Artificial taste avoidance memory induced by coactivation of NMDA and β-adrenergic receptors in the amygdala

Behavioural Brain Research 376 (2019) 112193

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Artificial taste avoidance memory induced by coactivation of NMDA and β-adrenergic receptors in the amygdala Daniel Osorio-Gómeza, Federico Bermúdez-Rattonia, Kioko Guzmán-Ramosb,

T ⁎

a

División de Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, 04510, Mexico City, Mexico b Departamento de Ciencias de la Salud, División de Ciencias Biológicas y de la Salud Universidad Autónoma Metropolitana, Unidad Lerma Av. de las Garzas No. 10, Col. El Panteón, Lerma de Villada, Estado de México, C.P. 52005, Mexico

ARTICLE INFO

ABSTRACT

Keywords: Amygdala Insular cortex Taste aversion Glutamate Memory induction

The association between a taste and gastric malaise allows animals to avoid the ingestion of potentially toxic food. This association has been termed conditioned taste aversion (CTA) and relies on the activity of key brain structures such as the amygdala and the insular cortex. The establishment of this gustatory-avoidance memory is related to glutamatergic and noradrenergic activity within the amygdala during two crucial events: gastric malaise (unconditioned stimulus, US) and the postacquisition spontaneous activity related to the association of both stimuli. To understand the functional implications of these neurochemical changes on avoidance memory formation, we assessed the effects of pharmacological stimulation of β-adrenergic and glutamatergic NMDA receptors through the administration of a mixture of L-homocysteic acid and isoproterenol into the amygdala after saccharin exposure on specific times to emulate the US and post-acquisition local signals that would be occurring naturally under CTA training. Our results show that activation of NMDA and β-adrenergic receptors generated a long-term avoidance response to saccharin, like a naturally induced rejection with LiCl. Moreover, the behavioral outcome was accompanied by changes in glutamate, norepinephrine and dopamine levels within the insular cortex, analogous to those displayed during memory retrieval of taste aversion memory. Therefore, we suggest that taste avoidance memory can be induced artificially through the emulation of specific amygdalar neurochemical signals, promoting changes in the amygdala-insular cortex circuit enabling memory establishment.

1. Introduction Classical conditioning has been used as a model to study how neurotransmitters are involved in the acquisition and storage of information [1,2]. The neurochemical basis of memory formation emerged through pharmacological manipulations of receptors’ activity before or after the learning phase. However, there is scarce information on how specific neurotransmitters participate in the encoding of both the conditioned stimulus (CS) and the unconditioned stimulus (US), triggering the mechanisms associated with the establishment of memory [3–7]. Taste aversion memory has been widely used as an associative learning paradigm in the search of the neurobiological mechanisms involved in memory processes. Animals’ survival depends on their capacity to adapt to the environment by efficiently store the consequences

of the ingestion of a specific food; specially the ones that might be toxic, this way, the animal will be able to display an avoidance response to evade aversive outcomes. In order to associate and store these memory traces, appropriate neurochemical representations on key brain structures have been described. Exposure to a novel taste produces increments of dopamine and norepinephrine in both the amygdala and the insular cortex (IC) [3–6]. Conversely, a malaise-inducing agent such as LiCl generates an elevation of glutamate [3,5,8] and norepinephrine [3] within the amygdala, suggesting that these neurotransmitters are related to the signaling of the aversive outcome. Furthermore, taste aversion memory can be enhanced by intra-amygdalar administration of glutamate when a weak US is given (low concentration of LiCl) [9,5]. Blockade of either noradrenergic [10] or glutamatergic [11] receptors within the amygdala before the induction of gastric malaise impairs aversive taste memory establishment. Altogether, this evidence suggests

Abbreviations: CS, conditioned stimulus; CTA, conditioned taste aversion; IC, insular cortex; ISO, isoproterenol; LHa, L-homocysteic acid; MIX, mixture of isoproterenol and homocysteic acid; SS, saline solution; US, unconditioned stimulus ⁎ Corresponding author. E-mail address: [email protected] (K. Guzmán-Ramos). https://doi.org/10.1016/j.bbr.2019.112193 Received 22 May 2019; Received in revised form 7 August 2019; Accepted 28 August 2019 Available online 29 August 2019 0166-4328/ © 2019 Elsevier B.V. All rights reserved.

