Computational design of bifaceted protein nanomaterials with tailorable properties.

Recent advances in computational methods have led to considerable progress in the design of self-assembling protein nanoparticles. However, nearly all nanoparticles designed to date exhibit strict point group symmetry, with each subunit occupying an identical, symmetrically related environment. This property limits the structural diversity that can be achieved and precludes anisotropic functionalization. Here, we describe a general computational strategy for designing multi-component bifaceted protein nanomaterials with two distinctly addressable sides. The method centers on docking pseudosymmetric heterooligomeric building blocks in architectures with dihedral symmetry and designing an asymmetric protein-protein interface between them. We used this approach to obtain an initial 30-subunit assembly with pseudo-D5 symmetry, and then generated an additional 15 variants in which we controllably altered the size and morphology of the bifaceted nanoparticles by designing de novo extensions to one of the subunits. Functionalization of the two distinct faces of the nanoparticles with de novo protein minibinders enabled specific colocalization of two populations of polystyrene microparticles coated with target protein receptors. The ability to accurately design anisotropic protein nanomaterials with precisely tunable structures and functions will be broadly useful in applications that require colocalizing two or more distinct target moieties.

Behavioral tests of the insulin-cholinergic-dopamine link in nucleus accumbens and inhibition by high fat-high sugar diet in male and female rats.

It was previously shown in striatal slices obtained from male rats that insulin excites cholinergic interneurons and increases dopamine (DA) release via α4ß2 nicotinic receptors on DA terminals. The effect of insulin on DA release was blocked either by maintaining rats on a high sugar-high fat (HS-HF) diet that induced hyperinsulinemia and nucleus accumbens (NAc) insulin receptor insensitivity, or applying the α4ß2 antagonist DHßE. In vivo, NAc shell insulin inactivation decreased a glucose lick microstructure parameter indicative of hedonic impact in male and female rats, and prevented flavor-nutrient learning, tested only in males. The HS-HF diet decreased hedonic impact in males but not females, and prevented flavor-nutrient learning, tested only in males. The present study extends testing to more fully assess the translation of brain slice results to the behaving rat. Insulin inactivation by antibody microinjection in NAc shell was found to decrease the number of lick bursts emitted and average lick burst size, measures of incentive motivation and hedonic impact respectively, for a wide range of glucose concentrations in male and female rats. In contrast, the HS-HF diet decreased these lick parameters in males but not females. Follow-up two-bottle choice tests for 10 % versus 40 % glucose showed decreased intake of both concentrations by males but increased intake of 40 % glucose by females. In a further set of experiments, it was predicted that α4ß2 receptor blockade would induce the same behavioral effects as insulin inactivation. In females, DHßE microinjection in NAc shell decreased both lick parameters for glucose as predicted, but in males only the number of lick bursts emitted was decreased. DHßE also decreased the number of lick bursts emitted for saccharin by females but not males. Finally, DHßE microinjection in NAc shell decreased flavor-nutrient learning in both sexes. The few discrepancies seen with regard to the hypothesized insulin-nicotinic-dopaminergic regulation of behavioral responses to nutritive sweetener, and its inhibition by HS-HF diet, are discussed with reference to sex differences in DA dynamics, female resistance to diet-induced metabolic morbidities, and extra-striatal cholinergic inputs to NAc.

Atomically accurate de novo design of single-domain antibodies.

Despite the central role that antibodies play in modern medicine, there is currently no way to rationally design novel antibodies to bind a specific epitope on a target. Instead, antibody discovery currently involves time-consuming immunization of an animal or library screening approaches. Here we demonstrate that a fine-tuned RFdiffusion network is capable of designing de novo antibody variable heavy chains (VHH's) that bind user-specified epitopes. We experimentally confirm binders to four disease-relevant epitopes, and the cryo-EM structure of a designed VHH bound to influenza hemagglutinin is nearly identical to the design model both in the configuration of the CDR loops and the overall binding pose.

Blueprinting extendable nanomaterials with standardized protein blocks.

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies, in comparison, has been much more complex, largely owing to the irregular shapes of protein structures1. Here we describe extendable linear, curved and angled protein building blocks, as well as inter-block interactions, that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight 'train track' assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not previously been possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank three-dimensional canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to 'back of an envelope' architectural blueprints.

