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In conditioned taste aversion (CTA), animals learn to avoid a flavored solution (conditioned stimulus, CS) previously paired with internal malaise (unconditioned stimulus, US). Metabotropic glutamate receptor 5 (mGlu5) has been implicated in learning and memory processes and is necessary for CTA. In the present study, local microinjections of a mGlu5-selective antagonist, 3-[2-methyl-1,3-thiazol-4yl)ethynyl]pyridine (MTEP, 0, 1 or 5 μg) into the insular cortex and basolateral amygdala were used in male, Sprague-Dawley rats to examine the role of mGlu5 receptors in the encoding of taste memory. MTEP was infused 20 min before saccharin intake during CTA conditioning. MTEP injection into the basolateral amygdala resulted in robust CTA, similar to the vehicle-treated animals but slowed extinction; that is, MTEP enhanced CTA. MTEP injection into the insular cortex resulted in an increased saccharin intake on the conditioning trial, which potentially influenced the performance on the test trials. MTEP had no effect on CTA learning when controlled access to saccharin was used on the conditioning trial. These results indicate that mGlu5 receptors are involved in taste memories in a region-specific manner.
Glutamate, the major excitatory neurotransmitter in the adult central nervous system, acts through ionotropic (NMDA, AMPA, kainate) and metabotropic glutamate receptors (mGlus: group I, mGlu1 and mGlu5; group II, mGlu2 and mGlu3; group III, mGlu4, mGlu6, mGlu7 and mGlu8). MGlu receptors belong to a family of G-protein coupled receptors linked to multiple intracellular signaling cascades. Group I mGlu receptors have been shown to be particularly important for synaptic plasticity, and learning and memory (reviewed by Riedel, Platt, & Micheau, 2003). The evaluation of the effects of mGlu5-selective antagonists in various behavioral paradigms has concluded that mGlu5 receptors play a critical role in aversive learning tasks and in hippocampal-dependent spatial learning (Riedel et al., 2003; Simonyi, Schachtman, & Christoffersen, 2005).
Previous research has also found that mGlu5 receptors are involved in conditioned taste aversion (CTA) learning. CTA is a form of aversive classical conditioning in which a flavored substance (the conditioned stimulus, CS) is paired with a drug or experience that produces internal malaise (the unconditioned stimulus, US). This pairing results in the conditioned response (CR)—that is, the subject’s avoidance of consuming the substance on a test trial. The acquisition of CTA is subserved by specific brain regions, including the insular cortex and the amygdala although their precise role in CTA is still unclear (Lamprecht, & Dudai, 2000; Yamamoto, Shimura, Sako, Yasoshima, & Sakai, 1994). The insular cortex and the basolateral amygdala are functionally and reciprocally interconnected and there are direct projections between these structures. Our studies using systemic administration of mGlu antagonists before conditioning have demonstrated that activation of mGlu5, but not mGlu1, receptors is required for CTA learning (Schachtman, Bills, Ghinescu, Murch, Serfozo, & Simonyi, 2003). Yasoshima, Morimoto, and Yamamoto (2000) and Berman, Hazvi, Neduva, and Dudai (2000) showed an attenuation of CTA by microinjection of a broad-spectrum mGlu antagonist into the basolateral amygdala and the insular cortex, respectively. However, the identity of the receptor subtype(s) mediating these effects is not known. In the present study, local microinjections of a mGlu5-selective antagonist, 3-[2-methyl-1,3-thiazol-4yl)ethynyl]pyridine (MTEP) (Kew & Kemp, 2005) into the basolateral amygdala (Experiment 1) and the insular cortex (Experiments 2 and 3) were used to examine the role of mGlu5 receptors in the encoding of taste memory.
Male, Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 190–220 g were housed individually with a 16 hr light/8 hr dark cycle (time on at 6:00 AM). Food and water were available ad libitum until the beginning of the behavioral procedures. In all experiments, animals were randomly assigned to groups except for counterbalancing of water consumption. All experiments were conducted blind to the treatment condition of the rats. Experiments were carried out in accordance with National Institutes of Health guidelines and with permission from the University of Missouri Animal Care and Use Committee (Protocol #4289). The rats were anesthetized with 87 mg/kg ketamine and 13 mg/kg xylazine (i.p.) and placed in a stereotactic apparatus (Kopf Instruments, Tujunga, CA). Twenty-six gauge stainless steel guide cannulas (C315G, Plastics One, Roanoke, VA) were implanted bilaterally into the insular cortex or into the basolateral amygdala. The stereotaxic coordinates were: A +1.2, L ±5.0, V −4.0 mm (insular cortex); A −2.8, L ±5.0, V −6.5 mm (basolateral amygdala) (Paxinos & Watson, 1998). A stylus was placed in the guide cannula to prevent clogging. Buprenex (0.03 mg/kg, s.c.) was given to the animals following surgery, and a one-week recovery period was allowed. Injections were made in a volume of 0.5 μl/side over 1 min via injection pump using 33 gauge infusion cannulas that extend 1 mm (basolateral amygdala) or 2 mm (insular cortex) from the base of the guide. The injection cannulas were left in position for an additional one min to minimize dragging of the drugs along the injection tract.