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that glutamatergic and noradrenergic activity within the amygdala could be related to the encoding of the necessary signals to generate an aversive taste memory trace. Memory consolidation involves post-acquisition processes whereby newly acquired information is maintained and established into longterm memory [12,13]. As part of these consolidation mechanisms, data suggest that electrical or neurochemical patterns of activity occur during post-acquisition wakefulness and during sleep in the absence of sensory information [3,4,14–17]. In this regard, it has been reported an increase in norepinephrine levels within the amygdala that remained elevated 2 h after inhibitory avoidance training [18]. Particularly, we demonstrated an increase of the extracellular levels of glutamate and norepinephrine within the amygdala [3], and dopamine and glutamate within the IC [4] about 45 min after taste and gastric malaise pairing. These changes of neurotransmitters levels are related to taste aversion consolidation, since amygdalar blockade of N-methyl-D-aspartate (NMDA) and β-adrenergic receptors during the timeframe of reactivation disrupts long-term memory consolidation [3]. Moreover, reversible inactivation of the amygdala at the time of reactivation impedes the release of glutamate and dopamine within the IC and impairs stabilization of conditioned taste aversion (CTA) [4]. These results strongly suggest that the amygdala modulates post-acquisition events within the IC required for CTA memory consolidation [19–21]. The aim of this study was to evaluate the effects of local amygdalar stimulation of noradrenergic β-receptors and glutamatergic NMDA receptors, in pursuance of emulating the neurochemical signals related to the US and to the post-acquisition spontaneous activity; that has been described within this structure during CTA formation and long-term stabilization. Thereby, we administrated a mixture of a NMDA receptor agonist (L-homocysteic acid) and a β-adrenergic receptor agonist (isoproterenol) into the amygdala to mimic the neurochemical signals triggered at different times. In addition, we monitored the neurochemical levels within the IC during memory retrieval as a result of the artificially induced taste avoidance and compared it with a naturallyinduced taste aversion retrieval as previously reported [21].

homocysteic acid (2 μg/μL; 18.315 μg/μL final concentrations respectively). There is evidence of central and basolateral nuclei participation in taste aversion formation, especially during LiCl administration [3,5,24,25]. Therefore, a total volume of 1 μL was injected per hemisphere in the amygdala (0.5 μL/min); the injector was left for another minute to allow diffusion into the tissue. Drug administration was made through 30-gauge dental needles that protruded 1 mm from the tip of the guide cannulae. Injection needles were connected via polyethylene tubing to two 10-mL Hamilton syringes, driven by an automated micro infusion pump (Carnegie Medicine). On the acquisition day, rats were divided in 7 groups counterbalanced according to their mean baseline consumption of tap water. Two graded bottles were presented, one with 15 mL of a 0.1% (wt/vol) saccharin solution (Sigma Aldrich) and the other with 15 mL of tap water. Rats were allowed to drink for 15 min. After a 15 min delay, rats received an intra-amygdalar injection and 30 min later rats were infused again according to the administration protocol. Experimental groups were separated as follows (final number of animals on each group is shown): saline solution - saline solution (SS-SS, n = 6); isoproterenol - isoproterenol (ISO - ISO, n = 6); L-homocysteic acid - Lhomocysteic acid (LHa-LHa, n = 6); saline solution-mix of isoproterenol and L-homocysteic acid (SS-MIX, n = 6); mix of isoproterenol and L-homocysteic acid-saline solution (MIX-SS, n = 6) and mix of isoproterenol and L-homocysteic acid-mix of isoproterenol and L-homocysteic acid (MIX-MIX, n = 7). We compared the behavioral response of an artificially induced taste avoidance to a naturally induced rejection through gastric malaise caused by LiCl; hence a positive control group received an i.p. injection of LiCl (0.15 M, 7.5 mL/kg) (LiCl 0.15 M, n = 5) 15 min after saccharin acquisition consumption during acquisition. Seventy-two hours after training, all animals could drink from two graded bottles, one with saccharin solution (0.1% (wt/ vol) and the other with tap water for 15 min; the consumption measurements of each bottle were obtained. Data is presented as Preference index (Preference index = Saccharin mL / Saccharin mL + tap water mL).

