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Biochemical Modulation of NMDA Receptors: Rolein Conditioned Taste Aversion*

Beatriz Jiménez1 and Ricardo Tapia1,2

(Accepted June 12, 2003)

Glutamate neurotransmission plays a crucial role in a variety of functions in the central nervoussystem, including learning and memory. However, little is known about the mechanisms underly-ing this process in mammals because of the scarceness of experimental models that permit corre-lation of behavioral and biochemical changes occurring during the different stages of learning andthe retrieval of the acquired information. One model that has been useful to study these mecha-nisms is conditioned taste aversion (CTA), a paradigm in which animals learn to avoid new tasteswhen they are associated with gastrointestinal malaise. Glutamate receptors of the N-methyl-D-aspartate (NMDA) type appear to be necessary in this process, because blockade of this receptorprevents CTA. Phosphorylation of the main subunits of the NMDA receptor is a well-establishedbiochemical mechanism for the modulation of the receptor response. Such modulation seems to beinvolved in CTA, because inhibitors of protein kinase C (PKC) block CTA acquisition and becausethe exposure to an unfamiliar taste results in an increased phosphorylation of tyrosine and serineresidues of the NR2B subunit of the receptor in the insular cortex, the cerebral region where gus-tatory and visceral information converge. In this work we review these mechanisms of NMDAreceptor modulation in CTA.

KEY WORDS: Synaptic plasticity; learning and memory; NMDA receptors; conditioned taste aversion; serinephosphorylation.

INTRODUCTION

Glutamate is the main excitatory neurotransmitterin the mammalian central nervous system (CNS). Twomajor classes of glutamate receptors have been identi-fied, the ionotropic and the metabotropic receptors.Ionotropic receptors are ligand-gated cationic channelsthat when activated cause neuronal depolarizationby generating fast excitatory postsynaptic poten-tials. These receptors are classified, according to theirsensitivity to nonphysiological glutamate analogs, as N-

methyl-D-aspartate (NMDA), �-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and kainicacid (KA) types. The metabotropic receptors are coupledto G-proteins, and the glutamate-induced signal is trans-duced to several kinds of intracellular messengers (1–3).

It is well established that glutamate-mediated neu-rotransmission plays a critical role in the function ofseveral neuronal circuits in the CNS. The activation ofglutamate receptors is involved in neuronal survival andin the refinement of neuronal connections during braindevelopment, as well as in the phenomenon of synapticplasticity underlying learning and memory (4–6). How-ever, the overactivity of glutamate receptors, particularlyof the NMDA type, results in massive Ca2� influx anda consequent elevation of the intracellular concentrationof the cation, producing hyperexcitation and neuronaldeath (excitotoxicity), a phenomenon that is probably

0364-3190/04/0100–0161/0 © 2004 Plenum Publishing Corporation

Neurochemical Research, Vol. 29, No. 1, January 2004 (© 2004), pp. 161–168

*Special issue dedicated to Dr. Herminia Pasantes-Morales.1 Departamento de Neurociencias, Instituto de Fisiología Celular, Uni-

versidad Nacional Autónoma de México, D.F., México.2 Address reprint requests to: Tel: 52-55-56225642; Fax: 52-55-56225607;

E-mail: rtapia@ifc.unam.mx

162 Jiménez and Tapia

involved in pathological states such as stroke, epilepsy,and neurodegenerative diseases (7,8).

The process of synaptic plasticity has been the sub-ject of a large number of publications, because it seemsto be the clue event underlying the molecular mecha-nisms of learning and memory. These mechanisms havebeen extensively studied in some invertebrates, such asAplysia califomica (9), but the analysis of synaptic plas-ticity in mammals has been more difficult to approach.One of the most studied models is the phenomenon oflong-term potentiation (LTP) of excitatory transmis-sion, which has provided much information regardingthe crucial role of NMDA receptor activation andcalcium influx in synaptic plasticity (10,11). This facil-itation of synaptic transmission is of relatively shortduration (1–3 h) and can be explained by increasedefficiency resulting from covalent modification of pre-existing proteins, such as phosphorylation of receptors,whereas the consolidation of the process leadingto long-lasting changes in synaptic efficiency dependson changes in gene expression and synthesis of newproteins (6,12).

