The suppressive effects of sucrose and cocaine, but not lithium chloride, are greater in Lewis than in Fischer rats: evidence for the reward comparison hypothesis

Rats suppress intake of a saccharin conditioned stimulus (CS) when it is paired with an aversive unconditioned stimulus (US), an appetitive US, or a drug of abuse such as morphine or cocaine. It is unclear, however, whether the reduction in intake induced by these drugs is mediated by their aversive or their rewarding properties. The present set of experiments addressed this question by comparing the suppressive effects of a known aversive US (LiCl), a known reinforcing US (sucrose), and a drug of abuse (cocaine) in two strains of rats (i.e., Lewis and Fischer 344 rats) that differ in their preference for rewarding stimuli. The results show that, although both strains readily acquired a LiCl-induced conditioned taste aversion (CTA), the suppressive effects of sucrose and cocaine were robust in the drug-preferring Lewis rats and absent in the Fischer rats. These data argue against a CTA account and in favor of the reward comparison hypothesis.     

Possible routes of visceral information in the rat brain in formation of conditioned taste aversion.

When ingestion of a taste stimulus is paired with internal malaise, the animal remembers the taste and rejects its ingestion thereafter. This learning is referred to as conditioned taste aversion (CTA). To establish CTA in adult male Wistar rats, 0.1% saccharin and an i.p. injection of 0.15 M LiCl were used as the conditioned and unconditioned stimuli, respectively. Neuroanatomical study using the tracer method was performed to examine the ascending routes from the lateral part of the parabrachial nucleus (PBlat) which receives general visceral information and suggested the three possible routes to the amygdala: (1) direct route to the central nucleus of the amygdala (CeA); (2) diencephalic route to the basolateral nucleus of the amygdala (BLA) involving the zona incerta (ZI) and the midline and intralaminar thalamic complex (MITC); and (3) cortical route to the BLA involving insular cortex (IC). Rats with excitotoxic lesions of each of the CeA, ZI, MITC or IC had only a small or negligible effect on the acquisition of CTA. However, single lesions of the BLA and combined lesions of the ZI and IC, but not CeA and IC, almost completely abolished the acquisition of CTA. These results together with previous findings suggest that visceral (or unconditioned stimulus) information in the PBlat is sent to the BLA which is essential for the acquisition of CTA via the functionally important two parallel routes, the diencephalic and cortical routes, with either being able to create the aversion.     

Memory-dependent c-Fos expression in the nucleus accumbens and extended amygdala following the expression of a conditioned taste aversive in the rat

Retrieving the memory of a conditioned taste aversion involves multiple forebrain areas. Although the amygdala clearly plays a role in the expression of a conditioned taste aversion, critical regions, downstream from the amygdala remain to be defined. To this end, Fos immunoreactivity was used in the rat to explore forebrain structures associated with retrieval that have an anatomical relationship with the amygdala. The results showed that expression of a conditioned taste aversion to 0.5 M sucrose elicited neuronal activation in the nucleus accumbens and in a complex of structures collectively referred to as the extended amygdala. The posterior hypothalamus and parasubthalamic nucleus, which receive inputs from the extended amygdala, were also activated upon re-exposure to the sucrose conditioned stimulus. Fos immunoreactivity did not increase in these regions in response to an innately aversive tastant, quinine hydrochloride (conditioned stimulus control), nor to LiCl-induced visceral stimulation in unconditioned animals (unconditioned stimulus control). In addition, these regions did not respond to the sucrose conditioned stimulus in sham-conditioned animals. These results suggest that conditioned and innately aversive tastes are differentially processed in the forebrain circuitry that includes the nucleus accumbens and extended amygdala.     