Sex difference in the effect of environmental enrichment on food restriction-induced persistence of cocaine conditioned place preference and mechanistic underpinnings.

Psychosocial and environmental factors, including loss of natural reward, contribute to the risk of drug abuse. Reward loss has been modeled in animals by removal from social or sexual contact, transfer from enriched to impoverished housing, or restriction of food. We previously showed that food restriction increases the unconditioned rewarding effects of abused drugs and the conditioned incentive effects of drug-paired environments. Mechanistic studies provided evidence of decreased basal dopamine (DA) transmission, adaptive upregulation of signaling downstream of D1 DA receptor stimulation, synaptic upscaling and incorporation of calcium-permeable AMPA receptors (CP-AMPARs) in medium spiny neurons (MSNs) of nucleus accumbens (NAc). These findings align with the still evolving 'reward deficiency' hypothesis of drug abuse. The present study tested whether a compound natural reward that is known to increase DA utilization, environmental enrichment, would prevent the persistent expression of cocaine conditioned place preference (CPP) otherwise observed in food restricted rats, along with the mechanistic underpinnings. Because nearly all prior investigations of both food restriction and environmental enrichment effects on cocaine CPP were conducted in male rodents, both sexes were included in the present study. Results indicate that environmental enrichment curtailed the persistence of CPP expression, decreased signaling downstream of the D1R, and decreased the amplitude and frequency of spontaneous excitatory postsynaptic currents (EPSCs) in NAc MSNs of food restricted male, but not female, rats. The failure of environmental enrichment to significantly decrease food restriction-induced synaptic insertion of CP-AMPARs, and how this may accord with previous pharmacological findings that blockade of CP-AMPARs reverses behavioral effects of food restriction is discussed. In addition, it is speculated that estrous cycle-dependent fluctuations in DA release, receptor density and MSN excitability may obscure the effect of increased DA signaling during environmental enrichment, thereby interfering with development of the cellular and behavioral effects that enrichment produced in males.

Involvement of the Receptor for Advanced Glycation End Products (RAGE) in high fat-high sugar diet-induced anhedonia in rats.

Clinical and basic science investigation indicates a link between insulin resistance and anhedonia. Previous results of this laboratory point to impaired nucleus accumbens (NAc) insulin signaling as an underpinning of diet-induced anhedonia, based on use of a glucose lick microstructure assay. The present study evaluated whether advanced glycation end products (AGEs) and their receptor (RAGE), known to mediate obesogenic diet-induced inflammation and pathological metabolic conditions, are involved in this behavioral change. Six weeks maintenance of male and female rats on a high fat-high sugar liquid diet (chocolate Ensure) increased body weight gain, and markedly increased circulating insulin and leptin, but induced anhedonia (decreased first minute lick rate and lick burst size) in males only. In these subjects, anhedonia correlated with plasma concentrations of insulin. Although the diet did not alter plasma or NAc AGEs, or the expression of RAGE in the NAc, marginally significant correlations were seen between anhedonia and plasma content of several AGEs and NAc RAGE. Importantly, a small molecule RAGE antagonist, RAGE229, administered twice daily by oral gavage, prevented diet-induced anhedonia. This beneficial effect was associated with improved adipose function, reflected in the adiponectin/leptin ratio, and increased pCREB/total CREB in the NAc, and a shift in the pCREB correlation with pThr34-DARPP-32 from near-zero to strongly positive, such that both phospho-proteins correlated with the rescued hedonic response. This set of findings suggests that the receptor/signaling pathway and cell type underlying the RAGE229-mediated increase in pCREB may mediate anhedonia and its prevention. The possible role of adipose tissue as a locus of diet-induced RAGE signaling, and source of circulating factors that target NAc to modify hedonic reactivity are discussed.

Actions and Consequences of Insulin in the Striatum.

