuated to stimuli at a high frequency (i.e., shortinterstimulus intervals, ISIs) recovers rapidly, whilean animal habituated to the same level to a low fre-quency recovers more slowly, this indicates that thetwo habituated animals are not the same, and somedifferent processes have been activated in the neuronsof the two animals to regulate recovery differently.From this observation
b0145
Rankin and Broster (1992)hypothesized that habituation was not mediated by asingle molecular mechanism, but that stimulation atdifferent frequencies recruited different cellularmechanisms. The observation that long ISIs can beused to produce long-term memory for habituation,while short ISIs cannot, provides further support forthis hypothesis (
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Beck and Rankin, 1997; Figures 2(b)and 2(c)).
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Broster and Rankin (1994) hypothesizedthat, if different ISIs recruited different molecularmechanisms, then studies of genes involved in habi-tuation should lead, at the very least, to the discoveryof genes that play a role in habituation to all frequen-cies, genes that play a role in habituation to highfrequencies, and genes that play a role in habituationto low frequencies. We have recently found supportfor this hypothesis and identified two genes that areinvolved in short-term habituation. One specificallyaffects habituation at high frequencies; the other spe-cifically affects habituation at low frequencies (Rankin,unpublished data, 2006).
s0040 4.04.5 Neural Circuit
s0045 4.04.5.1 Identifying Neurons Involvedin Habituation
p0060 Once the behavior was well characterized, the nextstep in discovering the molecular mechanismsinvolved in habituation to tap stimuli was to identifythe neural circuit responsible for this behavior. Theneural circuit underlying the behaviors of backwardswimming in response to head touch and forwardswimming in response to tail touch was characterizedby
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Chalfie et al. (1985).b0205
Wicks and Rankin (1995)investigated the neural circuit underlying the tap-withdrawal response by studying the effects of laserablating cells in the head and tail touch circuits on theresponse to tap and found that the response to tapinvolves integration of sensory input from both thehead and tail. The neural circuit for the response totap consists of five mechanosensory cells, two bilater-ally paired PLM neurons that transduce tail touch,and 2 bilaterally paired ALM neurons and a single
AVM neuron that transduce head touch; these sensoryneurons synapse onto four pairs of command inter-neurons that mediate forward (AVAs and AVDs) andbackward movement (AVBs and PVCs; Figure 3(a)).Ablation of all sensory cells completely abolished theresponse to tap (
b0205
Wicks and Rankin, 1995). Ablation ofonly the head touch neurons (ALMs and AVM)resulted in consistent forward swimming in responseto tap (termed an acceleration) in contrast to theconsistent reversal responses seen in intact worms.Similarly, backward swimming was always seen inresponse to tap in PLM ablated (PLM–) worms.These reversals were larger than the reversals of intactanimals, suggesting that in the intact worms the effectof stimulation of the tail cells by the tap competes withthe effect of stimulation of the head touch cells andmoderates the response size. Interestingly, ablation ofAVM only resulted in a decrease in reversal frequencyand magnitude of reversals and an increase in accel-eration (forward swimming) frequency. In a study ofthe response to tap across development, it wasobserved that, at younger stages, worms respondedby both reversing and accelerating at equal frequen-cies, similar to what is observed in AVM– worms(
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Chiba and Rankin, 1990). Since AVM is not presentat hatching and only becomes fully functional inyoung adults,
b0060
Chiba and Rankin (1990) suggestedthat the shift in adult worms to predominantly rever-sing response to tap may be mediated by thedevelopment of AVM. All these results combinedindicated that the response to tap was mediated bythe integration of inputs from two competing neuralcircuits, one driving forward movement and one driv-ing backward movement.
