Faculty Bibliography

Biological drive states exert homeostatic control in part by increasing the reinforcing effects of environmental incentive stimuli. An apparent by-product of this adaptive response is the enhanced acquisition of drug self-administration behavior in food-restricted (FR) animals. While previous research has demonstrated increased central sensitivity to rewarding effects of abused drugs and direct dopamine (DA) receptor agonists in FR subjects, the underlying neurobiology is not well understood. Recently, it was demonstrated that intracerebroventricular (i.c.v.) injection of the D-1 DA receptor agonist, SKF-82958 produces a stronger activation of striatal extracellular signal-regulated kinase (ERK) 1/2 and cyclic AMP response element-binding protein (CREB) in FR relative to ad libitum (AL) fed rats. The main purpose of the present study was to characterize the involvement and mechanisms of interaction between NMDA receptor function and the augmented cellular responses to D-1 DA receptor stimulation in nucleus accumbens (NAc) of FR rats. In experiment 1, Western immunoblotting was used to demonstrate that i.c.v. injection of SKF-82958 (20 microg) produces greater phosphorylation of the NMDA NR1 subunit and calcium-calmodulin kinase II (CaMK II) in NAc of FR as compared with AL rats. In experiment 2, pretreatment of subjects with the NMDA antagonist, MK-801 (1.0 mg/kg, i.p.) decreased SKF-82958-induced activation of CaMK II, ERK1/2 and CREB, and reversed the augmenting effect of FR on activation of all three proteins. In experiment 3, pretreatment with the mitogen-activated protein kinase/ERK kinase inhibitor SL-327 (60 mg/kg, i.p.) suppressed SKF-82958- induced activation of ERK1/2 and reversed the augmenting effect of FR on CREB activation. These results point to specific neuroadaptations in the NAc of FR rats whereby D-1 DA receptor stimulation leads to increased NMDA NR1 subunit phosphorylation and consequent increases in NMDA receptor-dependent CaMK II and ERK1/2 signaling, and increased NMDA receptor/ERK1/2-dependent phosphorylation of the nuclear transcription factor, CREB. The upregulated cellular responses to D-1 DA agonist challenge may play a role in the augmentation of drug reward and appetitive instrumental learning during periods of food restriction

Recently, attention has turned to the possibility that endocrine adiposity hormones, such as leptin, may regulate appetitively motivated behavior by modulating brain dopamine function. By extension, it has been hypothesized that the increased behavioral sensitivity of food-restricted, underweight rats to psychostimulant challenge may be triggered by the accompanying hypoleptinemia. The purpose of the present study was to determine whether two weeks of continuous intracerebroventricular (ICV) infusion of leptin alters the motor-activating effect of D-amphetamine (0.75 mg/kg, IP) in food-restricted rats. Lateral ventricular infusion of leptin, using a regimen that decreases food intake and body weight in ad libitum fed rats (12 microg/day), had no effect on the locomotor response to D-amphetamine in food-restricted rats that were maintained at 80% of prerestriction body weight. This result may indicate that hypoleptinemia is not involved in the induction/maintenance of neuroadaptations that mediate enhanced behavioral sensitivity to psychostimulant challenge. Interestingly, ad libitum fed rats treated with leptin displayed an increased locomotor response to D-amphetamine that was most prominent 3-5 days after termination of the infusion. Body weights and D-amphetamine sensitivity of these subjects returned to control values by 8-10 days postinfusion. The enhanced behavioral sensitivity to D-amphetamine in leptin-treated ad libitum fed rats may be a by-product of adipose depletion and, if so, would further support involvement of a peripheral signal other than hypoleptinemia in the modulation of central sensitivity to psychostimulant challenge