Insulin crosses the blood-brain barrier to enter the brain from the periphery. In the brain, insulin has well-established actions in the hypothalamus, as well as at the level of mesolimbic dopamine neurons in the midbrain. Notably, insulin also acts in the striatum, which shows abundant expression of insulin receptors (InsRs) throughout. These receptors are found on interneurons and striatal projections neurons, as well as on glial cells and dopamine axons. A striking functional consequence of insulin elevation in the striatum is promoting an increase in stimulated dopamine release. This boosting of dopamine release involves InsRs on cholinergic interneurons, and requires activation of nicotinic acetylcholine receptors on dopamine axons. Opposing this dopamine-enhancing effect, insulin also increases dopamine uptake through the action of insulin at InsRs on dopamine axons. Insulin acts on other striatal cells as well, including striatal projection neurons and astrocytes that also influence dopaminergic transmission and striatal function. Linking these cellular findings to behavior, striatal insulin signaling is required for the development of flavor-nutrient learning, implicating insulin as a reward signal in the brain. In this review, we discuss these and other actions of insulin in the striatum, including how they are influenced by diet and other physiological states.

Effects of nucleus accumbens insulin inactivation on microstructure of licking for glucose and saccharin in male and female rats.

Insulin of pancreatic origin enters the brain where several regions express a high density of insulin receptors. Functional studies of brain insulin signaling have focused predominantly on hypothalamic regulation of appetite and hippocampal regulation of learning. Recent studies point to involvement of nucleus accumbens (NAc) insulin signaling in a diet-sensitive response to glucose intake and reinforcement of flavor-nutrient learning. The present study used NAc shell microinjection of an insulin inactivating antibody (InsAb) to evaluate effects on the microstructure of licking for flavored 6.1% glucose. In both male and female rats, InsAb had no effect on the number of lick bursts emitted (a measure of motivation and/or satiety), but decreased the size of lick bursts (a measure of reward magnitude) in a series of five 30 min test sessions. This effect persisted beyond microinjection test sessions and was shown to depend on previous flavored glucose consumption under InsAb treatment rather than InsAb treatment alone. This suggests learning of diminished reward value and aligns with the previous finding that InsAb blocks flavor-nutrient learning. Specificity of the InsAb effect for nutrient reward was indicated by failure to affect any parameter of licking for flavored 0.25% saccharin solution. Finally, maintenance of rats on a 'Western' diet for twelve weeks produced a decrease in lick burst size for glucose in male rats, but an increase in lick burst size in females. Possible implications of these results for flavor-nutrient learning, maladaptive consequences of NAc insulin receptor subsensitivity, and the plausible involvement of distinct insulin-regulated mechanisms in NAc are discussed.

Modulatory Effects of Food Restriction on Brain and Behavioral Effects of Abused Drugs.

Energy homeostasis is achieved, in part, by metabolic signals that regulate the incentive motivating effects of food and its cues, thereby driving or curtailing procurement and consumption. The neural underpinnings of these regulated incentive effects have been identified as elements within the mesolimbic dopamine pathway. A separate line of research has shown that most drugs with abuse liability increase dopamine transmission in this same pathway and thereby reinforce self-administration. Consequently, one might expect shifts in energy balance and metabolic signaling to impact drug abuse risk. Basic science studies have yielded numerous examples of drug responses altered by diet manipulation. Considering the prevalence of weight loss dieting in Western societies, and the anorexigenic effects of many abused drugs themselves, we have focused on the CNS and behavioral effects of food restriction in rats. Food restriction has been shown to increase the reward magnitude of diverse drugs of abuse, and these effects have been attributed to neuroadaptations in the dopamine-innervated nucleus accumbens. The changes induced by food restriction include synaptic incorporation of calcium-permeable AMPA receptors and increased signaling downstream of D1 dopamine receptor stimulation. Recent studies suggest a mechanistic model in which concurrent stimulation of D1 and GluA2-lacking AMPA receptors enables increased stimulus-induced trafficking of GluA1/GluA2 AMPARs into the postsynaptic density, thereby increasing the incentive effects of food, drugs, and associated cues. In addition, the established role of AMPA receptor trafficking in enduring synaptic plasticity prompts speculation that drug use during food restriction may more strongly ingrain behavior relative to similar use under free-feeding conditions.

Homeostatic regulation of reward via synaptic insertion of calcium-permeable AMPA receptors in nucleus accumbens.