s00504.04.5.2 Roles of Identified Neuronsin Habituation
p0065To identify the sites of changes in the pathway(s)underlying habituation of the tap-withdrawalresponse,
b0215
Wicks and Rankin (1996b) laser ablatedspecific touch cells and observed any changes inhabituation rate or asymptotic level. Laser ablationof the PLM sensory neurons results in consistentbackward movement in response to tap. Whengiven the short-term habituation training at a 10-sand 60-s ISI, PLM– worms showed habituation atboth ISIs. The initial slope of the PLM– group wassmaller than that of the intact group (Figures 3(b)and 3(c)). When
b0215
Wicks and Rankin (1996b) investi-gated the role of the touch cells of the anteriormechanosensory field (AVM and ALM), they found
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results that were quite different from those of thePLM– group. Worms with ablations in the headsensory neurons accelerate forward in response totap. They found that both ALM– and ALM, AVM–groups habituated slower at a 10-s ISI than at a 60-sISI, and they showed an initial response facilitationprior to the decrement that was especially strongwhen habituated at a 10-s ISI (Figure 3(d)).Further, the ALM, AVM– group had a significantlyhigher asymptotic level at a 10-s ISI as opposed to the
60-s ISI. Finally, ablation of one of the pairs ofcommand interneurons (PVCs), thought to modulateforward movement, resulted in worms that reversed100% of the time; however, unlike the PLM– worms,the reversals seen in PVC– worms were not signifi-cantly different in magnitude from control worms.The results support the hypothesis of
b0045
Chalfie et al.(1985) that the PLM cells make inhibitory chemicalconnections to the head touch circuit interneurons(AVD and AVA); these connections remain intact
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f0015 Figure 3 (a) Simplified neural circuit underlying tap withdrawal. The circuit consists of five mechanosensory neurons
(triangles), eight command interneurons (circles), and two motor neuron pools (squares). All cells represent bilateral classes of
cells except ALM, which is a single cell. The arrows and dotted lines represent chemical synapses and gap junctions,respectively. The number of synaptic contacts is proportional to the width of the arrows. The red-colored arrows indicate the
synaptic connections that have been hypothesized to be the sites of plasticity that mediate habituation (b0220
Wicks and Rankin,
1997;b0095
Kitamura et al., 2001). (b, c) Scatter plot graphs fitted with best fit lines (bf) demonstrating the effect of ISI (10 s vs. 60 s)
on the kinetics of habituation of the reversal response in intact animals (control) and PLM-ablated animals (PLM–). (d) Scatterplot graph showing the effects of ablation of ALM and AVM on the kinetics if habituation of acceleration responses.
b0215
Wicks
and Rankin (1996b) hypothesized that the responses of both the PLM– and AVM, ALM– groups combine to make up the
behavior we see in the intact animal. Figure adapted from Wicks SR and Rankin CH (1996b). The integration of antagonisticreflexes revealed by laser ablation of identified neurons determines habituation kinetics of the Caenorhabditis elegans tap
withdrawal response. J. Comp. Physiol. A 179: 675–685, with permission from Springer-Verlag.
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following PVC ablation and continue to competewith the head sensory neuron stimulation. Takentogether, the results of ablation of cells important inboth the forward and backward motion pathwayshave shown that reversals and accelerations habituateat different rates; thus their relative contribution tothe intact response varies over the course of habitua-tion. For example, at a 10-s ISI the initial facilitationof the acceleration response competes more stronglywith the reversals and the reversals seen in intactworms decrease in amplitude very quickly. This sug-gests that to understand the habituation of behavior itis not sufficient to study the mechanisms underlyingthe decrement in a single cell, but it may be necessaryto understand the effect of repeated stimulation on allaspects of the neural circuits underlying the behavior.
s0055 4.04.5.3 Localizing the Site of Plasticityin the Neural Circuit
p0070 Two behaviors that share command interneurons andmotor neuron pools with the tap-withdrawalresponse are thermal sensation and spontaneousreversing. All of these behaviors differ from tap atthe level of sensory input.
b0220
Wicks and Rankin (1997)hypothesized that, if the site of plasticity that med-iates habituation is located at the sensory input level,then habituation to tap should not alter baselinelevels of both the response to a heat probe and spon-taneous reversing. On the other hand, if the site ofplasticity is at the level of the interneurons and motorneurons, then these behaviors should be altered byhabituation training in response to tap.
b0220
Wicks andRankin (1997) found that habituation to tap did notaffect other behaviors, thus providing data to supportthe hypothesis that the site of plasticity lies in thetouch cells and/or the synaptic connections theymake onto the interneurons.
p0075 Investigation into habituation of anterior bodytouch has also led to increased knowledge of howhabituation occurs in the neural circuit.