2. Material and methods

2.3. Collection of cortical neurotransmitters related to an artificiallyinduced taste avoidance

2.1. Animals

Unilateral microdialysis cannula (22-gauge, shaft length 14 mm. CMA Microdialysis) aiming the insular cortex (AP + 1.2 mm; L + 5.5 mm; DV -4.5 mm, [22]) and bilateral steel cannulas (12 mm length and 23 gauge) aiming the amygdala (AP -2.8 mm, L ± 4.8 mm, DV −6.5, [22]) were implanted using standard stereotaxic protocols. Six days after surgery, animals were deprived of water for 24 h and placed in the microdialysis chamber during 3 h for six days to let them habituate to the context and constant manipulation. Rats were allowed to drink 30 mL of tap water from a graded bottle for 15 min to obtain a water baseline consumption intake. Microinfused drugs were dissolved in saline solution (0.9% wt/vol) and a total volume of 1 μL was injected per hemisphere in the amygdala (0.5 μL/min) as described above. On the acquisition day, rats were divided into 3 groups counterbalanced according to their mean baseline tap water consumption and were exposed for 15 min to a graded bottle with 30 mL of a 0.1% (wt/vol) saccharin solution. After a 15 min delay, rats received a first intraamygdalar injection and 30 min later rats were infused again according to the administration protocol. Experimental groups were separated as follows: one group received saline solution-saline solution (SS-SS, n = 8); a second group received a mix of isoproterenol and L-homocysteic acid-mix of isoproterenol and L-homocysteic acid (MIX-MIX, n = 8) and a third group (positive control) that received a LiCl i.p. injection (0.15 M, 7.5 mL/kg) (LiCl 0.15 M, n = 6) 15 min after saccharin consumption. Seventy-two hours after training, a dialysis probe with a 3 mm membrane (CMA 12 MD Probe, CMA Microdialysis) was connected to the microinfusion pump system (CMA Microdialysis), which infused

Two-months old male Wistar rats, weighing 260–280 g at the time of surgery, were used in this study. Animals were obtained from the Instituto de Fisiología Celular animals production facility. Rats were housed individually and maintained on a 12 h light/12 h dark cycle with water and food ad libitum except when noted on the behavioral procedures. All procedures were completed during the light period and were approved by the Animal Care and Ethics Committee of the Instituto de Fisiología Celular (FBR 30-14) in accordance with the National Institutes of Health guidelines. 2.2. Intra-amygdalar drugs administration and taste avoidance assessment Rats were implanted bilaterally with 12 mm long stainless-steel guide cannulae (23 gauges) directed to the central and basolateral amygdalar nuclei using standard stereotaxic procedures (AP-2.8 mm, L ± 4.8 mm, DV-6.5 mm relative to Bregma, [22]. After six days of surgery recovery, animals were deprived of water for 24 h; rats were allowed to drink 15 mL of tap water from two graded bottles for 15 min between 10:00 and 11:00 in their home cage to obtain a baseline water consumption over 6 days. Additional water access was allowed for 15 min in the afternoon (around 5 pm) to ensure proper hydration of the animals. To diminish handling-associated stress, daily handling was performed during 3 min every day prior to the infusion protocol. All drugs were dissolved in saline solution (0.9% wt/vol). We administered isoproterenol (2 μg/μL, Sigma Aldrich), L-homocysteic acid (18.315 μg/ μL, Sigma Aldrich, [23]) or a mixture of isoproterenol and L2

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artificial cerebrospinal fluid (NaCl 125 mM, KCl 5 mM, NaH2PO4H2O 1.25 mM, MgSO4 •7H2O 1.5 mM, NaHCO3 26 mM, CaCl2 2.5 mM, glucose 10 mM) at a rate of 1 μL/min. After insertion of the probe, 1 h of fluid stabilization was allowed; samples were collected every 4 min in vials containing 1 μL of an antioxidant mixture (0.25 mM ascorbic acid, Na2EDTA 0.27 mM, 0.1 M acetic acid) and immediately frozen at −80 °C. Three fractions were collected as baseline samples, afterward, a 0.1% (wt/vol) saccharin solution was presented for 15 min. We obtained five more fractions after CS exposure onset. The saccharin consumption was measured during retrieval and it was reported as percentage of consumption of saccharin during the acquisition intake (acquisition consumption percentage = saccharin solution intake during retrieval × 100/saccharin intake during acquisition). During microdialysis experiments, animals were trained and evaluated with one bottle of saccharin to avoid changes in neurotransmitter levels associated with water preference.

Preference consumption indexes were analyzed using One-sample Student’s t-tests compared to a hypothetical value of 0.5 (chance level) to determine taste preference. Additionally, we used a One-way ANOVA with Dunnet’s post hoc test to compare preference consumption indexes of the different groups in comparison to the control group and effect size was calculated with Cohen’s d. In order to assure that treatments did not cause adipsia or a general reduction in consumption, we compared total fluid intake (mL saccharin + mL tap water) among groups with One-way ANOVA (Fig. 2). Changes in the extracellular levels of neurotransmitters within the insular cortex were analyzed using Repeated measures ANOVA with Bonferroni post hoc test. For the behavioral response during microdialysis protocol, one-way ANOVA with Dunnet’s post hoc test was used to compare avoidance response during test in contrast with the control group. The accepted level of significance was p-value ≤0.05. Data are presented as mean ± SEM. Statistical analysis was performed using StatView version 4.57 and graphs were obtained with GraphPad Prism version 6.00, GraphPad software.