The incentive effects of food and related cues are determined by stimulus properties and the internal state of the organism. Enhanced hedonic reactivity and incentive motivation in energy deficient subjects have been demonstrated in animal models and humans. Defining the neurobiological underpinnings of these state-based modulatory effects could illuminate fundamental mechanisms of adaptive behavior, as well as provide insight into maladaptive consequences of weight loss dieting and the relationship between disturbed eating behavior and substance abuse. This article summarizes research of our laboratory aimed at identifying neuroadaptations induced by chronic food restriction (FR) that increase the reward magnitude of drugs and associated cues. The main findings are that FR decreases basal dopamine (DA) transmission, upregulates signaling downstream of the D1 DA receptor (D1R), and triggers synaptic incorporation of calcium-permeable AMPA receptors (CP-AMPARs) in the nucleus accumbens (NAc). Selective antagonism of CP-AMPARs decreases excitatory postsynaptic currents in NAc medium spiny neurons of FR rats and blocks the enhanced rewarding effects of d-amphetamine and a D1R, but not a D2R, agonist. These results suggest that FR drives CP-AMPARs into the synaptic membrane of D1R-expressing MSNs, possibly as a homeostatic response to reward loss. FR subjects also display diminished aversion for contexts associated with LiCl treatment and centrally infused cocaine. An encompassing, though speculative, hypothesis is that NAc synaptic incorporation of CP-AMPARs in response to food scarcity and other forms of sustained reward loss adaptively increases incentive effects of reward stimuli and, at the same time, diminishes responsiveness to aversive stimuli that have potential to interfere with goal pursuit.

Interactions between insulin and diet on striatal dopamine uptake kinetics in rodent brain slices.

Diet influences dopamine transmission in motor- and reward-related basal ganglia circuitry. In part, this reflects diet-dependent regulation of circulating and brain insulin levels. Activation of striatal insulin receptors amplifies axonal dopamine release in brain slices, and regulates food preference in vivo. The effect of insulin on dopamine release is indirect, and requires striatal cholinergic interneurons that express insulin receptors. However, insulin also acts directly on dopamine axons to increase dopamine uptake by promoting dopamine transporter (DAT) surface expression, counteracting enhanced dopamine release. Here, we determined the functional consequences of acute insulin exposure and chronic diet-induced changes in insulin on DAT activity after evoked dopamine release in striatal slices from adult ad-libitum fed (AL) rats and mice, and food-restricted (FR) or high-fat/high-sugar obesogenic (OB) diet rats. Uptake kinetics were assessed by fitting evoked dopamine transients to the Michaelis-Menten equation and extracting Cpeak and Vmax . Insulin (30 nm) increased both parameters in the caudate putamen and nucleus accumbens core of AL rats in an insulin receptor- and PI3-kinase-dependent manner. A pure effect of insulin on uptake was unmasked using mice lacking striatal acetylcholine, in which increased Vmax caused a decrease in Cpeak . Diet also influenced Vmax , which was lower in FR vs. AL. The effects of insulin on Cpeak and Vmax were amplified by FR but blunted by OB, consistent with opposite consequences of these diets on insulin levels and insulin receptor sensitivity. Overall, these data reveal acute and chronic effects of insulin and diet on dopamine release and uptake that will influence brain reward pathways.

Food restriction induces synaptic incorporation of calcium-permeable AMPA receptors in nucleus accumbens.

Chronic food restriction potentiates behavioral and cellular responses to drugs of abuse and D-1 dopamine receptor agonists administered systemically or locally in the nucleus accumbens (NAc). However, the alterations in NAc synaptic transmission underlying these effects are incompletely understood. AMPA receptor trafficking is a major mechanism for regulating synaptic strength, and previous studies have shown that both sucrose and d-amphetamine rapidly alter the abundance of AMPA receptor subunits in the NAc postsynaptic density (PSD) in a manner that differs between food-restricted and ad libitum fed rats. In this study we examined whether food restriction, in the absence of reward stimulus challenge, alters AMPAR subunit abundance in the NAc PSD. Food restriction was found to increase surface expression and, specifically, PSD abundance, of GluA1 but not GluA2, suggesting synaptic incorporation of GluA2-lacking Ca2+-permeable AMPARs (CP-AMPARs). Naspm, an antagonist of CP-AMPARs, decreased the amplitude of evoked EPSCs in NAc shell, and blocked the enhanced locomotor response to local microinjection of the D-1 receptor agonist, SKF-82958, in food-restricted, but not ad libitum fed, subjects. Although microinjection of the D-2 receptor agonist, quinpirole, also induced greater locomotor activation in food-restricted than ad libitum fed rats, this effect was not decreased by Naspm. Taken together, the present findings are consistent with the synaptic incorporation of CP-AMPARs in D-1 receptor-expressing medium spiny neurons in NAc as a mechanistic underpinning of the enhanced responsiveness of food-restricted rats to natural rewards and drugs of abuse.