b0095
Kitamuraet al. (2001) performed gentle body touch using ahair at a 15-s ISI, using intact worms, and observedthe expected kinetics of habituation with an initial,rapid decrement of reversal magnitude followed byan asymptotic level of that response. Through sys-tematic ablations of combinations of neuronsinvolved in the response to anterior body touch(ALM, AVM, AVD, and PVC), they found thatlaser ablation of both AVD interneurons resulted insignificantly more rapid habituation than observed inintact animals. The ablation of any of the other
interneurons had no effect on the rate of habituation,suggesting that the AVD interneurons play a criticalrole in the habituation to anterior body touch.b0095
Kitamura et al. (2001) also found that coablating theright ALM and the AVM neurons resulted in rapidhabituation, suggesting that in these animals thechemical synapse that the left ALM makes with theright AVD and PVC interneurons is responsiblefor the behavior observed. Further investigation ledto the conclusion that the chemical synapses betweenthe left ALM sensory neurons and the PVC inter-neurons mediate the rapid habituation of thecoablated worms. The fact that more rapid habitua-tion can be attributed to at least two sites in theneural circuit suggests that each synapse in the cir-cuit may have the potential to be the site of plasticity.
p0080Taken together, the studies performed by bothb0205
Wicks and Rankin (1995)b0095
and Kitamura et al. (2001)show that habituation to mechanosensory stimuli inintact animals involves the integration of a variety ofinputs. However, the most likely site of plasticityappears to be situated at the level of the chemicalsynapses between the sensory neurons and their tar-get interneurons.
s00604.04.6 Genetic Dissection ofShort-Term Habituation
s00654.04.6.1 Role of Genes Involved inGlutamate Neurotransmission
p0085A number of genes involved in glutamate neurotrans-mission are expressed in the touch cells and thecommand interneurons, suggesting that glutamater-gic transmission plays a major role in the response totap. Presynaptically, in the sensory neurons (ALM,AVM, and PLM) a homologue of a mammalianglutamate vesicular transporter, known as EAT-4, isexpressed (
b0100
Lee et al., 1999). Postsynaptically, inthe command interneurons (AVA, AVB, AVD, andPVC) homologues of both the mammalian AMPA/Kainate-type and n-methyl-D-aspartate (NMDA)glutamatergic receptors, GLR-1 and NMR-1,respectively, are expressed (
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Hart et al., 1995;b0120
Maricqet al., 1995;
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Brockie et al., 2001). If glutamatergictransmission plays a critical role in habituation inC. elegans, then worms that lack, or have mutationsin, one or more of these genes should have alteredpatterns of habituation.
p0090b0150
Rankin and Wicks (2000) first examined eat-4
mutants using the short-term habituation paradigm.Initially, they found that, when compared to
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wild-type worms, there was no difference in theinitial response to the tap stimulus. When givenrepeated stimulation, at both 10- and 60-s ISIs, eat-4
worms habituated more rapidly and reached a lowerasymptotic level than wild-type worms. Similarly,eat-4 worms showed much slower spontaneous recov-ery following habituation training; however, thedependence on ISI was still present in both habitua-tion and recovery kinetics (faster decrement andfaster recovery from short ISI training as comparedto long ISI training). This result suggests that theabsence of EAT-4 disrupts one or more cellularmechanisms of habituation but leaves others (ISIdependent processes) intact. Interestingly, eat-4 isalso the first gene that has been shown to play arole in dishabituation. When given a dishabituatingstimulus (an electric shock), the eat-4 mutants did notshow facilitation of the response above the habituatedlevel, indicating that they did not dishabituate. Sinceeat-4 worms still show ISI-dependent spontaneousrecovery, the decrement seen in these worms is habi-tuation and not fatigue or adaptation. Because we donot know the relationship between the molecularmechanisms of habituation and the molecularmechanisms of dishabituation, these results illustratethe importance of having more than a single way todistinguish habituation from fatigue. Using a trans-genic rescue strain (DA1242) produced by
b0100
Lee et al.(1999),
b0150
Rankin and Wicks (2000) showed thatwild-type habituation and dishabituation behaviorswere also rescued. These studies with eat-4 wormssupport the hypothesis that glutamatergic transmis-sion plays an important role in habituation of thetap-withdrawal response and that the glutamate vesi-cular transporter is essential for dishabituation.
