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BackgroundEvidence supports a role for the gut-brain axis in Parkinson's disease (PD). Mice overexpressing human wild type α- synuclein (Thy1-haSyn) exhibit slow colonic transit prior to motor deficits, mirroring prodromal constipation in PD. Identifying molecular changes in the gut could provide both biomarkers for early diagnosis and gut-targeted therapies to prevent progression.ObjectiveTo identify early molecular changes in the gut-brain axis in Thy1-haSyn mice through gene expression profiling.MethodsGene expression profiling was performed on gut (colon) and brain (striatal) tissue from Thy1-haSyn and wild-type (WT) mice aged 1 and 3 months using 3' RNA sequencing. Analysis included differential expression, gene set enrichment and weighted gene co-expression network analysis (WGCNA).ResultsAt one month, differential expression (Thy1-haSyn vs. WT) of mitochondrial genes and pathways related to PD was discordant between gut and brain, with negative enrichment in brain (enriched in WT) but positive enrichment in gut. Linear regression of WGCNA modules showed partial independence of gut and brain gene expression changes. Thy1-haSyn-associated WGCNA modules in the gut were enriched for PD risk genes and PD-relevant pathways including inflammation, autophagy, and oxidative stress. Changes in gene expression were modest at 3 months.ConclusionsOverexpression of haSyn acutely disrupts gene expression in the colon. While changes in colon gene expression are highly related to known PD-relevant mechanisms, they are distinct from brain changes, and in some cases, opposite in direction. These findings are in line with the emerging view of PD as a multi-system disease.

We previously found that human heroin addicts and mice chronically exposed to morphine exhibit a significant increase in the number of detected hypocretin/orexin (Hcrt)-producing neurons. However, it remains unknown how this increase affects target areas of the hypocretin system involved in opioid withdrawal, including norepinephrine containing structures locus coeruleus (LC) and A1/A2 medullary regions. Using a combination of immunohistochemical, biochemical, imaging, and behavioral techniques, we now show that the increase in detected hypocretin cell number translates into a significant increase in hypocretin innervation and tyrosine hydroxylase (TH) levels in the LC without affecting norepinephrine-containing neuronal cell number. We show that the increase in TH is completely dependent on Hcrt innervation. The A1/A2 regions were unaffected by morphine treatment. Manipulation of the Hcrt system may affect opioid addiction and withdrawal.SIGNIFICANCE STATEMENT Previously, we have shown that the hypothalamic hypocretin system undergoes profound anatomic changes in human heroin addicts and in mice exposed to morphine, suggesting a role of this system in the development of addictive behaviors. The locus coeruleus plays a key role in opioid addiction. Here we report that the hypothalamic hypocretin innervation of the locus coeruleus increases dramatically with morphine administration to mice. This increase is correlated with a massive increase in tyrosine hydroxylase expression in locus coeruleus. Elimination of hypocretin neurons prevents the tyrosine hydroxylase increase in locus coeruleus and dampens the somatic and affective components of opioid withdrawal.

Impulsive behavior during adolescence may stem from developmental imbalances between motivational and cognitive-control systems, producing greater urges to pursue reward and weakened capacities to inhibit such actions. Here, we developed a Pavlovian-instrumental transfer (PIT) protocol to assay rats' ability to suppress cue-motivated reward seeking based on changes in reward expectancy. Traditionally, PIT studies focus on how reward-predictive cues motivate instrumental reward-seeking behavior (lever pressing). However, cues signaling imminent reward delivery also elicit countervailing focal-search responses (food-port entry). We first examined how reward expectancy (cue-reward probability) influences expression of these competing behaviors. Adult male rats increased rates of lever pressing when presented with cues signaling lower probabilities of reward but focused their activity at the food cup on trials with cues that signaled higher probabilities of reward. We then compared adolescent and adult male rats in their responsivity to cues signaling different reward probabilities. In contrast to adults, adolescent rats did not flexibly adjust patterns of responding based on the expected likelihood of reward delivery but increased their rate of lever pressing for both weak and strong cues. These findings indicate that control over cue-motivated behavior is fundamentally dysregulated during adolescence, providing a model for studying neurobiological mechanisms of adolescent impulsivity.

A multifunctional chemical neural probe fabrication process exploiting PDMS thin-film transfer to incorporate a microfluidic channel onto a silicon-based microelectrode array (MEA) platform, and enzyme microstamping to provide multi-analyte detection is described. The Si/PDMS hybrid chemtrode, modified with a nano-based on-probe IrOx reference electrode, was validated in brain phantoms and in rat brain.

