Risk preference following adolescent alcohol use is associated with corrupted encoding of costs but not rewards by mesolimbic dopamine.

Several emerging theories of addiction have described how abused substances exploit vulnerabilities in decision-making processes. These vulnerabilities have been proposed to result from pharmacologically corrupted neural mechanisms of normal brain valuation systems. High alcohol intake in rats during adolescence has been shown to increase risk preference, leading to suboptimal performance on a decision-making task when tested in adulthood. Understanding how alcohol use corrupts decision making in this way has significant clinical implications. However, the underlying mechanism by which alcohol use increases risk preference remains unclear. To address this central issue, we assessed dopamine neurotransmission with fast-scan cyclic voltammetry during reward valuation and risk-based decision making in rats with and without a history of adolescent alcohol intake. We specifically targeted the mesolimbic dopamine system, the site of action for virtually all abused substances. This system, which continuously develops during the adolescent period, is central to both reward processing and risk-based decision making. We report that a history of adolescent alcohol use alters dopamine signaling to risk but not to reward. Thus, a corruption of cost encoding suggests that adolescent alcohol use leads to long-term changes in decision making by altering the valuation of risk.

Long-term risk preference and suboptimal decision making following adolescent alcohol use.

Individuals who abused alcohol at an early age show decision-making impairments. However, the question of whether maladaptive choice constitutes a predisposing factor to, or a consequence resulting from, alcohol exposure remains open. To examine whether a causal link exists between voluntary alcohol consumption during adolescence and adult decision making the present studies used a rodent model. High levels of voluntary alcohol intake were promoted by providing adolescent rats with access to alcohol in a palatable gel matrix under nondeprivation conditions. A probability-discounting instrumental response task offered a choice between large but uncertain rewards and small but certain rewards to assess risk-based choice in adulthood either 3 weeks or 3 months following alcohol exposure. While control animals' performance on this task closely conformed to a predictive model of risk-neutral value matching, rats that consumed high levels of alcohol during adolescence violated this model, demonstrating greater risk preference. Evidence of significant risk bias was still present when choice was assessed 3 months following discontinuation of alcohol access. These findings provide evidence that adolescent alcohol exposure may lead to altered decision making during adulthood and this model offers a promising approach to the investigation of the neurobiological underpinnings of this link.

Biologically predisposed learning and selective associations in amygdalar neurons.

Modern views on learning and memory accept the notion of biological constraints-that the formation of association is not uniform across all stimuli. Yet cellular evidence of the encoding of selective associations is lacking. Here, conditioned stimuli (CSs) and unconditioned stimuli (USs) commonly employed in two basic associative learning paradigms, fear conditioning and taste aversion conditioning, were delivered in a manner compatible with a functional cellular imaging technique (Arc cellular compartmental analysis of temporal gene transcription by fluorescence in situ hybridization [catFISH]) to identify biological constraints on CS-US convergence at the level of neurons in basolateral amygdala (BLA). Results indicate coincident Arc mRNA activation within BLA neurons after CS-US combinations that yield rapid, efficient learning, but not after CS-US combinations that do not.

Visualizing stimulus convergence in amygdala neurons during associative learning.

A central feature of models of associative memory formation is the reliance on information convergence from pathways responsive to the conditioned stimulus (CS) and unconditioned stimulus (US). In particular, cells receiving coincident input are held to be critical for subsequent plasticity. Yet identification of neurons in the mammalian brain that respond to such coincident inputs during a learning event remains elusive. Here we use Arc cellular compartmental analysis of temporal gene transcription by fluorescence in situ hybridization (catFISH) to locate populations of neurons in the mammalian brain that respond to both the CS and US during training in a one-trial learning task, conditioned taste aversion (CTA). Individual neurons in the basolateral nucleus of the amygdala (BLA) responded to both the CS taste and US drug during conditioning. Coincident activation was not evident, however, when stimulus exposure was altered so as to be ineffective in promoting learning (backward conditioning, latent inhibition). Together, these data provide clear visualization of neurons in the mammalian brain receiving convergent information about the CS and US during acquisition of a learned association.

Neuronal representation of conditioned taste in the basolateral amygdala of rats.