p0095 Because eat-4 worms did not respond differentlyfrom wild-type in response to the initial tap stimulibut differed only after repeated stimuli,
b0150
Rankin andWicks (2000) concluded that EAT-4 (a glutamatevesicular transporter) is not required for glutamater-gic transmission but, rather, is required for sustainedsynaptic activity. The hypothesis is that the touchsensory neurons of eat-4 worms have fewer gluta-mate-filled vesicles than those of wild-type worms,and so they are quickly exhausted in response torepeated stimulation. The importance of neurotrans-mitter vesicles in habituation is supported by work inAplysia that has shown that there are fewer synapticvesicles in the active zones of sensory neurons fromhabituated animals than from the terminals of non-habituated animals (
b0005
Bailey and Chen, 1988). Theregulation of the amount of glutamate in vesicles in
the terminals of the sensory neurons and/or theregulation of vesicular release are the most likelymechanisms underlying the behavioral changesobserved during habituation.
p0100Since the deficits in presynaptic release of gluta-mate seen in eat-4 worms alter the kinetics ofhabituation to tap, it was suggested that postsynapticglutamate receptors should also play a significant rolein the same behavior. Genes for four ionotropicglutamate receptors are expressed on the commandinterneurons: glr-1, glr-2, nmr-1, and nmr-2 (
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Hart et al.,1995;
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Maricq et al., 1995;b0030
Brockie et al., 2001). glr-1
and glr-2 have been shown to form heteromericreceptors (
b0055
Chang and Rongo, 2005), and nmr-1 andnmr-2 are thought to form heteromeric receptors aswell. Studies to date have focused mainly on muta-tions in glr-1 and nmr-1. When given habituationtraining, glr-1 worms showed smaller initial responsesto tap than wild-type animals but showed relativelynormal short-term habituation to 10- and 60-s ISIswhen compared to wild-type worms (
b0165
Rose et al.,2002). nmr-1 worms showed normal short-termhabituation, indistinguishable from wild-type. Theresults thus far indicate that none of the glutamatereceptor genes tested alone gives the same pattern asEAT-4–deficient worms, which suggests that habi-tuation may be mediated postsynaptically by theactivation of an, as yet, unidentified glutamate recep-tor or by the simultaneous activation of multipleglutamate receptors.
s00704.04.6.2 Other Identified Componentsof Habituation
p0105b0175
Sanyal et al. (2004) showed that dopamine, whichplays an important role in behavioral plasticity inmany mammalian systems, also may play an impor-tant role in modulating C. elegans habituation.
b0175
Sanyalet al. (2004) studied mutations in two genes involvedin dopamine regulation and neurotransmission; dop-1
mutants, which do not express a dopamine receptoron neurons involved in the mechanosensory circuit,show altered patterns of habituation compared towild-type worms, as do cat-2 mutants, which lack anenzyme that is required for dopamine synthesis.Sanyal et al. found that dop-1 mutants and cat-2
mutants had a more rapid decrement in reversalfrequency (the number of worms that respond toeach tap) than wild-type worms. However, whenmeasuring reversal length (the dependent variablein all previous studies mentioned), there was nodifference among all three groups. This result
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suggests two alternative hypotheses: the first is thatdifferent mechanisms regulate the decrease in theprobability of a reversal response and the size of theresponse; the second is that dopamine may not beinvolved in habituation to tap, but instead maymodulate the integration of sensory stimuli fromthe head and tail touch circuits.
p0110b0230
Xu et al. (2002) performed a forward geneticscreen on habituation to tap and isolated hab-1, amutant which habituated more slowly and respondedat a higher asymptotic level than wild-type wormswhen tested at both 2- and 10-s ISIs. Further, thehab-1 mutants responded like wild-type worms to theinitial tap and also responded normally to a dis-habituating stimulus. Unfortunately, the geneproduct of hab-1 has yet to be identified. However,the observed pattern of slower habituation is oppositeto the results seen with both eat-4 and dop-1, suggest-ing that the gene product of hab-1 may play anantagonistic role to the molecular mechanisms ofglutamatergic and dopamine signaling that ishypothesized to underlie short-term habituation.