2.4. Analysis of glutamate, norepinephrine and dopamine in microdialysis samples

3. Results

Neurotransmitters levels were determined by capillary electrophoresis. Briefly, microdialysis samples were derivatized with 6 μL of 16.58 mM 3-(2-furoyl) quinoline-2-carboxaldehyde (Molecular Probes, Eugene, OR, USA) in the presence of 2 μL of KCN 25 mM in 10 mM borate buffer (pH 9.2) and 1 μL of internal standard (0.075 mM O-methyl-L-threonine). The mixture was allowed to react in the dark at 65 °C for 15 min. Separation and analysis were conducted in a capillary electrophoresis system (Beckman-Coulter PACE/MDQ, Glycoprotein System, Beckman Coulter, Brea, CA, USA) with laser-induced fluorescence detection; light at 488 nm from an argon ion laser was used to excite the 3-(2-furoyl) quinoline-2-carboxaldehyde-labeled analytes. A micellar electrokinetic chromatography buffer system was used to separate the dialyzed compounds. The borate buffer included 35 mM borates, sodium dodecylsulphate 25 mM, 13% (vol/vol) methanol HPLC grade, final pH 9.6. The samples were injected hydro-dynamically at 0.5 psi for 5 s in a 75 μm i.d. capillary (Beckman Coulter); then the separation was performed at 25 kV. After each sample, the capillary was flushed with 0.1 M NaOH, water and running buffer. The glutamate, norepinephrine and dopamine peaks were identified by matching the migration pattern with those in a spiked sample and corrected by relating the area under the curve of the unknown sample with the area under the curve of the internal standard. Data were analyzed using Karat System Gold (Beckman Coulter). Results are expressed as percentage of basal concentration (percentage of basal concentration = analyte concentration × 100/mean of the three first samples).

3.1. Emulation of amygdalar representation of the US and reactivation signals by pharmacological activation of NMDA and β-receptors elicits taste avoidance From the histological analysis the following rats with cannulae misplacement were removed from results and statistical analysis: ISOISO n = 1; MIX-SS n = 2; MIX-MIX n = 1. Fig. 1A represents the behavioral protocol previously described in the methodology. The twobottle protocol was used because is considered more sensitive since the animals have access to saccharin and water at the same time, enhancing the detection of the avoidance. The animals got accustomed to having two-bottles available for drinking during the baseline monitoring and they alternate between bottles throughout the experiment (Table 1). Table 2 shows the mean consumption of liquid during acquisition and test; no difference on total volume consumption (water + saccharin) was observed among groups during long-term memory test (F6,35) = 1.376, p = 0.253). Table 3 shows the raw consumption of the different groups during the long-term memory test; statistical analysis showed significant differences in the saccharin preference among groups (F6,35) = 6.629, p < 0.001), post hoc analysis revealed that both naturally (LiCl 0.15 M) and artificially induced (MIX-MIX) taste avoidance are reflected as a significantly lower saccharin preference when compared to the SS-SS group (p < 0.001, d = 4.721 and p = 0.003, d = 3.461, respectively). The ISO-ISO, LHa-LHa, MIX-SS and SS-MIX groups exhibited a similar saccharin preference compared to SS-SS groups and did not have significant differences (SS-SS vs ISOISO p = 0.970, d = 0.367; SS-SS vs Lha-Lha p = 0.269, d = 0.901; SSSS vs MIX-SS p = 0.995, d = 0.245; SS-SS vs SS-MIX p = 0.854, d = 0.879). Moreover, no differences were observed in saccharin preference between LiCl 0.15 M and MIX-MIX groups (p = 0.807 d = 1.4). The groups LiCl 0.15 M (p = 0.004) and MIX-MIX (p = 0.017) showed preference indexes below chance, indicating a clear avoidance of saccharin, whereas the SS-SS (p = 0.003) and SS-MIX (p = 0.018) groups showed a preference index above chance, demonstrating a predilection for saccharin, hence no avoidance. Therefore, only double intraamygdalar infusions of a mixture of isoproterenol and L-homocysteic acid, which resemble the US encoding and reactivation signaling, induced a long-term avoidance memory, which is like a naturally developed response after taste and malaise pairing (Fig. 1B).