Animals develop robust learning and long lasting taste aversion memory once they experience a new taste that is followed by visceral discomfort. A large body of literature has supported the hypothesis that basolateral amygdala (BLA) plays a critical role in the acquisition and extinction of such conditioned taste aversions (CTA). Despite the evidence that BLA is crucially engaged during CTA training, it is unclear how BLA neural activity represents the conditioned tastes. Here, we incorporated a modified behavioral paradigm suitable for single unit study, one which utilizes a sequence of pulsed saccharin and water infusion via intraoral cannulae. After conditioning, we investigated BLA unit activity while animals experience the conditioned taste (saccharin). Behavioral tests of taste reactivity confirmed that the utilized training procedure produced reliable acquisition and expression of the aversion throughout test sessions. When neural activity was compared between saccharin and water trials, half of the recorded BLA units (77/149) showed differential activity according to the types of solution. 76% of those cells (29/38) in the conditioned group showed suppressed activity, while only 44% of taste reactive cells (17/39) in controls showed suppressed activity during saccharin trials (relative to water trials). In addition, the overall excitability of BLA units was increased as shown by altered characteristics of burst activity after conditioning. The changes in BLA activity as a consequence of CTA were maintained throughout test sessions, consistent with the behavioral study. The current study suggests that the neuronal activity evoked by a sweet taste is altered as a consequence of CTA learning, and that the overall change might be related to the learning induced negative affect.

Sensitization of salt appetite is associated with increased "wanting" but not "liking" of a salt reward in the sodium-deplete rat.

To examine the role of incentive sensitization in the potentiation of salt appetite by prior depletions, the authors assessed the motivation to obtain salt ("wanting") and the palatability of salt ("liking") independently in salt-sensitized rats. Breakpoint on a progressive ratio reinforcement schedule was used to measure salt wanting and taste reactivity was used to measure salt liking in rats with and without a history of Na+ depletion. Salt-sensitized rats displayed higher breakpoints relative to controls. However, a history of Na+ depletion was not associated with a greater positive shift in taste reactivity measures. The data suggest that these components of reward are separable in this model and support the general proposition that sensitization may alter wanting but not liking.

Induction and expression of salt appetite: effects on Fos expression in nucleus accumbens.

Sodium depletion is a strong natural motivator that creates a pronounced sodium appetite and has been shown to activate neural regions associated with fluid and sodium balance. However, it is not known whether sodium appetite affects the mesolimbic circuitry associated with reward motivation. The present studies examined expression of the immediate early gene Fos in the nucleus accumbens (NAc) as a marker of neuronal activation following the induction and expression of furosemide-induced sodium appetite. During sodium appetite expression, sham-drinking and normal drinking were used to dissociate effects of NaCl taste stimulation from the repletion that follows absorption of sodium. These studies revealed that the combination of NaCl taste stimulation and persistent sodium depletion experienced by sham-drinking animals dramatically activates the NAc, while neither induction nor expression of sodium appetite alone is sufficient to increase Fos expression in this region. Results are discussed in terms of current theories of reward motivation.

Conditioning method determines patterns of c-fos expression following novel taste-illness pairing.

Conditioned taste aversions (CTAs) can be established by exposing rats to a novel taste CS through a bottle or through intra-oral (IO) infusion. Lesion studies suggest differences between the two methods in their engagement of brain circuits, as excitotoxic amygdala lesions have no effect on bottle-conditioned CTAs, but eliminate CTAs produced using IO infusion. Fos-like immunoreactivity (FLI) was used to compare patterns of brain activation after pairing CS taste and US drug using bottle and IO methods. Conditioning rats using the bottle method was associated with widespread elevations in FLI throughout the putative CTA circuit (basolateral and central nuclei of amygdala, insular cortex and nucleus of the solitary tract). In contrast, IO conditioning led to activation only in the central nucleus of amygdala. This supports the suggestion of differences in aversion processing as a function of conditioning method and may explain the greater reliance on amygdala of IO-conditioned CTAs due to engagement of a less distributed neural network.

A role for D2 but not D1 dopamine receptors in the cross-sensitization between amphetamine and salt appetite.

A history of sodium depletions has been found to potentiate the psychomotor as well as the rewarding effects of amphetamine, an indirect dopamine agonist. The present experiments were conducted to further define the role of dopamine receptor subtypes in this cross-sensitization effect. Rats with a history of sodium depletions were found to display psychomotor sensitization to a D2 but not a D1 direct agonist. Cross-sensitization between salt appetite and amphetamine was found to be blocked by a D2 but not a D1 antagonist. Together, these results implicate D2 but not D1 receptor function in the cross-sensitization seen after sodium depletions.

Induction of a salt appetite alters dendritic morphology in nucleus accumbens and sensitizes rats to amphetamine.

Sensitization to drugs, such as amphetamine, is associated with alterations in the morphology of neurons in the nucleus accumbens, a brain region critical to motivation and reward. The studies reported here indicate that a strong natural motivator, sodium depletion and associated salt appetite, also leads to alterations in neurons in nucleus accumbens. Medium spiny neurons in the shell of the nucleus accumbens of rats that had experienced sodium depletions had significantly more dendritic branches and spines than controls. In addition, a history of sodium depletions was found to have cross-sensitization effects, leading to enhanced psychostimulant responses to amphetamine. Thus, neuronal alterations common to salt and drug sensitization may provide a general mechanism for enhanced behavioral responses to subsequent exposures to these challenges.