Effects of diet and insulin on dopamine transporter activity and expression in rat caudate-putamen, nucleus accumbens, and midbrain.

Food restriction (FR) and obesogenic (OB) diets are known to alter brain dopamine transmission and exert opposite modulatory effects on behavioral responsiveness to psychostimulant drugs of abuse. Mechanisms underlying these diet effects are not fully understood. In this study, we examined diet effects on expression and function of the dopamine transporter (DAT) in caudate-putamen (CPu), nucleus accumbens (NAc), and midbrain regions. Dopamine (DA) uptake by CPu, NAc or midbrain synapto(neuro)somes was measured in vitro with rotating disk electrode voltammetry or with [3 H]DA uptake and was found to correlate with DAT surface expression, assessed by maximal [3 H](-)-2-ß-carbomethoxy-3-ß-(4-fluorophenyl)tropane binding and surface biotinylation assays. FR and OB diets were both found to decrease DAT activity in CPu with a corresponding decrease in surface expression but had no effects in the NAc and midbrain. Diet treatments also affected sensitivity to insulin-induced enhancement of DA uptake, with FR producing an increase in CPu and NAc, likely mediated by an observed increase in insulin receptor expression, and OB producing a decrease in NAc. The increased expression of insulin receptor in NAc of FR rats was accompanied by increased DA D2 receptor expression, and the decreased DAT expression and function in CPu of OB rats was accompanied by decreased DA D2 receptor expression. These results are discussed as partial mechanistic underpinnings of diet-induced adaptations that contribute to altered behavioral sensitivity to psychostimulants that target the DAT.

Effects of food restriction on expression of place conditioning and biochemical correlates in rat nucleus accumbens.

RATIONALE: When ad libitum-fed rats undergo cocaine place preference conditioning (CPP) but are switched to food restriction for testing, CPP becomes resistant to extinction and correlates with phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluA1 at Ser845 in nucleus accumbens (NAc) core. OBJECTIVES: This study tested whether food restriction increases persistence of morphine CPP and conditioned place aversions (CPA) induced by LiCl and naloxone-precipitated morphine withdrawal. MATERIALS AND METHODS: Ad libitum-fed rats were conditioned with morphine (6.0 mg/kg, i.p.), LiCl (50.0/75.0 mg/kg, i.p.), or naloxone (1.0 mg/kg, s.c.) 22 h post-morphine (20.0 mg/kg, s.c.). Half of the subjects were then switched to food restriction. Daily testing resumed 3 weeks later, and brains were harvested when one diet group met extinction criterion. Western analyses probed for pSer845-GluA1, pERK1, and pERK2 in NAc. RESULTS: Food restriction increased persistence of morphine CPP and preference scores correlated with pSer845-GluA1 in NAc core and shell. LiCl CPA was curtailed by food restriction, yet pSer845-GluA1 and pERK2 were elevated in NAc core of food-restricted rats. Food restriction increased persistence of naloxone CPA and elevated pSer845-GluA1 in NAc core and shell, and aversion scores were negatively correlated with pERK1 and pERK2 in NAc core. CONCLUSIONS: These results suggest that food restriction prolongs responsiveness to environmental contexts paired with subjective effects of both morphine and morphine withdrawal. A mechanistic scheme, attributing these effects to upregulation of pSer845-GluA1, but subject to override by CPA-specific, pERK2-mediated extinction learning, is explored to accommodate opposite effects of food restriction on LiCl and naloxone CPA.

Nucleus Accumbens AMPA Receptor Trafficking Upregulated by Food Restriction: An Unintended Target for Drugs of Abuse and Forbidden Foods.