Central gustatory lesions and learned taste aversions: unconditioned stimuli

The efficacy of two different unconditioned stimuli (US) in producing conditioned taste aversion (CTA) was tested in rats after bilateral ibotenic acid (IBO) lesions of the gustatory nucleus of thalamus (TTAx) and the medial and lateral parabrachial nuclei (mPBNx, lPBNx). An initial study determined an equivalent dose for the two USs, LiCl and cyclophosphamide (CY), using non-lesioned rats. Subsequently, using a separate set of lesioned animals and their sham controls (SHAM), injections of CY were paired 3 times with one of two taste stimuli (CSs), 0.1 M NaCl for half the rats in each group, 0.2 M sucrose for the other half. After these conditioning trials, the CS was presented twice more without the US, first in a 1-bottle test, then in a 2-bottle choice with water. The acquisition and test trials had 2 intervening water-only days to assure complete rehydration. Two weeks later, the same rats were tested again for acquisition of a CTA using LiCl as the US and the opposite CS as that used during the CY trials. The SHAM and TTAx groups learned to avoid consuming the taste associated with either CY or LiCl treatment. The two PBNx groups failed to learn an aversion regardless of the US.     

c-Fos Induction in the Rat Nucleus of the Solitary Tract by Intraoral Quinine Infusion Depends on Prior Contingent Pairing of Quinine and Lithium Chloride

Intraoral infusions of sucrose or saccharin induce c-Fos-like immunoreactivity (c-FLI) in the intermediate nucleus of the solitary tract (iNTS) of rats after acquisition of a conditioned taste aversion (CTA). The induction of c-FLI in the iNTS may be a consequence of the shift in behavioral response from ingestive to aversive behaviors that characterize acquisition and expression of a CTA. To test this hypothesis, rats were intraorally infused with 0.3 mM quinine sulfate, an aversive taste, 1. prior to conditioning, 2. after 3 noncontingent (unpaired) infusions of quinine and toxic lithium chloride (LiCl) injections, 3. after conditioning with 3 contingent pairings of quinine and LiCl, and 4. after extinction with repeated unpaired infusions of quinine. Intraoral infusions of quinine induced c-FLI in the iNTS only after acquisition of a CTA against quinine; quinine failed to induce c-FLI in the iNTS of unconditioned, noncontingently treated, or extinguished rats. The pattern of c-FLI in the iNTS induced by expression of a CTA against quinine was quantitatively and anatomically similar to that elicited by sucrose in rats expressing a CTA against sucrose. We conclude that aversive responses per se are not sufficient to induce c-FLI in the iNTS. Furthermore, contingent pairing of quinine and LiCl does not cause a shift in behavioral response from palatable, ingestive behaviors to aversive behaviors as in acquisition of a CTA against sucrose. Thus, we also conclude that a shift in behavior from ingestive to aversive responses is not required for increased c-FLI expression in the iNTS during CTA expression. Therefore, the induction of c-FLI in the iNTS during expression of a CTA may be correlated with neuronal processes specific to acquisition and expression of a CTA.     

Mechanical visceral pain model: chronic intermittent intestinal distention in the rat

Animal models designed to test the effectiveness of analgesic agents against viceral pain typically rely on a noxious chemical irritation of the peritoneum, e.g., acetic acid and phenylquinone writhing tests. While useful, this type of assay depends upon an acute inflammation and the release of local alogens. Further, ethical and scientific constraints prevent repeated assessments in a single animal, thereby compounding the difficulty of assessing tolerance development to analgesic agents. To overcome these constraints we developed a model for mechanical visceral pain (VPM) based on a repeatable and reversible duodenal distention in the rat. A chronic indwelling intraduodenal balloon catheter is well tolerated and upon inflation produces a writhing response graded in proportion to distention. This response is inhibited by morphine in a dose dependent manner.     

Wheel running produces conditioned food aversion

Previous investigations of conditioned taste aversion (CTA) induced by wheel running have used flavored liquids such as conditioned stimuli (CSs). Assuming that classical conditioning mediates activity anorexia, it is expected that CTA induced by physical activity should extend to food stimuli. The main purpose of the present experiment was to investigate this possibility. Rats were given a 60-min access to a running wheel [unconditioned stimulus (US)] either before or after being exposed to a novel distinctive flavored food (CS). An additional group had access to running wheels 4 h after receiving the CS food. Results from the present experiment indicate that regimented and contingent periods of wheel running decrease consumption of a food available before wheel running in nondeprived rats.     

Synergism of a compound unconditioned stimulus in taste aversion conditioning

Anti-lymphocyte serum (ALS) and lithium chloride have both previously been used successfully as unconditioned stimuli in taste aversion conditioning paradigms in rats. This report substantiates those findings but shows that when the two stimuli are given as a compound unconditioned stimulus in association with saccharin flavoured drinking solution, the conditioned taste aversion response following a second exposure to saccharin alone is more profound than that following conditioning with either ALS or LiCl alone. These results demonstrate synergism between the two stimuli when given together.     