One week after surgery, rats were water deprived for 24 hours. The animals were then acclimated to drinking from the drinking tubes for three days to obtain their daily water within 15 minutes in their home cages, and water consumption was measured. To ensure consistency, water was provided at the same time every day in the experiments. Animals were handled on these days. On the conditioning day, animals were injected with MTEP (1 or 5 μg, Ascent Scientific, Princeton, NJ; doses were chosen based on effective doses shown in other behavioral studies, e.g., Molina-Hernandez, Tellez-Alcántara, Pérez-García, Olivera-Lopez, & Jaramillo, 2006) or vehicle (1% v/v Tween-80 in physiological saline) and were presented with a 0.1% saccharin solution (CS) 20 minutes later. Forty minutes after the offset of the drinking period, LiCl (US) was injected i.p. (0.15M, 2% body weight). The animals were observed for behaviors that indicate internal malaise (e.g., “lying on belly”). In the 48-hour period between conditioning and testing, the rats received no treatment except for exposure to 15 min of water access. Two or three test trials were administered in each experiment (one in every 24 hours) in which saccharin was presented in drinking tubes for 15 min. Histological verification was performed as described in our earlier publication (Simonyi, Serfozo, Shelat, Dopheide, Coulibaly, & Schachtman, 2007). Animals with improper injection needle placements were excluded from further analysis. Data were analyzed by either one-way ANOVA or two-way repeated measures ANOVA followed by pairwise comparisons using Bonferroni posttests. P values of <0.05 were considered statistically significant.
In Experiment 1, during the three days of water baseline, there were no statistical differences among the groups (ns =8) in the mean water intake (data not shown). In addition, there were no statistical differences among groups in saccharin consumption during conditioning (Figure 1). MTEP injections into the basolateral amygdala resulted in robust CTA on the initial test trial, similar to the vehicle-treated animals (Figure 1). However, MTEP-treated rats showed slower extinction; that is, MTEP enhanced CTA, F4,42=3.19, p=0.0225 (Two-way repeated measures ANOVA, interaction). A rapid rate of extinction can be indicative of a weak association. The main effect of MTEP (F2,42=4.47, p=0.0241) and Trials (F2,42=90.31, p<0.0001) was also significant. Bonferroni posttest showed a significant difference (p<0.05) between the 5 μg MTEP group and vehicle group (0 μg MTEP) on the third test day. This attenuation in learning could be viewed as due to a state-dependent memory effect given that the rats were trained in the presence of drug and tested in the drug’s absence; and the current data do not rule out such an explanation. However, other CTA reports using mGlu5 receptor antagonists are not explicable in this way (Bills, Schachtman, Serfozo, Gasparini, Spooren, & Simonyi, 2005; Schachtman et al., 2003).
In Experiment 2, during the three days of water baseline, there were no statistical differences among groups in the mean water intake (data not shown). However, on the conditioning day, saccharin consumption of rats injected with 5 μg MTEP (n=10) into the insular cortex was significantly higher compared to the either 1 μg MTEP (n=5) or vehicle-treated control (0 μg MTEP, n=8) conditions (F2,20=5.94, p=0.0095, one-way ANOVA) (Figure 2A). This increased saccharin intake on the conditioning trial may have influenced performance on the test trials; 5 μg MTEP-treated animals consumed more saccharin (F2,20=3.68, p=0.0437, two-way repeated measures ANOVA, main effect of MTEP). The main effect of Trials was also significant (F1,20=115.36, p<0.0001).
In Experiment 3, controlled access to saccharin (maximum of 8 ml) was used during the conditioning trial and either vehicle (n=7) or 5 μg MTEP (n=6) was infused into the insular cortex 20 minutes before saccharin. Animals were tested for three successive days, once per day, beginning 48 hours after conditioning (Figure 2B). Two-way repeated measures ANOVA revealed no interaction or main effect of MTEP (F<1) but a significant effect of Trials (F2,22=22.24, p<0.0001) occurred. MTEP applied to the insular cortex had no effect on CTA learning.