Flexible neural probes have been pursued previously to minimize the mechanical mismatch between soft neural tissues and implants and thereby improve long-term performance. However, difficulties with insertion of such probes deep into the brain severely restricts their utility. We describe a solution to this problem using gallium (Ga) in probe construction, taking advantage of the solid-to-liquid phase change of the metal at body temperature and probe shape deformation to provide temperature-dependent control of stiffness over 5 orders of magnitude. Probes in the stiff state were successfully inserted 2 cm-deep into agarose gel "brain phantoms" and into rat brains under cooled conditions where, upon Ga melting, they became ultra soft, flexible, and stretchable in all directions. The current 30 μm-thick probes incorporated multilayer, deformable microfluidic channels for chemical agent delivery, electrical interconnects through Ga wires, and high-performance electrochemical glutamate sensing. These PDMS-based microprobes of ultra-large tunable stiffness (ULTS) should serve as an attractive platform for multifunctional chronic neural implants.

AimsLike drug addiction, cues associated with palatable foods can trigger food-seeking, even when sated. However, whether susceptibility to the motivating influence of food-related cues is a predisposing factor in overeating or a consequence of poor diet is difficult to determine in humans. Using a rodent model, we explored whether a highly palatable 'junk food' diet impacts responses to reward-paired cues in a Pavlovian-to-instrumental transfer test, using sweetened condensed milk (SCM) as the reward. The hedonic impact of SCM consumption was also assessed by analyzing licking microstructure.MethodsTo probe the effects of pattern and duration of junk food exposure, we provided rats with either regular chow ad libitum (controls) or chow plus access to junk food for either 2 or 24 h per day for 1, 3, or 6 weeks. We also examined how individual susceptibility to weight gain related to these measures.ResultsRats provided 24 h access to the junk food diet were insensitive to the motivational effects of a SCM-paired cue when tested sated even though their hedonic experience upon reward consumption was similar to controls. In contrast, rats provided restricted, 2 h access to junk food exhibited a cue generalization phenotype under sated conditions, lever-pressing with increased vigor in response to both a SCM-paired cue, and a cue not previously paired with reward. Hedonic response was also significantly higher in these animals relative to controls.ConclusionsThese data demonstrate that the pattern of junk food exposure differentially alters the hedonic impact of palatable foods and susceptibility to the motivating influence of cues in the environment to promote food-seeking actions when sated, which may be consequential for understanding overeating and obesity.

There is growing evidence that repeated consumption of highly palatable, nutritionally poor "junk food" diets can produce deficits in cognition and behavioral control. We explored whether long-term junk-food diet exposure disrupts rats' ability to make adaptive choices about which foods to pursue based on (1) expected reward value (outcome devaluation test) and (2) cue-evoked reward expectations (Pavlovian-to-instrumental test). Rats were initially food restricted and trained on two distinct response-outcome contingencies (e.g., left press chocolate pellets, and right press sweetened condensed milk) and stimulus-outcome contingencies (e.g., white noise chocolate pellets, and clicker sweetened condensed milk). They were then given 6 weeks of unrestricted access to regular chow alone (controls) or chow and either 1 or 24 h access to junk food per day. Subsequent tests of decision making revealed that rats in both junk-food diet groups were impaired in selecting actions based on either expected food value or the presence of food-paired cues. These data demonstrate that chronic junk food consumption can disrupt the processes underlying adaptive control over food-seeking behavior. We suggest that the resulting dysregulation of food seeking may contribute to overeating and obesity.

The dorsomedial striatum (DMS) has been strongly implicated in flexible, outcome-based decision making, including the outcome-specific Pavlovian-to-instrumental transfer effect (PIT), which measures the tendency for a reward-predictive cue to preferentially motivate actions that have been associated with the predicted reward over actions associated with different rewards. Although the neurochemical underpinnings of this effect are not well understood, there is growing evidence that striatal acetylcholine signaling may play an important role. This study investigated this hypothesis by assessing the effects of intra-DMS infusions of the nicotinic antagonist mecamylamine or the muscarinic antagonist scopolamine on expression of specific PIT in rats. These treatments produced dissociable behavioral effects. Mecamylamine infusions enhanced rats' tendency to use specific cue-elicited outcome expectations to select whichever action was trained with the predicted outcome, relative to their performance when tested after vehicle infusions. In contrast, scopolamine infusions appeared to render instrumental performance insensitive to this motivational influence of reward-paired cues. These drug treatments had no detectable effect on conditioned food cup approach behavior, indicating that they selectively perturbed cue-guided action selection without producing more wide-ranging alterations in behavioral control. Our findings reveal an important role for DMS acetylcholine signaling in modulating the impact of cue-evoked reward expectations on instrumental action selection.