2.5. Histology Cannulae placement was verified at the end of behavioral procedures, animals were sacrificed with an overdose of sodium pentobarbital and transcardially perfused with a physiological saline solution. Brains were removed and stored in paraformaldehyde (4% solution in phosphate buffered saline) for 24 h. After a sucrose gradient treatment, coronal sections of 40 μm were cut and stained with cresyl violet. Samples were then examined under a light microscope to corroborate the correct placement of cannulas; those tips that were not located in the aimed area were removed from the statistical analysis (Fig. 3). 2.6. Statistical analysis For all experiments, animals were assigned to each treatment group after balancing their average consumption of tap water. Preference consumption indexes were calculated as follows: mL saccharin consumption/mL saccharin + mL tap water consumption. A preference consumption index equal to 0.5 reflects a random choice for saccharin.

3.2. Artificially and naturally induced taste avoidance elicit similar neurochemical representations within the insular cortex During microdialysis experiments (protocol representation in Fig. 2A), regarding extracellular levels of glutamate, repeated measures 3

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Fig. 1. Effect of pharmacological manipulation in the amygdala over saccharin preference during taste avoidance retrieval. (A) Schematic representation of the behavioral and microinfusion protocols used. (B) Long-term memory (LTM) for saccharin preference, MIX-MIX and LiCl 0.15 M groups showed reduced preference for saccharin during taste avoidance retrieval, whereas SS-SS, ISO-ISO, LHa-LHa, MIX-SS, and SS-MIX did not. Dark arrows represent microinfusions into the amygdala (Amy); gray arrow represent LiCl i.p. administration. The graphic is expressed as means of saccharin preference index ± SEM. Dotted line represents consumption on chance level (0.5); ** p < 0.01 versus SS-SS group. LHa: L-homocysteic acid; ISO: isoproterenol; MIX: L-homocysteic acid + isoproterenol; SS: saline solution; LTM: Long-term memory.

ANOVA analysis revealed significant differences among fractions (F8,190) = 2.232, p = 0.030) and an interaction effect (F16,190) = 1.777, p = 0.044) with no groups differences (F2,190) = 1.058, p = 0.349). Bonferroni's post hoc test showed that artificially induced avoidance (MIX-MIX) and LiCl 0.15 M groups were different in comparison to SSSS group at fraction 6 (p = 0.006 and p = 0.028, respectively), i.e., when animals were exposed to the conditioned stimulus during retrieval. Our data demonstrated that a naturally or artificially induced taste avoidance caused a significant augmentation in the extracellular levels of glutamate within the IC during memory retrieval (Fig. 2B). For norepinephrine release, statistical analysis showed significant differences among fractions (F8,167) = 2.117, p = 0.041) with an interaction effect (F16,167) = 1.770, p = 0.043), without group effect (F2,167) = 0.477, p = 0.621). Bonferroni's post hoc analysis revealed that MIX-MIX (p < 0.001) and LiCl 0.15 M (p < 0.001) groups were different in comparison to SS-SS group at fraction 6 (saccharin exposure). Therefore, memory retrieval produces an elevation of norepinephrine levels in the IC during both artificial and natural-induced avoidance (Fig. 2C). Regarding the dopamine release (Fig. 2D), two-way ANOVA revealed differences in dopamine levels among fractions (F8,185) = 3.345, p = 0.015) with no group effect (F2,185) = 0.414, p = 0.668) and no interaction effect (F16,185) = 1.187, p = 0.288). Post-hoc analysis showed that MIX-MIX (p < 0.001) and LiCl 0.15 M (p = 0.01) groups were different in comparison to SS-SS group at fraction 6 when the animals were exposed to the conditioned stimulus. We observed a clear increase in dopamine extracellular levels within the IC when animals were exposed to the saccharin after artificial or natural induced avoidance. In the behavioral assessment of these animals we observed significant differences in saccharin consumption during taste avoidance