s0075 4.04.7 Analyses of Long-TermHabituation
s0080 4.04.7.1 Dependence on Protocol
p0115 Analysis of long-term memory for habituation to thetap-withdrawal response has led to increased knowl-edge and further understanding of the mechanismsthat govern habituation behavior.
b0140
Rankin et al. (1990)were the first to report long-term memory habituationto tap. Using a modified protocol from experimentswith Aplysia (
b0040
Carew et al., 1972), they were able toshow that if worms were given distributed habituationtraining they were able to retain memory for thattraining for at least 24 h. To investigate the behavioralparameters that reliably produce long-term memoryfor habituation,
b0020
Beck and Rankin (1997) examined ISIand type of training. They replicated the finding ofb0140
Rankin et al. (1990) that long-term memory could beproduced by distributed training, and they found that,if they used a massed training protocol where allstimuli were delivered without break periods insteadof a distributed training protocol, long-term memorywas not observed. In addition, they found that long-term memory could not be reliably produced fromtraining at a 10-s ISI, whereas it was produced usinga 60-s ISI. This result supports the hypothesis ofb0145
Rankin and Broster (1992) that there may be specificcellular mechanisms, which are recruited by training
with a 60-s ISI or inhibited by training with a 10-s ISI,to induce memory formation during habituation train-ing (Figures 2(b) and 2(c)). Taken together, these datashow that long-term memory of habituation training ismost reliably produced when stimuli are administeredat longer ISIs in a distributed or spaced manner. Thisparallels observations made in studies that found thatdistributed training is superior to massed training forthe induction of long-term memory in many species,including Aplysia, Drosophila, and humans (
b0070
Ebbinghaus,1885;
b0040
Carew et al., 1972;b0190
Tully and Quinn, 1985).
s00854.04.7.2 Molecular Correlates of Memoryfor Habituation Training
p0120Since distributed training was essential for the induc-tion of long-term memory for habituation training, itwas hypothesized by
b0015
Beck and Rankin (1995) that themechanisms that are responsible for consolidation ofthe memory were most likely occurring during theinterblock intervals. To assess this,
b0015
Beck and Rankin(1995) used heat shock (32 �C) to block proteinsynthesis and disrupt these cellular mechanisms.The cellular response to heat shock that was firstobserved in Drosophila has been shown to be same inevery organism studied (
b0180
Schlesinger et al., 1982). Theresponse is the termination of protein synthesis of allproteins other than a class of proteins called heatshock proteins, the production of which is signifi-cantly increased. To determine the timing ofmemory consolidation of habituation training, heatshock was delivered before, during, or after training.b0015
Beck and Rankin (1995) found that disruption ofmemory consolidation by heat shock occurred duringbut not before or after habituation training, whichsupported their hypothesis that some of mechanismsthat mediate the induction of long-term memory areoccurring during the interblock periods. As well, theyfound that neither the kinetics of habituation nor theinitial response to tap was affected by heat shocktreatment. These data suggest that repeated tap sti-mulation with intervals between blocks triggersmolecular mechanisms that involve protein synthesis.
p0125Since the research with eat-4 mutants suggestedthat glutamatergic neurotransmission plays a pivotalrole in short-term habituation,
b0165
Rose et al. (2002)hypothesized it might also play a role in long-termhabituation. To investigate this,
b0165
Rose et al. (2002)used the eat-4 mutants to test for the presence oflong-term memory for habituation training usingthe distributed training protocol. Interestingly, eventhough eat-4 mutants habituate faster and more
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completely than wild-type worms, they did notretain memory for this training 24 h later. This sug-gests that the sustained glutamate release that isimportant in short-term habituation is also criticalto the formation of long-term memory. Rose et al.used the eat-4 rescue strain, DA1242, and found thatDA1242 worms showed normal memory 24 h aftertraining; this supports the hypothesis that presynapticglutamate release is essential for the formation oflong-term memory.