It has been hypothesized that brain development during adolescence perturbs reward processing in a way that may ultimately contribute to the risky decision making associated with this stage of life, particularly in young males. To investigate potential reward dysfunction during adolescence, Experiment 1 examined palatable fluid intake in rats as a function of age and sex. During a series of twice-weekly test sessions, non-food-deprived rats were given the opportunity to voluntarily consume a highly palatable sweetened condensed milk (SCM) solution. We found that adolescent male, but not female, rats exhibited a pronounced, transient increase in SCM intake (normalized by body weight) that was centered around puberty. Additionally, adult females consumed more SCM than adult males and adolescent females. Using a well-established analytical framework to parse the influences of reward palatability and satiety on the temporal structure of feeding behavior, we found that palatability-driven intake at the outset of the meal was significantly elevated in adolescent males, relative to the other groups. Furthermore, although we found that there were some group differences in the onset of satiety, they were unlikely to contribute to differences in intake. Experiment 2 confirmed that adolescent male rats exhibit elevated palatable fluid consumption, relative to adult males, even when a non-caloric saccharin solution was used as the taste stimulus, demonstrating that these results were unlikely to be related to age-related differences in metabolic need. These findings suggest that elevated palatable food intake during adolescence is sex specific and driven by a fundamental change in reward processing. As adolescent risk taking has been hypothesized as a potential result of hypersensitivity to and overvaluation of appetitive stimuli, individual differences in reward palatability may factor into individual differences in adolescent risky decision making.

Through incentive learning, the emotional experience of a reward in a relevant need state (e.g. hunger for food) sets the incentive value that guides the performance of actions that earn that reward when the need state is encountered again. Opiate withdrawal has been proposed as a need state in which, through experience, opiate value can be increased, resulting in escalated opiate self-administration. Endogenous opioid transmission plays anatomically dissociable roles in the positive emotional experience of reward consumption and incentive learning. We, therefore, sought to determine if chronic opiate exposure and withdrawal produces a disruption in the fundamental incentive learning process such that reward seeking, even for non-opiate rewards, can become maladaptive, inconsistent with the emotional experience of reward consumption and irrespective of need. Rats trained to earn sucrose or water on a reward-seeking chain were treated with morphine (10-30 mg/kg, s.c.) daily for 11 days prior to testing in withdrawal. Opiate-withdrawn rats showed elevated reward-seeking actions, but only after they experienced the reward in withdrawal, an effect that was strongest in early (1-3 days), as opposed to late (14-16 days), withdrawal. This was sufficient to overcome a negative reward value change induced by sucrose experience in satiety and, in certain circumstances, was inconsistent with the emotional experience of reward consumption. Lastly, we found that early opiate withdrawal-induced inflation of reward value was blocked by inactivation of basolateral amygdala mu opioid receptors. These data suggest that in early opiate withdrawal, the incentive learning process is disrupted, resulting in maladaptive reward seeking.

Studies implicate opioid transmission in hedonic and metabolic control of feeding, although roles for specific endogenous opioid peptides have barely been addressed. Here, we studied palatable liquid consumption in proenkephalin knockout (PENK KO) and β-endorphin-deficient (BEND KO) mice, and how the body weight of these mice changed during consumption of an energy-dense highly palatable 'cafeteria diet'. When given access to sucrose solution, PENK KOs exhibited fewer bouts of licking than wild types, even though the length of bouts was similar to that of wild types, a pattern that suggests diminished food motivation. Conversely, BEND KOs did not differ from wild types in the number of licking bouts, even though these bouts were shorter in length, suggesting that they experienced the sucrose as being less palatable. In addition, licking responses in BEND, but not PENK, KO mice were insensitive to shifts in sucrose concentration or hunger. PENK, but not BEND, KOs exhibited lower baseline body weights compared with wild types on chow diet and attenuated weight gain when fed cafeteria diet. Based on this and related findings, we suggest endogenous enkephalins primarily set a background motivational tone regulating feeding behavior, whereas β-endorphin underlies orosensory reward in high need states or when the stimulus is especially valuable. Overall, these studies emphasize complex interplays between endogenous opioid peptides targeting μ-receptors, such as enkephalins and endorphins, underlying the regulation of feeding and body weight that might explain the poor efficacy of drugs that generally target μ-opioid receptors in the long-term control of appetite and body weight.