BACKGROUND: Chronic food restriction augments the rewarding effect of centrally administered psychostimulant drugs and this effect may involve a previously documented upregulation of D-1 dopamine receptor-mediated MAP kinase signaling in nucleus accumbens (NAc) and caudate-putamen (CPu). Psychostimulants are known to induce striatal glutamate release, and group I metabotropic glutamate receptors (mGluR) have been implicated in the cellular and behavioral responses to amphetamine. The purpose of the present study was to evaluate whether chronic food restriction increases striatal MAP kinase signaling in response to the group I mGluR agonist, DHPG. RESULTS: Western immunoblotting was used to demonstrate that intracerebroventricular (i.c.v.) injection of DHPG (500 nmol) produces greater activation of ERK1/2 and CREB in CPu and NAc of food-restricted as compared to ad libitum fed rats. Fos-immunostaining induced by DHPG was also stronger in CPu and NAc core of food-restricted relative to ad libitum fed rats. However, i.c.v. injection of saline-vehicle produced greater activation of ERK1/2 and CREB in CPu and NAc of food-restricted relative to ad libitum fed rats, and this difference was not seen when subjects received no i.c.v. injection prior to sacrifice. In addition, although DHPG activated Akt, there was no difference in Akt activation between feeding groups. To probe whether the augmented ERK1/2 and CREB activation in vehicle-injected food-restricted rats are mediated by one or more GluR types, effects of an NMDA antagonist (MK-801, 100 nmol), AMPA antagonist (DNQX, 10 nmol), and group I mGluR antagonist (AIDA, 100 nmol) were compared to saline-vehicle. Antagonist injections did not diminish activation of ERK1/2 or CREB. CONCLUSIONS: These results indicate that a group I mGluR agonist induces phosphorylation of Akt, ERK1/2 and CREB in both CPu and NAc. However, group I mGluR-mediated signaling may not be upregulated in food-restricted rats. Rather, a physiological response to 'i.c.v. injection stress' is augmented by food restriction and appears to summate with effects of the group I mGluR agonist in activating ERK1/2 and CREB. While the augmented cellular response of food-restricted rats to i.c.v. injection treatment represents additional evidence of enhanced CNS responsiveness in these subjects, the functional significance and underlying mechanism(s) of this effect remain to be elucidated

RATIONALE: Prior research indicates that psychostimulant-induced sensitization is not expressed in lateral hypothalamic electrical self-stimulation (LHSS)-based measures of drug reward, although the augmenting effect of chronic food restriction is. Neuroadaptations within the brain dopamine system have been identified in both psychostimulant-sensitized and food-restricted animals. Consequently, a variant of the LHSS paradigm in which responding is particularly sensitive to changes in dopaminergic tone may be best suited to detect and compare effects of chronic d-amphetamine and food restriction. Instrumental responding on a progressive ratio (PR) schedule is more sensitive to dopaminergic manipulations than is responding on a continuous reinforcement (CRF) schedule, but has not previously been used to examine chronic psychostimulant and food restriction effects on LHSS-based measures of drug reward. OBJECTIVE: The first aim of this study was to determine whether a regimen of d-amphetamine treatment, that produces locomotor sensitization (5 mg/kg per day x5 days), increases the reward-potentiating effect of d-amphetamine in a PR LHSS protocol. The second aim, was to determine whether chronic food restriction produces a marked increase in the reward-potentiating effect of d-amphetamine in the PR LHSS protocol and, if so, whether it is reversible in parallel with body weight recovery when free feeding is restored. METHOD: Reward-potentiating effects of a challenge dose of d-amphetamine (0.25 mg/kg, IP) were measured in terms of the break point of LHSS responding on a PR schedule of reinforcement, in ad libitum fed and food-restricted rats. RESULTS: A regimen of d-amphetamine treatment that produced locomotor sensitization did not increase the break point for LHSS in the presence or absence of d-amphetamine. Chronic food restriction produced a marked increase in the break point-increasing effect of d-amphetamine (3-fold), which returned to baseline in parallel with body weight recovery over a 4-week period of restored free-feeding. CONCLUSIONS: A locomotor-sensitizing regimen of d-amphetamine treatment does not increase the rewarding effect of LH electrical stimulation or the reward-potentiating effect of d-amphetamine in a PR LHSS protocol. The augmenting effect of chronic food restriction on drug reward is mechanistically and functionally different from psychostimulant sensitization and may be controlled by signals associated with adipose depletion and repletion