The present study demonstrates an important role of amygdalar mGlu5 receptors in CTA. MTEP injection into the basolateral amygdala affected the acquisition of CTA; MTEP enhanced CTA in a dose-dependent manner. Given that systemic injection of a mGlu5 receptor antagonist before conditioning has been shown to attenuate CTA (Schachtman et al., 2003), this enhancement of CTA is potentially unexpected. However, such divergence between systemic and local drug effects exist (Riedel et al., 2003). These studies highlight the importance of using local microinjection of drugs in order to clarify the distinct role of receptor subtypes and their anatomic loci in memory processing. Continuous infusion of a group I mGlu antagonist into the parabrachial nucleus by a microdialysis probe immediately after saccharin, during the CS-US interval and after LiCl injection dose-dependently attenuated CTA (Vales, Zach, & Bielavska, 2006). The authors suggested the contribution of mGlu5 receptors to this effect based on the results of anatomical studies. Information regarding the distribution of glutamatergic receptors in the insular cortex, amygdala and parabrachial nucleus is of great importance for the understanding of the mechanisms of novel taste acquisition and CTA. Using morphological methods in conjunction with biochemical, physiological and behavioral studies can help to elucidate the cellular and molecular mechanisms which underlie taste learning in the brain. When comparing the roles of NMDA receptors to mGlu5 receptors in CTA it appears that they serve opposite functions. Microinjection of NMDA receptor antagonists into the insular cortex or basolateral amygdala before or during CTA acquisition resulted in a strong disruption of CTA (Berman et al., 2000; Yashosima et al., 2000; Barki-Harrington, Belelovsky, Doron, & Rosenblum, 2009), but it had no effect in the parabrachial nucleus (Vales et al., 2006).
As basolateral amygdala-insular cortex interactions regulate the strength of CTA, amygdalar mGlu5 receptors might be involved in the mediation of emotional learning. Indeed, amygdalar mGlu5 receptors have been shown to be involved in the formation of aversive associative learning, including fear conditioning and fear-potentiated startle (reviewed by Simonyi et al., 2005). In both tasks, acquisition was controlled by mGlu5 receptors. In addition, physiological studies have also provided evidence that mGlu5 receptors are necessary for plasticity in the amygdala (Fendt, & Schmid, 2002; Rodrigues, Bauer, Farb, Schafe, & LeDoux, 2002). One of the mechanisms through which amygdalar mGlu5 receptors could participate in taste aversion memory formation is by influencing GABA receptor function. GABA(A) and mGlu5 receptors have been shown to be co-expressed in neurons in the basolateral amygdale, and activation of mGlu5 receptors stimulates GABA release (Besheer, & Hodge, 2005); although this interaction requires further investigations.
The present study demonstrates that mGlu5 receptor antagonism in the insular cortex produces an increase in consumption of saccharin, a novel taste. Similar to muscarinic acetylcholine receptors but dissimilar to NMDA receptors, mGlu5 receptor-mediated responses seem to be involved in the processing of new taste memory formation (including processing that reflects that a taste is “safe”) in the insular cortex (Bermudez-Rattoni, 2004; Bermudez-Rattoni, Ramirez-Lugo, Gutierrez, & Miranda, 2004; Barki-Harrington et al., 2009). It has been reported that novel taste consumption induces acetylcholine release in the insular cortex (Bermudez-Rattoni et al., 2004). Although further investigation is needed, it has been suggested that muscarinic acetylcholine receptors can initiate the activation of signaling pathways leading to the phosphorylation of specific NMDA receptor subunits in the insular cortex after consumption of a new, but not a familiar, taste; and intracortical infusion of carbachol can mimic this effect (Bermudez-Rattoni et al., 2004; Barki-Harrington et al., 2009). It is highly possible that mGlu5 receptors participate in taste learning similarly to muscarinic receptors. In vitro studies reported that stimulation of the mGlu5 receptor could lead to PKC phosphorylation and/or tyrosine kinase phosphorylation of the NMDA receptor (Collett, & Collingridge, 2004). However, this possibility needs to be explored experimentally and confirmed in the insular cortex for new taste learning. Furthermore, other mechanisms may also be important since mGlu5 receptor is coupled to a variety of second messenger cascades, and is known to modulate the function of different neurotransmitter systems as well as ion channels (see Hermans & Challiss, 2001; Simonyi et al., 2005 for reviews). The insular cortex has an established role in neophobia and our results are in accordance with this function (Bermudez-Rattoni et al., 2004). The insular cortex is also important in aversive taste memory formation although it is unclear if similar mechanisms are involved in neophobia and CTA. Our results suggest that aversive taste learning is a mGlu5 receptor-independent mechanism in the insular cortex.
In summary, while MTEP administration into the insular cortex did not influence CTA when controlled access to saccharin was used during conditioning, microinjection of MTEP into the basolateral amygdala enhanced CTA. These results indicate that mGlu5 receptors are involved in taste memories in a region-specific manner.
Supported in part by R03 MH64486-01A1 from NIH. We thank Dr. Phullara Shelat for her assistance.
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