Mouse models of neuropsychiatric diseases provide a platform for mechanistic understanding and development of new therapies. We previously demonstrated that knockout of the mouse homolog of CNTNAP2 (contactin-associated protein-like 2), in which mutations cause cortical dysplasia and focal epilepsy (CDFE) syndrome, displays many features that parallel those of the human disorder. Because CDFE has high penetrance for autism spectrum disorder (ASD), we performed an in vivo screen for drugs that ameliorate abnormal social behavior in Cntnap2 mutant mice and found that acute administration of the neuropeptide oxytocin improved social deficits. We found a decrease in the number of oxytocin immunoreactive neurons in the paraventricular nucleus (PVN) of the hypothalamus in mutant mice and an overall decrease in brain oxytocin levels. Administration of a selective melanocortin receptor 4 agonist, which causes endogenous oxytocin release, also acutely rescued the social deficits, an effect blocked by an oxytocin antagonist. We confirmed that oxytocin neurons mediated the behavioral improvement by activating endogenous oxytocin neurons in the paraventricular hypothalamus with Designer Receptors Exclusively Activated by Designer Drugs (DREADD). Last, we showed that chronic early postnatal treatment with oxytocin led to more lasting behavioral recovery and restored oxytocin immunoreactivity in the PVN. These data demonstrate dysregulation of the oxytocin system in Cntnap2 knockout mice and suggest that there may be critical developmental windows for optimal treatment to rectify this deficit.

Drug addiction is marked by pathological drug seeking and intense drug craving, particularly in response to drug-related stimuli. Repeated psychostimulant administration is known to induce long-term alterations in mesolimbic dopamine (DA) signaling that are hypothesized to mediate this heightened sensitivity to environmental stimuli. However, there is little direct evidence that drug-induced alteration in mesolimbic DA function underlies this hypersensitivity to motivational cues. In the current study, we tested this hypothesis using fast-scan cyclic voltammetry to monitor phasic DA signaling in the nucleus accumbens core of cocaine-pretreated (6 once-daily injections of 15 mg/kg, i.p.) and drug-naive rats during a test of cue-evoked incentive motivation for food-the Pavlovian-to-instrumental transfer task. We found that prior cocaine exposure augmented both reward seeking and DA release triggered by the presentation of a reward-paired cue. Furthermore, cue-evoked DA signaling positively correlated with cue-evoked food seeking and was found to be a statistical mediator of this behavioral effect of cocaine. Taken together, these findings provide support for the hypothesis that repeated cocaine exposure enhances cue-evoked incentive motivation through augmented phasic mesolimbic DA signaling. This work sheds new light on a fundamental neurobiological mechanism underlying motivated behavior and its role in the expression of compulsive reward seeking.

Monoamine neurotransmitters are stored in both synaptic vesicles (SVs), which are required for release at the synapse, and large dense-core vesicles (LDCVs), which mediate extrasynaptic release. The contributions of each type of vesicular release to specific behaviors are not known. To address this issue, we generated mutations in the C-terminal trafficking domain of the Drosophila vesicular monoamine transporter (DVMAT), which is required for the vesicular storage of monoamines in both SVs and LDCVs. Deletion of the terminal 23 aa (DVMAT-Δ3) reduced the rate of endocytosis and localization of DVMAT to SVs, but supported localization to LDCVs. An alanine substitution mutation in a tyrosine-based motif (DVMAT-Y600A) also reduced sorting to SVs and showed an endocytic deficit specific to aminergic nerve terminals. Redistribution of DVMAT-Y600A from SV to LDCV fractions was also enhanced in aminergic neurons. To determine how these changes might affect behavior, we expressed DVMAT-Δ3 and DVMAT-Y600A in a dVMAT null genetic background that lacks endogenous dVMAT activity. When expressed ubiquitously, DVMAT-Δ3 showed a specific deficit in female fertility, whereas DVMAT-Y600A rescued behavior similarly to DVMAT-wt. In contrast, when expressed more specifically in octopaminergic neurons, both DVMAT-Δ3 and DVMAT-Y600A failed to rescue female fertility, and DVMAT-Y600A showed deficits in larval locomotion. DVMAT-Y600A also showed more severe dominant effects than either DVMAT-wt or DVMAT-Δ3. We propose that these behavioral deficits result from the redistribution of DVMAT from SVs to LDCVs. By extension, our data suggest that the balance of amine release from SVs versus that from LDCVs is critical for the function of some aminergic circuits.