Mapping conditioned taste aversion associations using c-Fos reveals a dynamic role for insular cortex.

Novel tastes are more effective than familiar tastes as conditioned stimuli (CSs) in taste aversion learning. Parallel to this, a novel CS-unconditioned stimulus (US) pairing induced stronger Fos-like immunoreactivity (FLI) in insular cortex (IC), amygdala, and brainstem than familiar CS-US pairing, suggesting a large circuit is recruited for acquisition. To better define the role of IC, the authors combined immunostaining with lesion or reversible inactivation of IC. Lesions abolished FLI increases to novel taste pairing in amygdala, suggesting a role in novelty detection. Reversible inactivation during taste preexposure increased FLI to familiar taste pairing in amygdala and brainstem. The difference between temporary inactivation, which blocked establishment of "safe" taste memory, and lesions points to a dual role for IC in taste learning.

Polycose taste pre-exposure fails to influence behavioral and neural indices of taste novelty.

Taste novelty can strongly modulate the speed and efficacy of taste aversion learning. Novel sweet tastes enhance c-Fos-like immunoreactivity (FLI) in the central amygdala and insular cortex. The present studies examined whether this neural correlate of novelty extends to different taste types by measuring FLI signals after exposure to novel and familiar polysaccharide (Polycose) and salt (NaCl) tastes. Novel Polycose not only failed to elevate FLI expression in central amygdala and insular cortex, but also failed to induce stronger taste aversion learning than familiar Polycose. Novel NaCl, on the other hand, showed patterns of FLI activation and aversion learning similar to that of novel sweet tastes. Possible reasons for the resistance of Polycose to typical pre-exposure effects are discussed.

Novel tastes elevate c-fos expression in the central amygdala and insular cortex: implication for taste aversion learning.

Taste novelty strongly modulates the speed and strength of taste aversion conditioning. To identify molecular signals responsive to novel tastes, immunostaining for c-fos protein (Fos-like immunoreactivity [FLI]) was used to mark neurons that responded differentially to taste novelty. Novel saccharin induced larger increases in FLI than familiar saccharin. This pattern was seen in central amygdala and insular cortex, but not in basolateral amygdala, parabrachial nucleus, or nucleus of the solitary tract. Other parameters known to influence aversion learning were tested for effects on FLI. Manipulations known to reduce the strength of learning blunted the FLI response, supporting the idea that FLI marks neural pathways critical to taste processing during acquisition, and that c-fos expression is a key transcriptional event underlying this plasticity.

Inhibition of protein kinase A activity interferes with long-term, but not short-term, memory of conditioned taste aversions.

The present experiments examined whether inhibition of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activity interferes with conditioned taste aversion (CTA) memories. Rats were centrally infused with the selective PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS) before conditioning. Direct infusions of Rp-cAMPS into the amygdala showed no interference with short-term memory but did show significant attenuation of long-term memory and more rapid extinction. Results suggest that PKA activity is involved in the consolidation of long-term memory of CTAs, and that the amygdala may be 1 site that is important for this activity.

Inhibition of protein kinase A activity during conditioned taste aversion retrieval: interference with extinction or reconsolidation of a memory?

The involvement of the cAMP-dependent protein kinase A (PKA) signaling pathway in protein synthesis-dependent memory consolidation has been supported by studies of fear conditioning and conditioned taste aversion (CTA). The present experiment examined whether inhibition of PKA activity at the time of memory retrieval impedes or promotes subsequent extinction. When Rp-cAMPS was infused into the amygdala at the time of CTA testing (retrieval), extinction was accelerated. Results confirm recent findings that stored memories become more labile when they are retrieved and extend these findings to CTA memories.

Afferent and efferent connections of the parvicellular subdivision of iNTS: defining a circuit involved in taste aversion learning.

Conditioned taste aversion (CTA) expression is associated with strong increases in Fos-like immunoreactivity (FLI) in a region of the brainstem identified as the parvicellular subdivision of the intermediate nucleus of the solitary tract (iNTSpc). To identify the projections to and from cells in iNTSpc which display strong FLI in response to expression of a CTA, anterograde and retrograde tract tracing was used. When appropriate, tract tracing was combined with double labeling for FLI in animals which received CTA training as well as tracer injections and were re-exposed to the CS taste. With respect to afferent projections, iNTSpc receives a strong, direct, ipsilateral projection from amygdala and the distribution of the fiber terminals yields a striking match to that of cells expressing FLI after CTA expression. As for efferent projections, these cells in iNTSpc are characterized by a mixed, rather than homogeneous, projection pattern. Targets of these cells include pons and forebrain as well as local medullary sites, all of which are known to be involved in gastrointestinal function. Thus, activation of these cells may provide a circuit through which gastrointestinal/visceral responses are coordinated as a component of the conditioned aversion.