Taste reactivity as a dependent measure of the rapid formation of conditioned taste aversion: a tool for the neural analysis of taste-visceral associations

Several explanations may account for deficits in the ability of animals to form taste aversions following neural manipulations. These encompass impairments in conditioned stimulus (CS) and unconditioned stimulus (US) processing, conditioned response (CR) measurement, and expression, memory, and taste-visceral integration. A behavioral procedure that aids in the distinction between some of these possibilities is presented. In Experiment 1, 10 rats received seven intraoral (IO) infusions of sucrose (30 s, 0.55 ml) spaced every 5 min starting immediately after the injection of 3.0 mEq/kg of lithium chloride (LiCl). Control rats (n = 12) were treated identically except that they were injected with sodium chloride (NaCl). Oromotor and somatic taste reactivity behaviors were videotaped and analyzed. Lithium-injected rats systematically decreased their ingestive taste reactivity behavior over time, whereas aversive behavior increased. Control rats maintained high and stable levels of ingestive responding and demonstrated virtually no aversive behavior over the 30-min period following sodium injection. Rats were tested several days later for the presence of a conditioned taste aversion (CTA). Rats previously injected with lithium during sucrose infusions demonstrated significantly more aversive behavior than the control group, which demonstrated none. There were no differences in the level of ingestive behavior displayed by the two groups on the CTA test. Experiment 3 revealed that when similarly treated rats were tested for a CTA while in a lithium-induced state, a difference in the ingestive behavior between the two groups was observed. In Experiment 2, naive rats were injected with either NaCl or LiCl but did not receive their first sucrose infusion until 20 min later. These rats also received sucrose infusions at 25 and 30 min postinjection. There were no differences in the taste reactivity behavior displayed by lithium- or sodium-injected rats during any of the sucrose infusions. Collectively, these findings indicate that rats dramatically change their oromotor responses to sucrose during the period following LiCl administration, provided that the infusions start immediately after injection. Furthermore, this time-related behavioral change is predominantly attributable to associative processes. This paradigm can be useful in distinguishing between neural manipulations that affect the establishment of taste-visceral associations from others that affect the animal's ability to retain such associations over the commonly employed 24-hr conditioning-test interval.     

The suppressive effects of LiCl, sucrose, and drugs of abuse are modulated by sucrose concentration in food-deprived rats.

Suppression of intake of a gustatory conditioned stimulus (CS) occurs when paired with either an aversive or an appetitive unconditioned stimulus (US). Toxic substances, such as lithium chloride (LiCl), induce conditioned taste aversions while rewarding stimuli, such as high a concentration of sucrose, reduce intake through a comparison process referred to as anticipatory contrast. Drugs of abuse also suppress CS intake, but it is not known whether they do so via their rewarding or aversive properties. Using 0.1, 0.3, or 0.5 M sucrose solutions as the gustatory CS, we compared the suppressive effects of LiCl (5.29 mg/kg), morphine (15 mg/kg), cocaine (10 mg/kg), and a 1.0-M sucrose solution in rats that were food deprived. The doses of the three drugs have been equated in terms of their suppressive effects in water-deprived and free-feeding rats. The results showed that in food-deprived rats the sucrose US failed to suppress intake of any of the sucrose CSs, the drugs of abuse failed to suppress intake of the 0.3 and 0.5-M concentration of sucrose, and LiCl failed to suppress intake of the 0.5-M sucrose solution. When taken together, these findings reveal that the suppressive effects of all USs (aversive, appetitive, and drugs of abuse) can be offset by the use of a caloric CS when evaluated in food-deprived rats.     