Results of behavioral and c-fos immunohistochemical studies have suggested that chronic food restriction and maintenance of animals at 75-80% of free-feeding body weight may increase d-1 dopamine (DA) receptor function. The purpose of the present study was to determine whether D-1 DA receptor binding and/or mitogen-activated protein kinase (MAPK) signaling in caudate-putamen (CPu) and nucleus accumbens (NAc) are increased in food-restricted subjects. In the first experiment, saturation binding of the D-1 DA receptor antagonist [3H]SCH-23390 indicated no difference between food-restricted and ad libitum fed rats with regard to density or affinity of d-1 binding sites in CPu or NAc. In the second experiment, activation of extracellular signal-regulated kinases (ERK1/2) and cyclic AMP response element-binding protein (CREB) by i.c.v. injection of the D-1 DA receptor agonist SKF-82958 (20 microg) were markedly greater in food-restricted than ad libitum fed rats. Given a prior finding that SKF-82958 does not differentially stimulate adenylyl cyclase in CPu or NAc of food-restricted versus ad libitum fed subjects, the present results suggest that increased D-1 DA receptor-mediated ERK1/2 MAP kinase signaling may mediate the enhanced downstream activation of CREB, c-fos, and behavioral responses in food-restricted subjects. It is of interest that food restriction also increased the activation of c-Jun N-terminal protein kinase/stress-activated protein kinase, but this effect was no greater in rats injected with SKF-82958 than in those injected with saline vehicle. This represents additional evidence of increased striatal cell signaling in food-restricted subjects, presumably in response to the i.c.v. injection procedure, although the underlying receptor mechanisms remain to be determined. There were no differences between feeding groups in protein levels of the major phosphatases, MKP-2 and PP1. The upregulation of striatal MAP kinase signaling in food-restricted animals may adaptively serve to facilitate associative learning but, at the same time, increase vulnerability to the rewarding and addictive properties of abused drugs

Chronic food restriction increases the rewarding, motor-activating and immediate-early gene responses to centrally injected psychostimulants and direct dopamine (DA) receptor agonists. The purpose of the present study was to determine whether the food restriction regimen that produces these effects causes adaptive changes in D-1 DA receptors and their intracellular signaling pathways. In all experiments, rats were fed 10 grams of chow each day until a 20-25% weight loss was achieved. This was followed by maintenance of the target body weight for at least one additional week. D-1 receptor binding: saturation curves were obtained for specific 3H-SCH23390 binding in caudate-putamen (CPU) and nucleus accumbens (NAC). There were no differences in Kd or Bmax between food-restricted and control subjects. cA

Lateral hypothalamic self-stimulation (LHSS) and the threshold-lowering effects of abused drugs are increased by chronic food restriction. LHSS is also modulated by the endocrine adiposity hormones insulin and leptin. The endogenous melanocortin receptor (MCR) ligands, AGRP (antagonist) and alpha-MSH (agonist) mediate opponent physiological and behavioral actions in response to changes in mediobasal hypothalamic leptin and insulin exposure. The purpose of the present study was to evaluate whether chronic stimulation or blockade of central MCRs alters the rewarding effect of LH stimulation or the threshold-lowering effect of d-amphetamine. Thirty rats were trained in a rate-frequency LHSS paradigm and the threshold-lowering effect of d-amphetamine (0.25 mg/kg, i.p.) was determined. Rats were then divided into 3 groups of 10 and implanted with subcutaneous osmotic minipumps that delivered either the MCR agonist MTII (0.5 mug/12 mul/24 hr), the MCR antagonist SHU9119 (0.5 mug/12 mul/24 hr), or vehicle (12 mul/24 hr) via a lateral ventricular cannula for 12 days. LHSS and the threshold-lowering effect of d-amphetamine were tested again on days 3, 8 and 12 of icv infusions. MTII and SHU9119 produced a sustained decrease and increase, respectively, in food intake. SHU9119 had no effect on LHSS threshold or the threshold-lowering effect of d-amphetamine. MTII, however, lowered LHSS thresholds and augmented the threshold-lowering effect of d-amphetamine. It is not yet clear whether these effects result directly from MCR stimulation or arise secondarily as a consequence of the progressive weight loss produced by MTII infusion