Reward-seeking actions can be guided by external cues that signal reward availability. For instance, when confronted with a stimulus that signals sugar, rats will prefer an action that produces sugar over a second action that produces grain pellets. Action selection is also sensitive to changes in the incentive value of potential rewards. Thus, rats that have been prefed a large meal of sucrose will prefer a grain-seeking action to a sucrose-seeking action. The current study investigated the dependence of these different aspects of action selection on cholinergic transmission. Hungry rats were given differential training with two unique stimulus-outcome (S1-O1 and S2-O2) and action-outcome (A1-O1 and A2-O2) contingencies during separate training phases. Rats were then given a series of Pavlovian-to-instrumental transfer tests, an assay of cue-triggered responding. Before each test, rats were injected with scopolamine (0, 0.03, or 0.1 mg/kg, intraperitoneally), a muscarinic receptor antagonist, or mecamylamine (0, 0.75, or 2.25 mg/kg, intraperitoneally), a nicotinic receptor antagonist. Although the reward-paired cues were capable of biasing action selection when rats were tested off-drug, both anticholinergic treatments were effective in disrupting this effect. During a subsequent round of outcome devaluation testing-used to assess the sensitivity of action selection to a change in reward value--we found no effect of either scopolamine or mecamylamine. These results reveal that cholinergic signaling at both muscarinic and nicotinic receptors mediates action selection based on Pavlovian reward expectations, but is not critical for flexibly selecting actions using current reward values.

μ-opioid receptors (MORs) are necessary for the analgesic and addictive effects of opioids such as morphine, but the MOR-expressing neuronal populations that mediate the distinct opiate effects remain elusive. Here we devised a new conditional bacterial artificial chromosome rescue strategy to show, in mice, that targeted MOR expression in a subpopulation of striatal direct-pathway neurons enriched in the striosome and nucleus accumbens, in an otherwise MOR-null background, restores opiate reward and opiate-induced striatal dopamine release and partially restores motivation to self administer an opiate. However, these mice lack opiate analgesia or withdrawal. We used Cre-mediated deletion of the rescued MOR transgene to establish that expression of the MOR transgene in the striatum, rather than in extrastriatal sites, is needed for the restoration of opiate reward. Our study demonstrates that a subpopulation of striatal direct-pathway neurons is sufficient to support opiate reward-driven behaviors and provides a new intersectional genetic approach to dissecting neurocircuit-specific gene function in vivo.

BackgroundRecent theories addressing mesolimbic dopamine's role in reward processing emphasize two apparently distinct functions, one in reinforcement learning (i.e., prediction error) and another in incentive motivation (i.e., the invigoration of reward seeking elicited by reward-paired cues). Here, we evaluate the latter.MethodsUsing fast-scan cyclic voltammetry, we monitored, in real time, dopamine release in the nucleus accumbens core of rats (n = 9) during a Pavlovian-to-instrumental transfer task in which the effects of a reward-predictive cue on an independently trained instrumental action were assessed. Voltammetric data were parsed into slow and phasic components to determine whether these forms of dopamine signaling were differentially related to task performance.ResultsWe found that a reward-paired cue, which increased reward-seeking actions, induced an increase in phasic mesolimbic dopamine release and produced slower elevations in extracellular dopamine. Interestingly, phasic dopamine release was temporally related to and positively correlated with lever-press activity generally, while slow dopamine changes were not significantly related to such activity. Importantly, the propensity of the reward-paired cue to increase lever pressing was predicted by the amplitude of phasic dopamine release events, indicating a possible mechanism through which cues initiate reward-seeking actions.ConclusionsThese data suggest that those phasic mesolimbic dopamine release events thought to signal reward prediction error may also be related to the incentive motivational impact of reward-paired cues on reward-seeking actions.