retrieval among groups (F2,18) = 6.017, p = 0.039), post hoc analysis showed that saccharin consumption in LiCl 0.15 M (p = 0.021, d = 1.89) and MIX-MIX (p = 0.044, d = 1.046) groups were different from SS-SS group, whereas no difference was observed between LiCl 0.15 M and MIX-MIX groups (p = 0.588, d = 0.290) (Fig. 2E). 4. Discussion During the last decades, memory research has focused on how cellular and molecular mechanisms are involved in the codification and consolidation of memory. In CTA memory establishment, the amygdalar glutamatergic activity has been related to visceral signaling of the US [3,5,8,11,25]; whereas noradrenergic transmission is probably implicated with a stress response due to the administration of the malaiseinducing agent [3]. Specifically, β-adrenergic and NMDA receptors within the amygdala play an essential role in the US processing [5,9–11]. However, it has been reported that microinfusions of glutamate into the amygdala did not generate taste aversion or avoidance, unless it was paired with a subthreshold dose of LiCl [10,5]. Our experiments confirm that concomitant activation of β-adrenergic and NMDA receptors at 15 min post-intake is not enough to induce an avoidance response during retrieval, and subsequent reactivation of these receptors appears to prompt the long-term memory. Long-term memory stabilization involves post-acquisition reactivation patterns of activity [17] that can be reflected as extracellular changes in neurotransmitters after the CS-US pairing [3,4]. Coactivation of NMDA and β-adrenergic receptors during the post-acquisition reactivation timeframe has demonstrated to be necessary for taste aversion memory consolidation within the amygdala [3]. The present results further support the idea that glutamatergic and noradrenergic activities during the post-acquisition period are part of the 4

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Group

Saccharin

10.51 ± 0.68

Water

8.41 ± 0.68

11.07 ± 0.74 9.16 ± 0.59

Test

SS-SS ISO-ISO LHa-LHa MIX-SS SS-MIX MIX-MIX LiCl 0.15 M SS-SS ISO-ISO LHa-LHa MIX-SS SS-MIX MIX-MIX LiCl 0.15 M SS-SS ISO-ISO LHa-LHa MIX-SS SS-MIX MIX-MIX LiCl 0.15 M

Acquisition

Bottle 1 Bottle 2

Bottle 2

9.14 ± 0.74 11.55 ± 0.87 10.79 ± 0.88 8.01 ± 0.82

Bottle 1 Bottle 2 Bottle 1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.48 1.37 1.22 1.24 1.15 1.68 1.99 2.48 1.37 1.22 1.24 1.15 1.68 1.99 2.48 1.37 1.22 1.24 1.15 1.68 1.99

17.50 18.00 17.33 16.33 22.17 21.29 16.83 17.50 18.00 17.33 16.33 22.17 21.29 16.83 17.50 18.00 17.33 16.33 22.17 21.29 16.83

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.50 1.52 1.45 1.60 1.55 1.01 0.94 2.50 1.52 1.45 1.60 1.55 1.01 0.94 2.50 1.52 1.45 1.60 1.55 1.01 0.94

Group

Saccharin consumption (mL) during test

Water consumption (mL) during test

Preference index

SS-SS ISO-ISO Lha-Lha MIX-SS SS-MIX MIX-MIX LiCl

12.83 ± 1.24 12.17 ± 1.53 9.83 ± 2.12 11.67 ± 1.68 14.33 ± 0.42 8.00 ± 1.17 5.16 ± 1.60

4.66 ± 1.38 5.83 ± 2.023 7.5 ± 2.094 4.66 ± 1.94 7.83 ± 1.64 13.29 ± 0.68 11.67 ± 1.46

0.76 ± 0.05 0.69 ± 0.09 0.56 ± 0.12 0.72 ± 0.09 0.66 ± 0.04 0.36 ± 0.04 ** 0.29 ± 0.09**

neurobiological mechanisms involved in the consolidation of taste aversive memory. According to our observations, it is indispensable to generate both the US signaling and the post-acquisition signaling in the amygdala to induce a reliable taste avoidance response reflecting or representing the establishment of a taste memory trace. Therefore, it is possible that US signaling through the activation of NMDA and βadrenergic receptors initiates molecular pathways involved in synaptic modifications, whereas post-learning reactivation activity patterns underlies long-term memory persistence. Although the period between administrations was calculated according to the US and post-acquisition events reported previously, it is possible that kinetics of drug’s action may be altered by the co-administration of isoproterenol and LHa. It has been reported that isoproterenol promotes LHa release from astrocytes, as it is an endogenous excitatory aminoacid [26]. Therefore, it is possible that β-adrenergic activation leads to a higher concentration of LHa within the amygdala altering the kinetics of NMDA activation. The activation of β-adrenergic and NMDA receptors leads to a synergistic effect for the establishment of taste avoidance memory. It has been demonstrated that synaptic plasticity is facilitated by β-adrenergic activation, probably as a result of the enhancement of P/Q type calcium currents in amygdalar neurons, generating an increase in intracellular calcium levels [27]. In addition, isoproterenol enhances synaptic transmission through an increase in presynaptic Ca2+ influx in the amygdala [28], producing a higher response mediated by AMPA [27] and NMDA currents [29]. Activation of β-adrenergic receptors