Differential involvement of medial prefrontal cortex and basolateral amygdala extracellular signal-regulated kinase in extinction of conditioned taste aversion is dependent on different intervals of extinction following conditioning

Extinction reflects a decrease in the conditioned response (CR) following non-reinforcement of a conditioned stimulus. Behavioral evidence indicates that extinction involves an inhibitory learning mechanism in which the extinguished CR reappears with presentation of an unconditioned stimulus. However, recent studies on fear conditioning suggest that extinction erases the original conditioning if the time interval between fear acquisition and extinction is short. The present study examined the effects of different intervals between acquisition and extinction of the original memory in conditioned taste aversion (CTA). Male Long-Evans rats acquired CTA by associating a 0.2% sucrose solution with malaise induced by i.p. injection of 4 ml/kg 0.15 M LiCl. Two different time intervals, 5 and 24 h, between CTA acquisition and extinction were used. Five or 24 h after CTA acquisition, extinction trials were performed, in which a bottle containing 20 ml of a 0.2% sucrose solution was provided for 10 min without subsequent LiCl injection. If sucrose consumption during the extinction trials was greater than the average water consumption, then rats were considered to have reached CTA extinction. Rats subjected to extinction trials lasting 24 h, but not 5 h, after acquisition re-exhibited the extinguished CR following injection of 0.15 M LiCl alone 7 days after acquisition. Extracellular signal-regulated kinase (ERK) in the medial prefrontal cortex (mPFC) and basolateral nucleus of the amygdala (BLA) was examined by Western blot after the first extinction trial. ERK activation in the mPFC was induced after the extinction trial beginning 5 h after acquisition, whereas the extinction trial performed 24 h after acquisition induced ERK activation in the BLA. These data suggest that the original conditioning can be inhibited or retained by CTA extinction depending on the time interval between acquisition and extinction and that the ERK transduction pathway in the mPFC and BLA is differentially involved in these processes.     

Impaired conditioned taste aversion learning in APP transgenic mice

Cognition in transgenic mouse models of Alzheimer's disease (AD) has been predominantly characterized in explicit spatial orientation tasks. However, dementia in AD encompasses also implicit memory systems. In the present study a line of transgenic mice (TgCRND8) encoding a double mutated allele of the human amyloid precursor protein (APP) genes was evaluated in an implicit associative learning task of conditioned taste aversion (CTA). CTA is a form of Pavlovian classical conditioning, in which a mouse learns to avoid a novel taste of saccharine (conditioned stimulus) paired with an experimentally induced (systemic injection of lithium chloride) nausea (unconditioned stimulus). In contrast to conditioned non-Tg mice, TgCRND8 APP mice developed weaker aversion against saccharine and quickly increased its consumption in repeated tests. These results indicate that TgCRND8 mice show a significant impairment not only in explicit spatial memory, as has been previously shown [Nature 408 (2000) 979], but also in implicit memory. Control experiments confirmed that TgCRND8 and non-Tg mice had comparable taste sensitivities in response to appetitive as well as aversive tastes. The study suggests that the CTA paradigm can be a sensitive tool to evaluate deficits in implicit associative learning in APP transgenic mouse models of AD.     

Once is too much: conditioned changes in accumbens dopamine following a single saccharin-morphine pairing

The present study tested whether presentation of a taste cue would support conditioned suppression of dopamine in the nucleus accumbens (NAcc) following a single taste-drug pairing. Nondeprived male Sprague-Dawley rats were given 20-min access to a 0.15% saccharin conditioned stimulus (CS). Immediately thereafter, experimental rats were injected with morphine (15 mg/kg ip); standard controls were injected with saline; and explicitly unpaired controls were injected with morphine, but approximately 24 hr later. All rats were then given one 20-min CS-only test. Microdialysis samples from the NAcc were measured over 20-min intervals before, during, and after CS access on the conditioning and test trial. The results showed that a single saccharin-morphine pairing led to a marked reduction in CS intake, and the reduction in intake was accompanied by a conditioned blunting of the accumbens dopamine response to the saccharin reward cue. In turn, a single exposure to the saccharin cue also blunted the unconditioned dopamine response to morphine. Reward comparison effects, then, are cross-modal, bidirectional, and immediate, resulting in both unconditioned and conditioned changes in brain and behavior.     

Taste effectiveness of some D- and L-amino acids in mice

To measure the hedonic effectiveness of some D-amino acids two-bottle preference tests were carried out on ddy mice, and subsequently conditioned taste aversion (CTA) experiments were performed to determine to what degree hedonic responses to D- and L-amino acids are related to sweet taste in mice. Mice showed preferences for D-Trp, D-His, and D-Leu over certain concentration ranges, but were behaviorally neutral to D-Ser and D-Val up to 0.1 M. The preference magnitudes for D-amino acids increased with increasing molecular weight (MW) or the bulk of the side chain. CTA experiments using 0.2 M sucrose as a conditioning stimulus and 4 D- and 6 L-amino acids as test stimuli indicated that generalization of sucrose taste became stronger with increasing MW of D-amino acids and decreasing MW of L-amino acids. There was a highly significant positive correlation between the degree of generalization of sucrose taste and the preference magnitude for D- and L-amino acids. The results indicate that preferences for D- and L-amino acids in mice result from sweet taste produced by these chemicals. Comparison of the results with the human psychophysical data reveal similarity between humans and ddy mice in taste sensitivity to amino acids.     