Cocaine-and Amphetamine Regulated Transcript (CART) was first identified as a mRNA upregulated in striatum in response to acute injection of cocaine and amphetamine. The CART transcript encodes for a protein (Pro-CART) which is processed to smaller forms of bioactive peptides, CART 55-102 and CART 62-102. The subcellular localization of CART peptides within dense core vesicles of axon terminals suggests a neurotransmitter role for these peptides. However, CART receptors have not been identified. In the present study, saturable specific binding of (125I) CART was demonstrated in GH3 cells (a rat pituitary adenoma cell line) and bovine anterior pituitary. Further, it was shown by confocal microscopy in live cells that activation of these binding sites results in translocation of beta-arrestin2/green fluorescent protein (beta-arr2-GFP) to the plasma membrane, suggesting that CART binding sites are G-protein coupled receptors. Analysis of receptor-mediated signal transduction revealed that CART peptides inhibit forskolin-stimulated cAMP production in a pertussis toxin-sensitive manner, suggesting that the CART binding site is coupled to Gi/o proteins. CART peptides also activated Mitogen Activated Protein Kinase (MAPK) in a time and dose dependent manner, with significantly different time courses for the 55-102 and 62-102 fragments. Together, these results indicate that cellular effects of CART peptides are mediated by membrane G-protein coupled receptors with negative and positive coupling to cAMP and MAPK signaling pathways, respectively

Cocaine and amphetamine regulated transcript (CART) was originally discovered as a novel up-regulated mRNA in a drug abuse paradigm. CART is one of the most abundantly expressed mRNA in the hypothalamus and nucleus accumbens. In rat, differential splicing gives rise to two different CART mRNAs encoding precursors of either 116 or 129 amino acids. CART precursor processing is tissue dependent and two forms of peptide predominate in the brain, CART 55-102 and CART 62-102. Aside from the first report on amphetamine and cocaine as inducers of CART mRNA expression, other studies found modest gender-dependent effects on expression level or no changes at all. The aim of the present study was to re-investigate effects of cocaine on ir-CART peptide levels in the brain using a 'binge' cocaine paradigm. Male rats were treated with 'binge' cocaine for 14 days (3x15 mg/kg/day, i.p.) . One hour after the final injection, levels of ir-CART were quantified by RIA and immunofluorescence. CART immunoreactivity was detected using polyclonal CART antibody as primary and Cy-3-conjugated donkey anti-rabbit secondary antibody. High levels of expression were found in hypothalamic areas and nucleus accumbens and very low levels in caudate putamen, frontal cortex and cerebellum. 'Binge' cocaine caused an increase in the cerebellum, pons, medulla and paraventricular hypothalamic nucleus. Pro-CART processing in 'binge' cocaine treated rats was compared with controls by Western blot analysis. In untreated rats the dominant peptide in all brain structures is CART 55-102. Data on the ratio between the two forms of peptides in cocaine treated rats will be presented

It is well established that chronic food restriction enhances sensitivity to the rewarding and motor-activating effects of abused drugs. However, neuroadaptations underlying these behavioral effects have not been characterized. The purpose of the present study was to explore the possibility that food restriction produces increased dopamine (DA) receptor function that is evident in behavior, signal transduction, and immediate early gene expression. In the first two experiments, rats received intracerebroventricular (i.c.v.) injections of the D1 DA receptor agonist SKF-82958, and the D2/3 DA receptor agonist quinpirole. Both agonists produced greater motor-activating effects in food-restricted than ad libitum-fed rats. In addition, Fos-immunostaining induced by SKF-82958 in caudate-putamen (CPu) and nucleus accumbens (Nac) was greater in food-restricted than ad libitum-fed rats, as was staining induced by quinpirole in globus pallidus and ventral pallidum. In the next two experiments, neuronal membranes prepared from CPu and Nac were exposed to SKF-82958 and quinpirole. Despite the documented involvement of cyclic AMP (cAMP) signaling in D1 DA receptor-mediated c-fos induction, stimulation of adenylyl cyclase (AC) activity by SKF-82958 in CPu and Nac did not differ between groups. Food restriction did, however, decrease AC stimulation by the direct enzyme stimulant, forskolin, but not NaF or MnCl(2), suggesting a shift in AC expression to a less catalytically efficient isoform. Finally, food restriction increased quinpirole-stimulated [(35)S]guanosine triphosphate-gammaS binding in CPu, suggesting that increased functional coupling between D2 DA receptors and G(i) may account for the augmented behavioral and pallidal c-Fos responses to quinpirole. Results of this study support the hypothesis that food restriction leads to neuroadaptations at the level of postsynaptic D1 and D2 receptor-bearing cells which, in turn, mediate augmented behavioral and transcriptional responses to DA. The signaling pathways mediating these augmented responses remain to be fully elucidated