One of the problems in the study of synaptic plastic-ity in mammals is the scarceness of experimental modelsthat permit correlation of a measurable behavioral para-digm with the biochemical changes occurring in theinvolved cerebral regions. One of the models that hasbeen found to be very useful in this regard is conditionedtaste aversion (CTA), an associative learning process thatpermits separate study of the recognition of a novelstimulus (new taste) and the different stages of the acqui-sition and consolidation of the aversion (through the asso-ciation of the new taste with gastric malaise, see below)(13–15). As in other models of learning and memory,NMDA receptors seem to be involved in CTA, and thepurpose of the present article is to briefly review the avail-able evidence regarding the biochemical modulation ofNMDA receptor sensitivity in this paradigm, includingnew data from our laboratory.

DISCUSSION

Modulation of NMDA Receptor Activity

The NMDA receptor is a structurally and func-tionally complex molecule, because the gating of itschannel is sensitive to both the ligand and the mem-brane voltage. It consists of three different types of sub-units: NR1, which can have eight variants as a result ofalternative splicing, four NR2 subunits (A, B, C, andD), and two NR3 subunits. The channels are formed by

tetrameters or pentameters, and the prevailing view isthat they are assembled by the NR1, which is funda-mental for receptor function, together with one or moretypes of NR2, which can modulate the receptor activ-ity. Glutamate binds exclusively to NR2 subunits andthe coagonist glycine binds only to the NR1 subunit.The NMDA receptor has a site located inside the ionchannel to which Mg2� binds and as a consequenceblocks the channel, and possesses several other modu-latory sites for polyamines, Zn2�, protons, and redoxactive reagents (1,2,3,16,17). NR3 subunits (A and B)also seem to have a modulatory role on the receptor,because when associated with NR1 and NR2 subunits,the Ca2� current induced by receptor activation isdecreased (18,19).

Whereas the AMPA/KA receptor channels open anddesensitize rapidly in response to the agonist and themajority are selective for Na� and K�, the NMDA recep-tor permits the influx of Na�, Ca2�, and K�, and its acti-vation/deactivation is slow (1). At the resting membranepotential the NMDA receptor channel is blocked byMg2�, and when the neuron is depolarized, Mg2� isexpelled, thus allowing the activation of the receptor bythe binding of both the agonist glutamate and the coag-onist glycine (17). The resulting influx of Ca2� throughNMDA receptors can trigger intracellular signalingmechanisms by activating protein kinases and phos-phatases, inducing long-lasting changes in neuronal func-tion (3,10).

Several lines of evidence indicate that an import-ant mechanism of NMDA receptor modulation isthrough phosphorylation of its subunits, which possesstyrosine, serine, and threonine residues at their intracel-lular loops. The balance of phosphorylation/dephos-phorylation depends on the activity of protein kinasesand protein phosphatases (20–24). Thus it has beendemonstrated that activation of protein kinase A (PKA)or protein kinase C (PKC) phosphorylate NR1 and NR2subunits (25–27), that such phosphorylation enhances theresponse to NMDA receptor agonists (28–32), and that asimilar effect is observed by inhibition of serine/threonine protein phosphatases (23,33,34). In addition,serine/threonine protein phosphatase inhibition in vivoleads to serine hyperphosphorylation of NR2B subunit,which results in epileptic seizures that are prevented byNMDA receptor antagonists (24,35,36). Serine/threoninephosphorylation of NR1, NR2A, and NR2B subunits bycalcium/ calmodulin-dependent protein kinase (CAMKII)(37–39) and tyrosine phosphorylation by tyrosine kinases(40–42) have also been shown, and these phosphoryla-tions can also modulate NMDA receptor function(43–45).

NMDA Receptors in Conditioned Taste Aversion 163

Fig. 1. Simplified scheme of the ERK cascade resulting from PKCand PKA activation, consequent to Ca2� influx through NMDA recep-tors, and/or to the activation of metabotropic receptors such as mus-carinic or �-adrenergic.