Induction of a brainstem correlate of conditioned taste aversion expression: role of the pontine parabrachial nucleus.

Increases in Fos-like immunoreactivity (FLI) in the intermediate division of the nucleus of the solitary tract (iNTS) are seen following the expression of a conditioned taste aversion (CTA). In studies limited to behavioral assessment, the pontine parabrachial nucleus (PBN) has been demonstrated to play a critical role in the acquisition, but not the expression, of CTAs. To better define the role of the PBN in taste aversion learning, the present study examined the effects of PBN lesions on FLI in iNTS in animals with lesions placed either before or after CTA training. As is the case with behavioral expression of a CTA, timing of PBN lesions was found to be critical. Lesions placed prior to conditioning blocked evidence of conditioning, including both taste rejection and FLI in iNTS. Lesions placed after conditioning, but before testing, did not interfere with either taste rejection or FLI. These results support and extend prior claims that PBN is critical for CTA acquisition but not expression. They also demonstrate that input from PBN to iNTS is not necessary for the FLI seen there during CTA expression.

Conditioned taste aversion memory and c-Fos induction are disrupted in RIIbeta-protein kinase A mutant mice.

The cAMP-dependent protein kinase (PKA) signaling pathway has been implicated in many forms of learning. The present studies examined conditioned taste aversion (CTA) learning, an amygdala-dependent task, in mice with a targeted disruption of a gene for a specific regulatory subunit of PKA (RIIbeta), which is selectively expressed in amygdala. Null mutant (RIIbeta(-/-)) mice and littermate controls (RIIbeta(+/+)) were tested for protein synthesis-independent short-term memory (STM) and protein synthesis-dependent long-term memory (LTM) for CTAs. The ability of the unconditioned stimulus (US) drug, LiCl, to induce c-Fos in regions thought to be important in this learning was also determined. RIIbeta(-/-) mice showed significant impairment in CTA memory when tested 24h after training (LTM). In contrast, STM was normal. With regard to the c-Fos response to LiCl, the US drug, significant elevations were evident in brainstem (nucleus of the solitary tract) and pontine (parabrachial nucleus) regions, in mutants as well as wild-type controls. However, in amygdala, elevations were seen in controls but were absent in the mutants. These findings suggest that disruption of PKA signaling interferes with LTM consolidation of CTA and that a possible mediator of this effect is interference with c-Fos expression in amygdala which may be necessary for CTA memory.

Reciprocal cross-sensitization between amphetamine and salt appetite.

Previous work in our laboratory has demonstrated a potentiation of the psychomotor effects of amphetamine in animals with a history of sodium depletion, a process referred to as cross-sensitization. The present studies were done to further develop this finding by assessing multiple effects of amphetamine in rats with and without a history of sodium depletion. For Experiments 1-3, rats were depleted of sodium twice then subjected to one of three experimental procedures [open-field activity, conditioned place preference (CPP) and conditioned taste aversion (CTA)]. A history of depletion produced an elevation in the psychomotor effects of amphetamine. CPP, used to assess the rewarding properties of amphetamine, developed in rats with a history of depletion but not in controls. The aversive component of amphetamine as measured by CTA was unaffected by previous experience with sodium depletion. Finally, acute salt appetite after depletion was assessed in rats exposed to a sensitizing regimen of amphetamine. Animals with a drug history demonstrated a significant elevation in NaCl solution intake after depletion in comparison to controls. Together, the data provide strong evidence for the reciprocal cross-sensitization of salt appetite and response to amphetamine.

Altered risk-based decision making following adolescent alcohol use results from an imbalance in reinforcement learning in rats.

Alcohol use during adolescence has profound and enduring consequences on decision-making under risk. However, the fundamental psychological processes underlying these changes are unknown. Here, we show that alcohol use produces over-fast learning for better-than-expected, but not worse-than-expected, outcomes without altering subjective reward valuation. We constructed a simple reinforcement learning model to simulate altered decision making using behavioral parameters extracted from rats with a history of adolescent alcohol use. Remarkably, the learning imbalance alone was sufficient to simulate the divergence in choice behavior observed between these groups of animals. These findings identify a selective alteration in reinforcement learning following adolescent alcohol use that can account for a robust change in risk-based decision making persisting into later life.