The neurochemical changes underlying human emotions and social behaviour are largely unknown. Here we report on the changes in the levels of two hypothalamic neuropeptides, hypocretin-1 and melanin-concentrating hormone, measured in the human amygdala. We show that hypocretin-1 levels are maximal during positive emotion, social interaction and anger, behaviours that induce cataplexy in human narcoleptics. In contrast, melanin-concentrating hormone levels are minimal during social interaction, but are increased after eating. Both peptides are at minimal levels during periods of postoperative pain despite high levels of arousal. Melanin-concentrating hormone levels increase at sleep onset, consistent with a role in sleep induction, whereas hypocretin-1 levels increase at wake onset, consistent with a role in wake induction. Levels of these two peptides in humans are not simply linked to arousal, but rather to specific emotions and state transitions. Other arousal systems may be similarly emotionally specialized.

There is growing evidence that mere exposure to drugs can induce long-term alterations in the neural systems that mediate reward processing, motivation, and behavioral control, potentially causing the pathological pursuit of drugs that characterizes the addicted state. The incentive sensitization theory proposes that drug exposure potentiates the influence of reward-paired cues on behavior. It has also been suggested that drug exposure biases action selection towards the automatic execution of habits and away from more deliberate goal-directed control. The current study investigated whether rats given repeated exposure to peripherally administered cocaine would show alterations in incentive motivation (assayed using the Pavlovian-to-instrumental transfer (PIT) paradigm) or habit formation (assayed using sensitivity to reward devaluation). After instrumental and Pavlovian training for food pellet rewards, rats were given 6 daily injections of cocaine (15 mg/kg, IP) or saline, followed by a 10-d period of rest. Consistent with the incentive sensitization theory, cocaine-treated rats showed stronger cue-evoked lever pressing than saline-treated rats during the PIT test. The same rats were then trained on a new instrumental action with a new food pellet reward before undergoing a reward devaluation testing. Although saline-treated rats exhibited sensitivity to reward devaluation, indicative of goal-directed performance, cocaine-treated rats were insensitive to this treatment, suggesting a reliance on habitual processes. These findings, when taken together, indicate that repeated exposure to cocaine can cause broad alterations in behavioral control, spanning both motivational and action selection processes, and could therefore help explain aberrations of decision-making that underlie drug addiction.

BackgroundSequential reward-seeking actions are readily learned despite the temporal gap between the earliest (distal) action in the sequence and the reward delivery. Fast dopamine signaling is hypothesized to mediate this form of learning by reporting errors in reward prediction. However, such a role for dopamine release in voluntarily initiated action sequences remains to be demonstrated.MethodsUsing fast-scan cyclic voltammetry, we monitored phasic mesolimbic dopamine release, in real time, as rats performed a self-initiated sequence of lever presses to earn sucrose rewards. Before testing, rats received either 0 (n = 11), 5 (n = 11), or 10 (n = 8) days of action sequence training.ResultsFor rats acquiring the action sequence task at test, dopamine release was strongly elicited by response-contingent (but unexpected) rewards. With learning, a significant elevation in dopamine release preceded performance of the proximal action and subsequently came to precede the distal action. This predistal dopamine release response was also observed in rats previously trained on the action sequence task, and the amplitude of this signal predicted the latency with which rats completed the action sequence. Importantly, the dopamine response to contingent reward delivery was not observed in rats given extensive pretraining. Pharmacological analysis confirmed that task performance was dopamine-dependent.ConclusionsThese data suggest that phasic mesolimbic dopamine release mediates the influence that rewards exert over the performance of self-paced, sequentially-organized behavior and sheds light on how dopamine signaling abnormalities may contribute to disorders of behavioral control.

Abstract Hedonic reward, dependence and addiction are unwanted effects of opioid analgesics, linked to the phasic cycle of μ opioid receptor activation, tolerance and withdrawal. In vitro studies of recombinant G protein coupled receptors (GPCRs) over expressed in cell lines reveal an alternative tonic signaling mechanism that is independent of agonist. Such studies demonstrate that constitutive GPCR signaling can be inhibited by inverse agonists but not by neutral antagonists. However, ligand-independent activity has been difficult to examine in vivo, at the systems level, due to relatively low levels of constitutive activity of most GPCRs including μ receptors, often necessitating mutagenesis or pharmacological manipulation to enhance basal signaling. We previously demonstrated that the absence of β-arrestin 2 (β-arr2) augments the constitutive coupling of μ receptors to voltage-activated Ca2+ channels in primary afferent dorsal root ganglion neurons from β-arr2-/- mice. We used this in vitro approach to characterize neutral competitive antagonists and inverse agonists of the constitutively active wild type μ receptors in neurons. We administered these agents to β-arr2-/- mice to explore the role of constitutive μ receptor activity in nociception and hedonic tone. This study demonstrates that the induction of constitutive μ receptor activity in vivo in β-arr2-/- mice prolongs tail withdrawal from noxious heat, a phenomenon that was reversed by inverse agonists, but not by antagonists that lack negative efficacy. By contrast, the aversive effects of inverse agonists were similar in β-arr2-/- and β-arr2+/+ mice, suggesting that hedonic tone was unaffected.