p0130 Because release of presynaptic glutamate appearedto be essential for the induction of long-term memory,b0165
Rose et al. (2002) hypothesized that postsynaptic glu-tamatergic receptors might be involved in aspects oflong-term memory for habituation as well. To exam-ine this hypothesis
b0170
Rose et al. (2003) tested wormswith mutations in glr-1, a homologue of mammalianAMPA/Kainate glutamate receptor subunit (GluR1)and worms with a mutation in nmr-1, a homologue ofmammalian NMDA-type glutamate receptor subunit(NR1). They found that worms with a mutationin nmr-1 showed normal long-term memory forhabituation. In contrast, glr-1 worms showed nolong-term memory for habituation. To confirm theimportance of glutamate receptors in the formationof long-term memory, wild-type worms were treatedwith DNQX, a competitive non-NMDA glutamatereceptor antagonist, during training. Treatment withDNQX had no effect on short-term habituation, butit did block the formation of long-term memory(
b0170
Rose et al., 2003). These data suggest that glr-1
plays a critical role in the induction of long-termmemory but is not an essential component forshort-term habituation.
p0135 The role of glutamate receptors in synaptic plas-ticity and memory formation has been extensivelystudied in mammalian systems. The most prominentand well-characterized forms of synaptic plasticitythat may underlie mammalian memory formationare long-term potentiation (LTP) and long-termdepression (LTD). Both LTP and LTD involvechanges in synaptic expression levels of GluR1-containing AMPA-type glutamate receptors and traf-ficking of the receptors to and from the postsynapticmembrane (
b0110
Malinow and Malenka, 2002). To exam-ine whether similar processes were involved in theinduction of memory for habituation training
b0170
Roseet al. (2003) tested whether habituation trainingaffected the expression pattern of the GluR1 homo-logue, GLR-1. Using worms carrying chimericreceptors made up of GLR-1 tagged with GFP(GLR-1::GFP), they were able to visualize changes
in punctate glr-1 expression (b0160
Rongo and Kaplan,1999) along the ventral nerve cord (an area thatcorresponds to important synaptic sites reported inelectron microscopy studies;
b0200
White et al., 1986).They found that, following distributed habituationtraining, the number of GLR-1::GFP puncta alongthe ventral nerve cord did not change, but the size ofthe puncta in trained worms was significantly smallerthan in control worms (Figures 4(a) and 4(b)). Thisresult suggests that the distributed habituation train-ing did not alter the number of synapses along thenerve cord but rather reduced the number of recep-tors expressed per synapse on the interneurons.Further, blocking protein synthesis by applying heatshock during training blocked the downregulation ofGLR-1::GFP expression. These data suggest that,although glr-1 does not appear to play an importantrole in short-term habituation, its regulation criti-cally mediates long-term memory for habituation.
s00904.04.8 Summary
p0140The evidence discussed has shown that C. elegans canhabituate to both olfactory stimuli and mechanosen-sory stimuli. Little is known about olfactoryhabituation other than that it appears to require lowdoses of the chemical stimuli and is dependent on theglr-1 gene. On the other hand, mechanosensory habi-tuation has been studied at the level of behavior,neural components, and genes involved. Using thetap-withdrawal paradigm, the dependence upon ISIappears to mediate a large number of aspects of habi-tuation, including the rate of habituation, the level ofhabituation, and spontaneous recovery from habitua-tion. The neural circuit that mediates habituation totap has been thoroughly investigated, and this has ledto the hypothesis that the most likely sites of plasticityare the synaptic connections between the sensoryneurons and the command interneurons. Thus far,the molecular components that affect habituationinclude presynaptic glutamate release and dopaminer-gic neurotransmission, and are independent of theactivation of glr-1. However, distributed habituationtraining that induces long-term memory recruits othercellular processes. These processes have been shownto be protein synthesis dependent and rely heavilyupon the alterations in the expression of glr-1.
p0145Taken together, these data suggest that habitua-tion is not a simple or singular process but, rather, ismediated by a complex set of events that incorporatesa variety of molecular mechanisms. These events
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Fintegrate multiple neurotransmitters, multiple recep-tors, and multiple subcircuits, each mediatingdifferent aspects of habituation. Rather than therebeing a single mechanism of habituation, the datafrom C. elegans lead to the hypothesis that there aremultiple mechanisms that can underlie this see-mingly simple response decrement. Because of itswell-studied genetics, physiology, and behavior,C. elegans is now, and will continue to be, a verypowerful model system in which to elucidate thecellular events and processes that underlie key com-ponents involved in learning and memory.
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