Distinct Patterns of Gene Expression Changes in the Colon and Striatum of Young Mice Overexpressing Alpha-Synuclein Support Parkinson's Disease as a Multi-System Process.

BACKGROUND: Evidence 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. OBJECTIVE: To identify early molecular changes in the gut-brain axis in Thy1-haSyn mice through gene expression profiling. METHODS: Gene 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). RESULTS: At 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. CONCLUSIONS: Overexpression 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.

The proSAAS Chaperone Provides Neuroprotection and Attenuates Transsynaptic α-Synuclein Spread in Rodent Models of Parkinson's Disease.

BACKGROUND: Parkinson's disease involves aberrant aggregation of the synaptic protein alpha-synuclein (aSyn) in the nigrostriatal tract. We have previously shown that proSAAS, a small neuronal chaperone, blocks aSyn-induced dopaminergic cytotoxicity in primary nigral cultures. OBJECTIVE: To determine if proSAAS overexpression is neuroprotective in animal models of Parkinson's disease. METHODS: proSAAS- or GFP-encoding lentivirus was injected together with human aSyn-expressing AAV unilaterally into the substantia nigra of rats and motor asymmetry assessed using a battery of motor performance tests. Dopamine neuron survival was assessed by nigral stereology and striatal tyrosine hydroxylase (TH) densitometry. To examine transsynaptic spread of aSyn, aSyn AAV was injected into the vagus of mice in the presence of AAVs encoding either GFP or proSAAS; the spread of aSyn-positive neurites into rostral nuclei was quantified following immunohistochemistry. RESULTS: Coinjection of proSAAS-encoding lentivirus profoundly reduced the motor asymmetry caused by unilateral nigral AAV-mediated human aSyn overexpression. This was accompanied by significant amelioration of the human aSyn-induced loss of both nigral TH-positive cells and striatal TH-positive terminals, demonstrating clear proSAAS-mediated protection of the nigrostriatal tract. ProSAAS overexpression reduced human aSyn protein levels in nigra and striatum and reduced the loss of TH protein in both regions. Following vagal administration of human aSyn-encoding AAV, the number of human aSyn-positive neurites in the pons and caudal midbrain was considerably reduced in mice coinjected with proSAAS-, but not GFP-encoding AAV, supporting proSAAS-mediated blockade of transsynaptic aSyn transmission. CONCLUSION: The proSAAS chaperone may represent a promising target for therapeutic development in Parkinson's disease.

Hypocretin/Orexin Interactions with Norepinephrine Contribute to the Opiate Withdrawal Syndrome.

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.

Reward-predictive cues elicit excessive reward seeking in adolescent rats.

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.

An implantable multifunctional neural microprobe for simultaneous multi-analyte sensing and chemical delivery.

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, multifunctional neural probe with liquid metal enabled, ultra-large tunable stiffness for deep-brain chemical sensing and agent delivery.

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.

Junk Food Exposure Disrupts Selection of Food-Seeking Actions in Rats.

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.

Pattern of access determines influence of junk food diet on cue sensitivity and palatability.

AIMS: Like 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. METHODS: To 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. RESULTS: Rats 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. CONCLUSIONS: These 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.

Sex-specific enhancement of palatability-driven feeding in adolescent rats.

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.

Modulation of cue-triggered reward seeking by cholinergic signaling in the dorsomedial striatum.

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.

Effects of decreased dopamine transporter levels on nigrostriatal neurons and paraquat/maneb toxicity in mice.

How genetic variations in the dopamine transporter (DAT) combined with exposure to environmental toxins modulate the risk of Parkinson's disease remains unclear. Using unbiased stereology in DAT knock-down mice (DAT-KD) and wild-type (WT) littermates, we found that decreased DAT caused a loss of tyrosine hydroxylase-positive (dopaminergic) neurons in subregions of the substantia nigra pars compacta at 3-4 days, 5 weeks, and 18 months of age. Both genotypes lost dopaminergic neurons with age and remaining neurons at 11 months were resilient to paraquat/maneb. In 5-week-old mice, the toxins decreased substantia nigra pars compacta dopaminergic neurons in both genotypes but less in DAT-KD. Regional analysis revealed striking differences in the subsets of neurons affected by low DAT, paraquat/maneb, and aging. In particular, we show that a potentially protective effect of low DAT against toxin exposure is not sufficient to reduce death of all nigrostriatal dopaminergic neurons. Thus, different regional vulnerability of nigrostriatal dopaminergic neurons may contribute to an increased risk of developing Parkinson's disease when multiple factors are combined.