The role of gamma amino butyric acid (GABA) release and inhibitory neurotransmission in regulating most behaviors remains unclear. The vesicular GABA transporter (VGAT) is required for the storage of GABA in synaptic vesicles and provides a potentially useful probe for inhibitory circuits. However, specific pharmacologic agents for VGAT are not available, and VGAT knockout mice are embryonically lethal, thus precluding behavioral studies. We have identified the Drosophila ortholog of the vesicular GABA transporter gene (which we refer to as dVGAT), immunocytologically mapped dVGAT protein expression in the larva and adult and characterized a dVGAT(minos) mutant allele. dVGAT is embryonically lethal and we do not detect residual dVGAT expression, suggesting that it is either a strong hypomorph or a null. To investigate the function of VGAT and GABA signaling in adult visual flight behavior, we have selectively rescued the dVGAT mutant during development. We show that reduced GABA release does not compromise the active optomotor control of wide-field pattern motion. Conversely, reduced dVGAT expression disrupts normal object tracking and figure-ground discrimination. These results demonstrate that visual behaviors are segregated by the level of GABA signaling in flies, and more generally establish dVGAT as a model to study the contribution of GABA release to other complex behaviors.

We assessed whether the presence of contextual cues paired with alcohol would disrupt rats' capacity to express appropriate goal-directed action control. Rats were first given differential context conditioning such that one set of contextual cues was paired with the injection of ethanol and a second, distinctive set of cues was paired with the injection of saline. All rats were then trained in a third, neutral context to press one lever for grain pellets and another lever for sucrose pellets. They were then given two extinction tests to evaluate their ability to choose between the two actions in response to the devaluation of one of the two food outcomes with one test conducted in the alcohol-paired context and the other conducted in the control (saline-paired) context. In the control context, rats exhibited goal-directed action control; i.e., they were able selectively to withhold the action that previously earned the now devalued outcome. However, these same rats were impaired when tested in the alcohol-paired context, performing both actions at the same rate regardless of the current value of their respective outcomes. Subsequent testing revealed that the rats were capable of overcoming this impairment if they were giving response-contingent feedback about the current value of the food outcomes. These results provide a clear demonstration of the disruptive influence that alcohol-paired cues can exert on decision-making in general and goal-directed action selection and choice in particular.

Unlike other monoamine neurotransmitters, the mechanism by which the brain's histamine content is regulated remains unclear. In mammals, vesicular monoamine transporters (VMATs) are expressed exclusively in neurons and mediate the storage of histamine and other monoamines. We have studied the visual system of Drosophila melanogaster in which histamine is the primary neurotransmitter released from photoreceptor cells. We report here that a novel mRNA splice variant of Drosophila VMAT (DVMAT-B) is expressed not in neurons but rather in a small subset of glia in the lamina of the fly's optic lobe. Histamine contents are reduced by mutation of dVMAT, but can be partially restored by specifically expressing DVMAT-B in glia. Our results suggest a novel role for a monoamine transporter in glia that may be relevant to histamine homeostasis in other systems.

Using Micro-Electro-Mechanical-Systems (MEMS) technologies, we have developed silicon wafer-based platinum microelectrode arrays (MEAs) modified with glutamate oxidase (GluOx) for electroenzymatic detection of glutamate in vivo. These MEAs were designed to have optimal spatial resolution for in vivo recordings. Selective detection of glutamate in the presence of the electroactive interferents, dopamine and ascorbic acid, was attained by deposition of polypyrrole and Nafion. The sensors responded to glutamate with a limit of detection under 1muM and a sub-1-second response time in solution. In addition to extensive in vitro characterization, the utility of these MEA glutamate biosensors was also established in vivo. In the anesthetized rat, these MEA glutamate biosensors were used for detection of cortically-evoked glutamate release in the ventral striatum. The MEA biosensors also were applied to the detection of stress-induced glutamate release in the dorsal striatum of the freely-moving rat.