Day 5

mL of liquid intake

17.50 18.17 16.83 17.17 19.00 16.14 18.33 17.50 18.17 16.83 17.17 19.00 16.14 18.33 17.50 18.17 16.83 17.17 19.00 16.14 18.33

Consumption during test (mL)

** p < 0.01 vs SS-SS. Preference index = Sac mL/(Sac mL + H2O mL).

Day 6 7.28 ± 0.81 9.66 ± 0.66 6.11 ± 0.84

Bottle 1 Bottle 2 Bottle 1

Consumption during acquisition (mL)

Table 3 Mean consumption ± standard error and preference index values during test.

8.012 ± 0.82

Saccharin

8.53 ± 0.65 9.29 ± 0.61

Bottle 2

Bottle 1

Day 3 Day 2 Day 1 mL of liquid intake

Table 1 Mean consumption ± standard error of the two bottles presented throughout the experiments.

Table 2 Total fluid intake during acquisition and test stages. (Mean ± standard error).

8.04 ± 0.75

Water

8.57 ± 0.47

Day 4

Bottle 2

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Fig. 2. Taste avoidance responses and extracellular levels of glutamate, norepinephrine, and dopamine in the insular cortex during taste avoidance retrieval. (A) Schematic representation of the microdialysis protocol used. Changes in (B) glutamate, (C) norepinephrine, and (D) dopamine levels in SS-SS, LiCl 0.15 M and MIXMIX groups during retrieval of either naturally or artificially induced avoidance. Exposure to saccharin elicits an augmentation in glutamate, norepinephrine, and dopamine in the LiCl 0.15 M and MIX-MIX groups during taste avoidance retrieval. (E) Saccharin consumption during microdialysis procedure as percentage of the first saccharin exposure. MIX-MIX and LiCl 0.15 M groups display a taste avoidance response during memory retrieval. Dark arrows represent micro-infusions into the amygdala (Amy); gray arrow represents LiCl i.p. administration; and dotted arrow represent the insertion of the microdialysis probe in the insular cortex. Graphics are expressed as means of percentage of basal concentration ± SEM. (∗) p < 0.05 and (∗∗) p < 0.01 versus SS-SS group. Behavioral response graph is expressed as the mean of percentage of saccharin intake during acquisition phase ± SEM. (#) p < 0.05 versus SS-SS group. MIX: L-homocysteic acid + isoproterenol; SS: saline solution. LTM: Long-term memory.

promotes the stimulation of adenylate cyclase that triggers cAMP formation and subsequent activation of cyclic-AMP-dependent kinase (PKA) and mitogen-activated protein kinase (MAPK) [30]. The

activated PKA phosphorylates many synaptic and intracellular targets including cAMP response element binding pathway (CREB) [31,32]. The β-adrenergic activation causes the strengthening of intracellular 6

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Fig. 3. Summary of injector tips and microdialysis probes. (A) Representation of the tips of the injectors aiming the amygdala in the pharmacological activation experiment. (B) Representation of the tips of the injectors aiming the amygdala and the microdialysis probes within the insular cortex.

processes, resulting in a greater response to a given input, in this case the activation of glutamatergic receptors. PKA also facilitates AMPA receptors trafficking [33] and phosphorylation of NMDA receptors by PKA enhances Ca2+ conductivity [34,35]. The increased intracellular flow of Ca2+ promotes the activation of adenylate cyclase through calcium / calmodulin kinase by improving cAMP production [36] and the subsequent intracellular events described before. Therefore, the synergistic activation of adenylate cyclase, as a consequence of the activation of β-adrenergic receptors and NMDA, improves the activity of MAPK, CREB and transcription factors; that determine in a coordinated way the transcription and translation of genes. All these processes culminate in the increase in protein synthesis required for neuronal remodeling within the neural networks necessary to acquire and consolidate taste avoidance memories [37]. In this regard, it has been proposed that a single molecular event triggered during acquisition may not be sufficient to maintain longterm memory; and the persistence of memory requires rounds of consolidation-like processes during different time windows [38–41]. Therefore, we suggest that emulation of US amygdalar events is necessary to initiate the molecular pathways involved in modifications of the amygdala-insular network and the coactivation of NMDA and βadrenergic receptors during the post-learning period facilitates the taste avoidance memory consolidation through protein synthesis-dependent mechanisms.