The visceromotor response to colorectal distention fluctuates with the estrous cycle in rats

The existence of a sex difference in several chronic pain syndromes and the fluctuation of symptoms during the menstrual cycle strongly suggest sex hormones are involved in pain processing. The mechanisms underlying these changes are not well understood. Using the colorectal distention model in the rat, we previously reported a sex difference in the response to distention [Ji Y, Murphy AZ, Traub RJ (2006) Sex differences in morphine induced analgesia of visceral pain are supraspinally and peripherally mediated. Am J Physiol Regul Integr Comp Physiol 291:R307-R314] and that ovariectomy decreased the responses to distention while estrogen replacement reversed the decrease [Ji Y, Murphy AZ, Traub RJ (2003) Estrogen modulates the visceromotor reflex and responses of spinal dorsal horn neurons to colorectal stimulation in the rat. J Neurosci 23:3908-3915], suggesting estrogen increases visceral nociception. In the present study we tested the hypothesis that the visceromotor response to colorectal distention fluctuates with the estrous cycle. Three measurements (vaginal smears, uterine tube weight and plasma estrogen concentration) were used to determine the estrous phase. Comparison of the visceromotor response threshold and magnitude was made between proestrus and metestrus/diestrus. Our experiment demonstrated that the distention threshold was significantly lower in proestrus (median: 15 mm Hg) as compared with metestrus/diestrus (median: 25 mm Hg); and the magnitude of the visceromotor response to graded intensities of colorectal distentions (20, 40, 60, 80 mm Hg) was significantly higher in proestrus. The results indicate that the visceromotor response fluctuates with estrous phase, providing evidence for endogenous estrogen modulation of visceral nociceptive processing that could contribute to sex differences.     

Water-deprivation prevents morphine-, but not LiCl-induced, suppression of sucrose intake.

Intake of a saccharin-conditioned stimulus (CS) can be suppressed following pairing with an aversive agent such as lithium chloride (LiCl) or x-rays (referred to as a conditioned taste aversion or CTA), a highly rewarding sucrose solution (referred to as an anticipatory contrast effect), or a drug of abuse such as morphine or cocaine. Although the suppressive effects of LiCl and sucrose are clear examples of aversive and appetitive conditioning, respectively, it is not certain which properties (aversive or appetitive) mediate the suppressive effects of drugs of abuse. It is known, however, that the suppressive effects of a rewarding sucrose US are attenuated when using a caloric sucrose CS in food deprived rats, while LiCl induced CTAs are much less effected. Standard CTA testing typically is conducted in water-deprived rather than food-deprived rats and, although LiCl is known to suppress intake of a sucrose CS in water-deprived rats, the suppressive effects of drugs of abuse have not been evaluated under these conditions. The present experiment, then, compared the suppressive effects of a standard dose of morphine (15 mg/kg) and a matched dose of LiCl (0.009 M) on intake of a sucrose CS in water-deprived and free-feeding rats. The results showed that both drugs suppressed intake in free-feeding subjects, but only the aversive agent, LiCl, reduced CS intake in the water-deprived rats. This finding dissociates the suppressive effects of morphine and LiCl and, in so doing, aligns the suppressive effects of morphine with those of an appetitive sucrose US.     

Electrophysiology of appetitive taste and appetitive taste conditioning in humans

The present study examines two novel aspects of appetitive processing and conditioning: the electrophysiological response to (a) the appetitive taste of a sweet fluid and (b) appetitively/taste conditioned visual stimuli. Event-related potentials (ERPs) were recorded from 32 subjects while they performed a taste conditioning task, in which two symbols were paired with a sweet or a neutral fluid. The results show a clear P1/N1/P2 complex, and a late positive potential (P3) with maxima at right fronto-central electrode sites, in response to the taste stimuli. Of these components particularly the P3 showed robust differences between the sweet and neutral taste. In addition, the electrophysiological response to the taste conditioned stimuli (CS), also showed the expected differences for both P2 and P3 at frontocentral electrodes. The present data demonstrate that the employed paradigm is a useful methodology to study the electrophysiological responses to unconditioned appetitive stimuli. Since appetitive conditioning is assumed to be disturbed in many psychopathological conditions, such as substance dependence, depression and eating disorders, this paradigm could be employed to get more insight in these conditions in humans.     