The neural chaperone proSAAS blocks α-synuclein fibrillation and neurotoxicity.

Emerging evidence strongly suggests that chaperone proteins are cytoprotective in neurodegenerative proteinopathies involving protein aggregation; for example, in the accumulation of aggregated α-synuclein into the Lewy bodies present in Parkinson's disease. Of the various chaperones known to be associated with neurodegenerative disease, the small secretory chaperone known as proSAAS (named after four residues in the amino terminal region) has many attractive properties. We show here that proSAAS, widely expressed in neurons throughout the brain, is associated with aggregated synuclein deposits in the substantia nigra of patients with Parkinson's disease. Recombinant proSAAS potently inhibits the fibrillation of α-synuclein in an in vitro assay; residues 158-180, containing a largely conserved element, are critical to this bioactivity. ProSAAS also exhibits a neuroprotective function; proSAAS-encoding lentivirus blocks α-synuclein-induced cytotoxicity in primary cultures of nigral dopaminergic neurons, and recombinant proSAAS blocks α-synuclein-induced cytotoxicity in SH-SY5Y cells. Four independent proteomics studies have previously identified proSAAS as a potential cerebrospinal fluid biomarker in various neurodegenerative diseases. Coupled with prior work showing that proSAAS blocks ß-amyloid aggregation into fibrils, this study supports the idea that neuronal proSAAS plays an important role in proteostatic processes. ProSAAS thus represents a possible therapeutic target in neurodegenerative disease.

Parsing the hedonic and motivational influences of nociceptin on feeding using licking microstructure analysis in mice.

Opioid peptides are implicated in processes related to reward and aversion; however, how specific opioid peptides are involved remains unclear. We investigated the role of nociceptin (NOC) in voluntary licking for palatable and aversive tastants by studying the effect of intracerebroventricularly administered NOC on licking microstructure in wild-type and NOC receptor knockout (NOP KO) mice. Compared with the wild-type mice, NOP KO mice emitted fewer bouts of licking when training to lick for a 20% sucrose solution. Correspondingly, intracerebroventricular administration of NOC increased the number of licking bouts for sucrose and sucralose in wild-type, but not in NOP KO mice. The ability of NOC to initiate new bouts of licking for sweet solutions suggests that NOC may drive motivational aspects of feeding behavior. Conversely, adulterating a sucrose solution with the aversive tastant quinine reduced licking bout lengths in wild-type and NOP KOs, suggesting that NOC signaling is not involved in driving voluntary consumption of semiaversive tastants. Interestingly, when consuming sucrose following 20 h of food deprivation, NOP KO mice emitted longer bouts of licking than wild types, suggesting that under hungry conditions, NOC may also contribute toward hedonic aspects of feeding. Together, these results suggest differential roles for NOC in the motivational and hedonic aspects of feeding.

Inflated reward value in early opiate withdrawal.

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.

Involvement of Endogenous Enkephalins and ß-Endorphin in Feeding and Diet-Induced Obesity.

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 ß-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 ß-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.

Exogenous and evoked oxytocin restores social behavior in the Cntnap2 mouse model of autism.

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.

Phasic mesolimbic dopamine signaling encodes the facilitation of incentive motivation produced by repeated cocaine exposure.

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.

The redistribution of Drosophila vesicular monoamine transporter mutants from synaptic vesicles to large dense-core vesicles impairs amine-dependent behaviors.

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.

Targeted expression of µ-opioid receptors in a subset of striatal direct-pathway neurons restores opiate reward.

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.

Differential effects of systemic cholinergic receptor blockade on Pavlovian incentive motivation and goal-directed action selection.

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.