There is a high rate of comorbidity between eating disorders and substance abuse, and specific evidence that weight-loss dieting can increase risk for binge pathology, rebound excessive weight gain, and initiation and relapse to drug abuse. The present overview discusses basic science findings indicating that chronic food restriction induces dopamine conservation, compensatory upregulation of D-1 dopamine receptor signaling, and synaptic incorporation of calcium-permeable glutamatergic AMPA receptors in nucleus accumbens. Evidence is presented which indicates that these neuroadaptations account for increased incentive effects of food, drugs, and associated environments during food restriction. In addition, these same neuroadaptations underlie upregulation of sucrose- and psychostimulant-induced trafficking of AMPA receptors to the nucleus accumbens postsynaptic density, which may be a mechanistic basis of enduring maladaptive behavior.

Insulin enhances striatal dopamine release by activating cholinergic interneurons and thereby signals reward.

Insulin activates insulin receptors (InsRs) in the hypothalamus to signal satiety after a meal. However, the rising incidence of obesity, which results in chronically elevated insulin levels, implies that insulin may also act in brain centres that regulate motivation and reward. We report here that insulin can amplify action potential-dependent dopamine (DA) release in the nucleus accumbens (NAc) and caudate-putamen through an indirect mechanism that involves striatal cholinergic interneurons that express InsRs. Furthermore, two different chronic diet manipulations in rats, food restriction (FR) and an obesogenic (OB) diet, oppositely alter the sensitivity of striatal DA release to insulin, with enhanced responsiveness in FR, but loss of responsiveness in OB. Behavioural studies show that intact insulin levels in the NAc shell are necessary for acquisition of preference for the flavour of a paired glucose solution. Together, these data imply that striatal insulin signalling enhances DA release to influence food choices.

Nucleus accumbens AMPA receptor involvement in cocaine-conditioned place preference under different dietary conditions in rats.

RATIONALE: When ad libitum-fed (AL) rats undergo cocaine place preference conditioning (CPP) but are switched to food restriction (FR) for testing, CPP is enhanced and preference scores correlate with phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluA1 at Ser845 in nucleus accumbens (NAc) core. OBJECTIVES: The present study tested whether a similar association exists in AL rats and whether an inhibitor of Ca(2+)-permeable AMPARs blocks CPP expression in either diet group. MATERIALS AND METHODS: In experiments 1-3, AL rats were conditioned with cocaine (12.0 mg/kg, i.p.). Three weeks later, CPP was tested daily and brains were harvested after the fifth test. Western analyses were used to probe for levels of AMPA receptors in NAc. In experiment 4, AL rats were conditioned, half were switched to FR for testing, and half in each diet group received NAc core microinjection of 1-naphthylacetyl spermine (NASPM (NASPM) (25.0 µg) prior to each test. RESULTS: In experiment 1, CPP expression in AL rats was associated with elevated pSer845-GluA1, GluA1, and GluA2 in NAc. In experiment 2, the correlation between pSer845-GluA1 and CPP was localized to NAc core. In experiment 3, pSer845-GluA1 following a CPP test was higher in NAc synaptic membranes of FR relative to AL rats. In experiment 4, NASPM blocked CPP expression in both diet groups. CONCLUSIONS: Results support a scheme in which pSer845-GluA1 in NAc core underlies expression of cocaine CPP and does so by stabilizing or trafficking Ca(2+)-permeable AMPARs to the synaptic membrane. The more robust CPP of FR rats may result from upregulation of stimulus-induced pSer845-GluA1.

Involvement of nucleus accumbens AMPA receptor trafficking in augmentation of D- amphetamine reward in food-restricted rats.

RATIONALE: Chronic food restriction (FR) increases behavioral responsiveness to drugs of abuse and associated environments. Pre- and postsynaptic neuroadaptations have been identified in the mesoaccumbens dopamine pathway of FR subjects but the mechanistic basis of increased drug reward magnitude remains unclear. OBJECTIVES: Effects of FR on basal and D-amphetamine-induced trafficking of AMPA receptor subunits to the nucleus accumbens (NAc) postsynaptic density (PSD) were examined, and AMPA receptor involvement in augmentation of D-amphetamine reward was tested. MATERIALS AND METHODS: FR and ad libitum fed (AL) rats were injected with D-amphetamine (2.5 mg/kg, i.p.) or vehicle. Brains were harvested and subcellular fractionation and Western analyses were used to assess AMPA receptor abundance in NAc homogenate and PSD fractions. A follow-up experiment used a curve-shift protocol of intracranial self-stimulation to assess the effect of 1-naphthylacetyl spermine (1-NASPM), a blocker of Ca(2+)-permeable AMPA receptors, on rewarding effects of D-amphetamine microinjected in NAc shell. RESULTS: FR increased GluA1 in the PSD, and D-amphetamine increased p-Ser845-GluA1, GluA1, GluA2, but not GluA3, with a greater effect in FR than AL rats. D-amphetamine lowered reward thresholds, with greater effects in FR than AL rats, and 1-NASPM selectively reversed the enhancing effect of FR. CONCLUSIONS: Results suggest that FR leads to increased synaptic incorporation of GluA1 homomers to potentiate rewarding effects of appetitive stimuli and, as a maladaptive byproduct, D-amphetamine. The D-amphetamine-induced increase in synaptic p-Ser845-GluA1, GluA1, and GluA2 may contribute to the rewarding effect of D-amphetamine, but may also be a mechanism of synaptic strengthening and behavior modification.