Participation NMDA Receptor in Synaptic Plasticity

The key role of NMDA receptors in various learning andmemory processes has been mentioned in the Introduction.Available evidence indicates that such processes require short-or long-lasting changes in synaptic efficacy, produced by prioruse of the synapses involved or by association of diversesynaptic inputs to the participating neurons. Whereas therepetitive use of a synapse or its exposure to new stimulican cause local plastic changes leading to facilitatedresponses, the association involves interaction amongdiverse neurotransmitter systems in such a way that thefunctional modifications produced in one synapse enhancethe efficacy of other synapses. This is generally accom-plished by the activation of metabotropic receptors, whichtriggers a variety of biochemical reactions inside the neu-ron, generating intracellular messengers that modulate theresponse of the receptors at other synapses. Several find-ings suggest that this kind of associative change occurs inglutamatergic synapses with NMDA receptors, through thephosphorylation reactions reviewed in the previous section.Thus the activation of muscarinic acetylcholine (ACh)receptors produces tyrosine phosphorylation of the NR2Bsubunit (46), and this phosphorylation enhances the responseof NMDA receptors (47); similarly, stimulation of PKA orPKC activity consequent to the activation of metabotropic glu-tamate, �-adrenergic, and dopamine D1 receptors alsoenhances NMDA receptor function (30,48–50).

The facilitation of NMDA receptor response resultsin the influx of Ca2�, which in turn triggers a series ofbiochemical events in the postsynaptic neuron. Amongthem, the extracellular signal-regulated kinase (ERK) cas-cade has been the most studied, given the prominent roleof these kinases in regulating gene expression and inNMDA receptor–dependent plastic changes such as LTP(51–55). Three steps distinguish the ERK cascade. Thefirst is the activation of MAP kinase kinase kinases (Raf 1and B-Raf), which occurs as a response to PKA and PKCactivity and also to growth factors such as brain derivedgrowth factor. These kinases in turn activate a secondkinase, MAP kinase kinase (MEK), by serine/threoninephosphorylation. MEK activates MAP kinases ERK1 andERK2, by phosphorylating threonine and tyrosineresidues. Finally, ERKs phosphorylation can lead to theactivation of several nuclear transcription factors such asCREB, Elk-1, and c-Myc (Fig. 1) (56).

The cascade of phosphorylation reactions involvingNMDA receptors also seems to play a fundamental rolein the biochemical mechanisms underlying gustatorylearning and memory. Before reviewing these mechan-isms, in the next section we will address the main char-acteristics of taste discrimination and CTA.

Conditioned Taste Aversion

As mentioned in the Introduction, CTA has provedto be an extremely useful paradigm for studying thesynaptic modifications during the acquisition and con-solidation of a conditioned response. This is due to thefact that CTA is an example of a natural adaptativelearning that protects animals against the deleteriouseffects of toxic food. Thus, when a new, unfamiliar fla-vor is followed by nausea or gastric malaise, the newtaste is signaled as dangerous and the animals refrainfrom eating the food. To do so, they need to comparethe new taste with existing memory contents of famil-iar tastes, and during this process the consumption offood with a novel taste is decreased as a precautionaryreaction. This conduct is called neophobia and is relatedto the formation of a memory trace for the novel taste,which, depending of the consequences that follow theconsumption during the next hours, will be labeled assafe or not safe. In the former case, when successivetaste consumptions occur the neophobia is attenuated,whereas in the latter case the animal will show an aver-sive conduct toward the new taste. This aversion has aclear phylogenetic advantage, because it ensures thattoxic or poisonous food will not be approached again

164 Jiménez and Tapia

and implies that a single harmful experience is sufficientto imprint a long-term memory of the new flavor andits adverse consequences (14,15).

In the laboratory, CTA is considered a variant ofclassical conditioning. In this paradigm, the taste stim-ulus becomes the conditioned stimulus (CS) and thesickness-inducer substance is the unconditioned stimu-lus (US, gastric malaise generally induced by iP injec-tion of LiCl). Upon reexposure to the CS, the animalwill avoid ingestion of what is considered to be a toxicsubstance, and such a behavior indicates that the animalhas developed a CTA. This associative learning occurseven though a relatively long delay (several hours) sep-arates the ingestion of food and the gastric malaise, andthis facilitates the study of the different processes under-lying CTA (15,57).