Acid taste thresholds assessed by conditioned taste aversion and two-bottle preference in rats

The conditioned taste aversion (CTA) threshold for either citric acid (CA) or HCl solutions and the two-bottle taste preference (TBP) threshold were determined in rats that are familiarized to the odor of conditioning solutions or that are naive. The CTA method appeared to be more sensitive than the TBP test, particularly when rats were not familiarized to the odor of the conditioning solution. The CTA threshold for HCl-conditioned rats and familiarized to the odor of conditioning solution lies between 1.00 and 2.00 mmol; in unconditioned rats, it lies between 4.00 and 5.00 mmol. In CA-conditioned and odor-familiarized rats, the threshold lies between 0.09 and 0.20 mmol; in unconditioned rats, it lies between 7.00 and 10.00 mmol. In rats not familiarized to the odor of the conditioning solution, the threshold for HCl-conditioned rats lies between 0.90 and 1.00 mmol; in unconditioned rats, it lies between 2.00 and 3.00 mmol. In CA-conditioned rats, the CTA threshold lies between 0.03 and 0.05 mmol; in unconditioned rats, it lies between 4.00 and 7.00 mmol. The two-bottle test is less sensitive than the CTA method. The TBP threshold lies between HCl 4.00 and 5.00 mmol, and between CA 4.00 and 7.00 mmol. The odor of a solution may potentiate the ability of rats to detect the concentration of CA and HCl solutions.     

Dissociation of conditioned and unconditioned factors in the running-induced feeding suppression

In adult rats, running wheel introduction induces a 7 to 10 day feeding suppression, either due to a learned conditioned taste avoidance or to the direct unconditioned effects of wheel running. The three experiments investigated the effects of wheel introduction on familiar (rat chow) and novel (24% sucrose solution) food consumption (Experiment 1), and then explored how alternate-day wheel access affected sucrose consumption when it was novel (Experiment 2) or familiar (Experiment 3). When paired with wheel introduction the consumption of a novel sucrose solution was completely suppressed for an extended period, whether the rats had continuous or alternate-day wheel access. In contrast, familiar food consumption was suppressed, for a limited period, only on wheel days. When rats were pre-exposed to the sucrose, consumption was suppressed only on wheel days. The results suggest that in addition to the direct unconditioned effects of wheel running on feeding, learning factors may influence the feeding suppression observed and thus wheel introduction supports a learned conditioned taste avoidance.     

Metabotropic glutamate receptor antagonists but not NMDA antagonists affect conditioned taste aversion acquisition in the parabrachial nucleus of rats

The effect of glutamate receptor antagonists on conditioned taste aversion (CTA) was studied in rats. The association of the short-term memory of a gustatory conditioned stimulus (CS) with visceral malaise (unconditioned stimulus, US) in the CTA paradigm takes place in the parabrachial nuclei (PBN) of the brainstem. The first direct evidence of participation of glutamatergic neurotransmission in the PBN during CTA demonstrated that the extracellular level of glutamate rises during saccharin drinking (Bielavska et al. in Brain Res 887:413–417, 2000). Our results show an effect of microdialysis administration of selective GluR antagonists into the PBN on the formation of CTA engram. We used four glutamate receptor (GluR) antagonists of different types (D-AP5, MK-801 as antagonists of ionotropic GluR and L-AP3, MSPG as antagonists of metabotropic GluR). The disruptive effect of MK-801 on CTA formation in the PBN is concentration-dependent, with the greatest inhibition under the higher concentrations eliciting significant disruption. The application of D-AP5 (0.1, 1, 5 mM) did not elicit a statistically significant blockade of CTA acquisition. This indicates that the association of the US–CS in the PBN is not dependent on NMDA receptors. On the contrary, application of L-AP3 (0.1, 1, 5 mM) blocked the CS–US association.