Sucrose ingestion induces rapid AMPA receptor trafficking.

The mechanisms by which natural rewards such as sugar affect synaptic transmission and behavior are largely unexplored. Here, we investigate regulation of nucleus accumbens synapses by sucrose intake. Previous studies have shown that AMPA receptor (AMPAR) trafficking is a major mechanism for regulating synaptic strength, and that in vitro, trafficking of AMPARs containing the GluA1 subunit takes place by a two-step mechanism involving extrasynaptic and then synaptic receptor transport. We report that in rat, repeated daily ingestion of a 25% sucrose solution transiently elevated spontaneous locomotion and potentiated accumbens core synapses through incorporation of Ca(2+)-permeable AMPA receptors (CPARs), which are GluA1-containing, GluA2-lacking AMPARs. Electrophysiological, biochemical, and quantitative electron microscopy studies revealed that sucrose training (7 d) induced a stable (>24 h) intraspinous GluA1 population, and that in these rats a single sucrose stimulus rapidly (5 min) but transiently (<24 h) elevated GluA1 at extrasynaptic sites. CPARs and dopamine D1 receptors were required in vivo for elevated locomotion after sucrose ingestion. Significantly, a 7 d protocol of daily ingestion of a 3% solution of saccharin, a noncaloric sweetener, induced synaptic GluA1 similarly to 25% sucrose ingestion. These findings identify multistep GluA1 trafficking, previously described in vitro, as a mechanism for acute regulation of synaptic transmission in vivo by a natural orosensory reward. Trafficking is stimulated by a chemosensory pathway that is not dependent on the caloric value of sucrose.

Effects of time of feeding on psychostimulant reward, conditioned place preference, metabolic hormone levels, and nucleus accumbens biochemical measures in food-restricted rats.

RATIONALE: Chronic food restriction (FR) increases rewarding effects of abused drugs and persistence of a cocaine-conditioned place preference (CPP). When there is a single daily meal, circadian rhythms are correspondingly entrained, and pre- and postprandial periods are accompanied by different circulating levels of metabolic hormones that modulate brain dopamine function. OBJECTIVES: The present study assessed whether rewarding effects of d-amphetamine, cocaine, and persistence of cocaine-CPP differ between FR subjects tested in the pre- and postprandial periods. MATERIALS AND METHODS: Rats were stereotaxically implanted with intracerebral microinjection cannulae and an electrode in lateral hypothalamus. Rewarding effects of d-amphetamine and cocaine were assessed using electrical self-stimulation in rats tested 1-4 or 18-21 h after the daily meal. Nonimplanted subjects acquired a cocaine-CPP while ad libitum fed and then were switched to FR and tested for CPP at these same times. RESULTS: Rewarding effects of intranucleus accumbens (NAc) d-amphetamine, intraventricular cocaine, and persistence of cocaine-CPP did not differ between rats tested 18-21 h food-deprived, when ghrelin and insulin levels were at peak and nadir, respectively, and those tested 1-4 h after feeding. Rats that expressed a persistent CPP had elevated levels of p-ERK1, GluA1, and p-Ser845-GluA1 in NAc core, and the latter correlated with CPP expression. CONCLUSIONS: Psychostimulant reward and persistence of CPP in FR rats are unaffected by time of testing relative to the daily meal. Further, NAc biochemical responses previously associated with enhanced drug responsiveness in FR rats are associated with persistent CPP expression.