Clearly, to understand the mechanisms of CTA it isnecessary to know the gustatory neural pathways involved.These pathways are well known in rodents (for review,see ref [58]). Briefly, gustatory information from thetongue and other structures of the mouth cavity reachesthe nucleus of the solitary tract, which is the first relay ofthe pathway and receives also the visceral gastrointestinalinput through the vagus nerve. Gustatory neurons fromthis nucleus project to the parabrachial nucleus of thepons, where the initial gustatory and visceral integrationoccurs, and axons from its neurons ascend to the thala-mic ventral posteromedial nucleus. From this thalamicnucleus the gustatory information reaches the gustatoryneocortex, called the insular cortex (IC). Other projectionsfrom both the parabrachial and the thalamic nuclei trans-mit information to the amygdala and to the lateral hypo-thalamus (15,58).

Glutamatergic and Cholinergic Neurotransmissionin CTA

Among these structures involved in taste memoryformation, the IC and the amygdala seem to play a keyrole, because lesions or pharmacological blockade ofthese regions impair CTA (58–60), although the func-tional roles of these two structures seem to be differentduring taste memory formation. Tetrodotoxin (TTX),which allows blockade of the activation of a particularregion during a specific stage of CTA acquisition butleaves it intact during retrieval tests, has been used toblock IC and amygdala in CTA. When IC injections ofTTX are applied before CS (novel taste) presentation,but not between CS and US (malaise-induction drug),CTA is prevented, suggesting that IC is involved in tasteprocessing but not in visceral signaling processing. Con-

versely, although inactivation of the amygdala with TTXbefore the CS does not prevent CTA acquisition, itsblockade before the US impedes CTA (13). These resultssuggest that the amygdala does not play an important rolein the processing of taste signal, but seems to be indis-pensable for processing the visceral stimulus, through aneural pathway that most probably uses glutamate asneurotransmitter (13,61).

IC is an essential structure for CTA memory becauseit is an associative region where visceral and gustatoryinformation converge (15,62). Different neurotransmittersystems in IC are involved in CTA. Neurons in this regionare capable of releasing noradrenaline, GABA, dopamine,glutamate, and ACh, and/or possess receptors for theseneurotransmitters (60,63). Particularly in the case of glu-tamate and ACh, there is strong evidence that choliner-gic and glutamatergic synapses in the IC are cruciallyimportant in the acquisition (short-term memory) and inthe consolidation (long-term memory) of taste memory(60,64–69).

Microdialysis experiments have shown that AChrelease in rat frontal cortex is stimulated simply by expo-sure of rats to auditory, visual, and gustatory stimulation(70,71). In the IC, ACh release is induced by presentationof novel taste stimuli (saccharin or quinine), in compari-son with rats that drank a familiar taste (water) (67,72),and this increment is attenuated as the new flavor becomesfamiliar, indicating an inverse relationship between tastefamiliarity and ACh release in this structure (67).

These data clearly suggest an involvement ofcholinergic transmission in the IC in the recognition ofnew tastes and in the processing of CTA. This has beenfurther substantiated by studies with muscarinic receptorantagonists. IC microinjections of scopolamine, beforethe presentation of a novel stimulus (saccharine, CS),impaired the acquisition of CTA: rats did not developaversion to the new taste after the presentation of the US(injection of LiCl as a malaise-inducing drug) (60,65).In another recent study it was demonstrated that scopo-lamine application in the IC before, but not after, thepresentation of saccharin, blocked both short-term mem-ory (4 h) and long-term memory (72 h) of CTA (69).

