Faculty Bibliography

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.

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.

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.

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.

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.

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.

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 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.

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 increases dopamine uptake by promoting dopamine transporter (DAT) surface expression, which counteracts 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 C_peak and V_max . 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 V_max caused a decrease in C_peak . Diet also influenced V_max , which was lower in FR versus AL. The effects of insulin on C_peak and V_max 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 (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 the present study we examined diet effects on expression and function of the dopamine transporter (DAT) in caudate-putamen (CPu), nucleus accumbens (NAc), and midbrain regions. 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 DA transporter (DAT) surface expression, assessed by maximal [3 H]CFT 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 (InsR) expression, and OB producing a decrease in NAc. The increased expression of InsR in NAc of FR rats was accompanied by increased DA D2 receptor (D2 R) expression, and the decreased DAT expression and function in CPu of OB rats was accompanied by decreased D2 R 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

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. The present study 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 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