Although unspecific modifications between the amygdala and different brain structures could be induced by our pharmacological manipulations, we focused on the response in the IC considering that taste aversion memory involves the functional integrity of this structure [42–46]; it is also possible that the communication of the amygdalainsular cortex had been modified affecting the trace of the taste in a way that an avoidance response is displayed in a second encounter. The amygdala has afferents to the IC [47,48] and receives feedback information from the insula [49]. The functional connectivity between the amygdala and the IC has been demonstrated by the tetanic stimulation of the amygdala that generates a long-term potentiation in the IC, promoting an improved retention of CTA [19,50]. Although the amygdala-IC pathway appears to be predominantly glutamatergic [9,43,50], it can also be modulated by dopaminergic activity from the ventral area tegmental [51], and norepinephrine from the locus coeruleus [52]. During the time frame following the acquisition of CTA, levels of dopamine and glutamate involved in the maintenance of longterm memory also increase within the IC. Interestingly, when the amygdala is transiently inactivated, there is an impairment of the reactivation signaling within the IC and CTA memory consolidation [4]. All these results suggest that the interaction of the amygdala-IC plays an important role in the formation and stabilization of taste memories where an aversive outcome is avoided. Recently, it has been demonstrated that during the taste aversion 7

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References

retrieval, the presentation of a CS stored as aversive induces an elevation of glutamate, dopamine and norepinephrine levels within the IC [21]. As shown, the artificial induction of taste avoidance promoted a similar increase in glutamate during memory retrieval. Glutamatergic neurotransmission seems to be relevant for the retrieval and maintenance of CTA since the infusions of α-amino-3-hidroxi-5-metilo-4isoxazolpropionic (AMPA) receptor antagonists in the IC hinders taste aversion retrieval [21,53]. Moreover, the presentation of an aversive or stressful stimulus causes an increase in norepinephrine levels in the forebrain [54], including the IC [55]. Similarly, injections of the malaise-inducing agent (the US) induce an increase in norepinephrine levels within the IC [4]. Thus, exposure to an aversively-conditioned taste stimulus generates an elevation of norepinephrine within the IC as reported previously [56]. In relation to dopamine release, a novel or salient stimulus cause activation of dopaminergic neurons [57,58]. Hence, consumption of an aversively conditioned taste stimulus promotes an increase in dopamine levels within the IC. This elevation of dopamine probably reflects the response to the salient stimulus generated by the relevance of the aversive taste. Therefore, the consumption of a taste stimulus associated with aversive signals, artificially stimulated or naturally associated with gastric malaise, produces an elevation of the extracellular levels of glutamate, norepinephrine and dopamine inside the IC. Our results demonstrate that the pharmacological activation of NMDA and β-adrenergic receptors in the amygdala could induce behavioral and neurochemical responses like a naturally-induced taste avoidance memory. Even though other neurotransmitter systems are involved in CTA formation, such as acetylcholine [59,60], cortical extracellular changes of this neurotransmitter have been related mostly to the novelty of the stimulus [6] and apparently is not required during CTA retrieval [59,61]. Other monoamines, such as serotonin participate in the CTA formation within the IC or the amygdala, but have been less studied during the retrieval phase in the amygdala [62] or the insular cortex [63].

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5. Conclusion The confirmation of the functional role of the neurochemical signals that have been described during and after the acquisition of an aversive taste memory allow us to better understand the mechanisms that underly memory formation. We suggest that the induction of an artificial taste aversion memory, through pharmacological activation of NMDA and β-adrenergic receptors within the amygdala, promotes changes in the amygdala-insular cortex circuit enabling aversive memory establishment. It is important to note that during memory retrieval, behavioral and neurotransmitters patterns response within the IC are indistinguishable from an artificially induced or true avoidance memory. Author contributions Conceptualization and writing DOG, FBR, KGR; data curation and methodology DOG and KRG; funding acquisition FBR and KGR. The authors declare no competing financial interests to disclose. Funding and disclosures This work was supported by CONACyT (CB-250870), DGAPA PAPIIT-UNAM (IN212919), FOSSIS-CONACyT (273308) and SEPPROMEP (UAM-PTC-585) grants. Acknowledgments Authors thank Dr. Luis Rodríguez-Durán for his technical assistance. Special thanks to Auraly Axell Luyen Díaz and Luz del Carmen Medellín Cruz for their excellent assistance during the experiments. 8

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