With regard to glutamate, a microdialysis study inthe amygdala and the IC has shown a notable enhance-ment of glutamate release in the amygdala and a modestincrease in the IC during the presentation of the US (LiClinjection) (61). In addition, similarly to the effect ofscopolamine, pretraining microinjections in the IC ofNMDA receptor blockers, such as 2-amino-5-phospho-novaleric acid (AP-5), impair CTA (60,66). However, dif-ferently from scopolamine, AP-5 treatment impaired onlylong-term memory but left short-term memory intact (69).

NMDA Receptors in Conditioned Taste Aversion 165

Fig. 2. Preliminary results of experiments designed to test the hypoth-esis of serine phosphorylation of the NMDA receptor NR2B subunitin the IC, induced by the exposure of rats to a novel taste. Rats weretrained to get their daily water from a graded tube during 15 min for3 days (baseline value), and on the fourth day they received 0.1%saccharin solution instead of water for 1, 2, or 3 days consecutively.Liquid consumption was recorded. Fifteen minutes after sacchariningestion, animals were decapitated, and the IC excised and homoge-nized in a lysis buffer containing a protease and phosphatase inhibitorscocktail. After pooling the homogenates of two rats, an aliquot wastaken for protein determination by the Lowry method, and immuno-precipitation of phosphoserine proteins was carried out by incubatingovernight 2 mg protein with protein G-agarose and with a phospho-serine monoclonal antibody in lysis buffer. After five washes, theprecipitated fraction was recovered and subjected to electrophoresis,transferred to a nitrocellulose membrane, and incubated with a poly-clonal antibody against NR2B subunit and an IgG horseradish perox-idase conjugate. Immunodetection was made by a chemoluminescenceenhancer and detected on Kodak X-Omat film. Optical densities per�m2 were measured with an image analyzer. Top: Representative blotof the phosphorylated NR2B (p-NR2B) immunostaining, detected asdescribed above, in the IC of control rats (H2O lane) and of rats tast-ing a 0.1% saccharin solution once, twice, or thrice on consecutivelydays. The line at the right indicates the location of the 180-kDa marker.Bottom graph: The black bars and left ordinate show densitometricanalysis of the anti–p-NR2B subunit blots, expressed as percent of thecontrol (water tasting). The white bars and right ordinate represent thevolume of saccharin solution consumed on the first, second, and thirdday of saccharin sampling, expressed as percent of the base line con-sumption. Mean values � SEM of three experiments in each group,with two rats for each condition.

These results provide evidence that cholinergic andglutamatergic systems in the IC are involved in differ-ent but interrelated memory processes of CTA. Thecholinergic system plays an important role in the encod-ing of taste stimuli, whereas NMDA receptors areinvolved mainly in US processing. However, as will bediscussed next, the interaction between the two neuro-transmitter systems at a postsynaptic level seems deter-minant for the establishment of CTA.

NMDA Receptor Phosphorylation in CTA

Muscarinic receptors of the type M1, M3, and M5,which are present throughout the cerebral cortex, trans-duce their signals through their coupled G-proteins,which activate phospholipase C and consequently PKC(73). In view of the role of PKC in the phosphorylationof NMDA receptors and the consequent enhancement ofits response (reviewed in previous sections), and con-sidering the close functional relationships between themuscarinic and the NMDA receptors in CTA process-ing, it seems reasonable to postulate that a new tastestimulation results in the activation of muscarinic recep-tors in the IC. This activation triggers several proteinkinases that may phosphorylate NMDA receptors, thusmaking them sensitive to the glutamate released fromendings of axons originated in the amygdala as aresponse to the visceral US (69).

This postulate is also supported by the finding thatthe blockade of PKC activity in the IC within the CS-US interval, but not after the US, impairs CTA, whichsuggests an interference with information processingnecessary for CTA acquisition, but not for the retentionor retrieval phases of CTA (74). Two additional find-ings give further support to this postulate. First, the ICmicroinjections of the muscarinic agonist carbacholinduced tyrosine phosphorylation of the NR2B subunitof NMDA receptor (46). Second, when rats wereexposed to a new taste (saccharin) the NR2B subunit inthe IC was also phosphorylated at tyrosine residues andthis phosphorylation diminished when the rats werereexposed to the taste 24 h later (75).

The serine and threonine residues of the NR2Bsubunit of the NMDA receptor are also substrates ofphosphorylation, particularly by PKC, which, as alreadymentioned, is activated by muscarinic receptorsresponse. In fact, it has been shown that the serineS1323 and S1354 residues of the NR2B subunit arephosphorylated by PKC (26) and that such phosphory-lation enhances the NMDA receptor current (27). Wehave therefore studied whether the exposure to a novel

taste induces the phosphorylation of serine residues ofthis subunit in the IC and whether this can be relatedto the decrease of consumption of the new taste (neo-phobia). In addition, we tested the course of serine phos-phorylation of the NR2B subunit during the attenuationof neophobia occurring when the new taste becomesfamiliar. As shown in Fig. 2, we have found that thereis an increment to approximately 185% in NR2B sub-unit serine phosphorylation of rats sampling a new taste(saccharin) for the first time, in comparison with control

166 Jiménez and Tapia

animals (rats that drank water). This phosphorylationdecreases as the taste becomes familiar, to 78% of thecontrol on the second day, and to about 50% of the con-trol the third day. As expected, saccharin consumptionfollowed an inverse course: it decreased about 50% thefirst day of presentation of saccharin (neophobia), incomparison with their own baseline, augmented to 82%during the second presentation and reached the controlbaseline value the third day (Fig. 2).

These results indicate that the NR2B subunit in theIC becomes highly phosphorylated in serine residueswhen the rat is confronted with a novel gustatory stimu-lus and the behavioral neophobia is evident. In addition,as the familiarity with the taste stimulus is established,serine phosphorylation is less evident, in parallel with theattenuation of the neophobia along 3 days. Thus, takentogether with the data reviewed previously, these experi-ments strongly support the hypothesis that NMDA recep-tor sensitization by serine phosphorylation, consequent tothe activation of muscarinic receptors, is involved in thememory processing of the new taste, which when asso-ciated with visceral malaise results in CTA. Therefore wesuggest that NR2B phosphorylation in the IC may be akey process in memory signaling of unfamiliar tastes,which determines the successive responses of the animalto such novel gustatory stimuli.

The facilitation of the ERK kinases cascade subse-quent to NMDA receptor activation has been already men-tioned. The memory processing of unfamiliar tastes mayalso involve this cascade, because PKC is known to phos-phorylate the mitogen-activated protein kinase kinasekinase (Raf1 and B-Raf in the ERK cascade) and the MAPkinases ERK1-2 via Raf1 and B-Raf (76). ERK1-2 has animportant role in CTA learning because it seems to regu-late gene expression (51,52,55,76,77). Thus it has beendemonstrated that an unfamiliar taste activates ERK1-2 andtwo of its downstream substrates, the ELK-1 which increasesc-fos induction, and JNK1-2, which activates ELK-1. Fur-thermore, IC microinjections of ERK inhibitors beforeexposure to novel taste inhibited the activation of ERK1-2 and impaired CAT, whereas a familiar taste did not acti-vate ERK1-2 (78). It has also been demonstrated that ICmicroinjections of glutamate and muscarinic antagonistsbefore exposure to a novel taste significantly reducesERK1-2 activation (79).

CONCLUSIONS

The analysis of the biochemical mechanismsinvolved in learning and memory in vivo is only begin-ning. The present knowledge of the modulation of neu-

rotransmitter receptors response and of gene expressionby phosphorylation reactions has greatly opened the wayfor understanding such mechanisms. CTA, because itssimplicity, reproducibility, and duration and because ofthe well-identified neural structures involved, is an excel-lent model for studying them. Although CTA is an asso-ciative learning process requiring US and CS, the firststep in this process, the recognition and memory of anunfamiliar taste, represents a good model to study the par-ticipation of synaptic signaling, both at the postsynapticreceptor level and at the intracellular changes mediatedby second messengers. The data reviewed in this workprovide a basis for future investigations that can unravelthe link between the biochemical and the physiologicalevents underlying such important learning behavior.

ACKNOWLEDGMENTS

This work was supported by CONACYT (project MillenniumW8072-35806N) and DGAPA, UNAM (IN206100).

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