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Current antipsychotics provide symptomatic relief for patients suffering from schizophrenia and related psychoses; however, their effectiveness is variable and many patients discontinue treatment due to side effects. Although the etiology of schizophrenia is still unclear, a leading hypothesis implicates an imbalanced dopaminergic system. Muscarinic acetylcholine (ACh) receptors regulate dopamine levels in key areas of the brain involved in psychosis, with the M4 subtype emerging as a key regulator of dopaminergic hyperactivity. Unfortunately, no selective small molecule tools exist to provide pharmacological validation of this hypothesis. Here, we describe the discovery of a small molecule modulator, LY2033298, that is highly selective for human M4 receptors by virtue of targeting an allosteric site on this receptor. Pharmacological assays confirmed the selectivity of LY2033298 for the M4 receptor and revealed the highest degree of positive allosteric enhancement of ACh potency thus far identified. Radioligand binding assays also show this compound to directly potentiate agonist binding while having minimal effects on antagonist binding. Mutational analysis identified a key amino acid (D432) in the third extracellular loop of the human M4 receptor to be critical for selectivity and agonist potentiation by LY2033298. Importantly, LY2033298 was active in animal models predictive of clinical antipsychotic drug efficacy indicating its potential use as a first-in-class, selective, allosteric muscarinic antipsychotic agent. [ABSTRACT FROM AUTHOR]

Background: Previous studies indicated that, under continuous access conditions, the 5-HT3 antagonist MDL 72222 (MDL) effectively reduced ethanol drinking of alcohol-preferring P rats. However, MDL was without effect when similar doses were tested under scheduled access conditions, unless the ethanol access period was randomly presented. This study examined the effects of MDL on operant responding for ethanol and water by adult male alcohol-preferring P rats. Methods: During the dark cycle, subjects in the first experiment were trained to respond concurrently for 15% ethanol and water on a fixed-ratio 5 (FR-5) and FR-1 schedule of reinforcement, respectively. Approximately 30 min before the 4-hr operant session, rats were injected subcutaneously (sc) with saline or MDL (1,3, or 5 mg/kg). A second experiment tested the effects of 1 mg/kg MDL on operant responding for 15% ethanol in 1-hr sessions when operant access was given at a fixed time each day (fixed scheduled access, FSA group) or at variable time periods throughout the dark cycle (variable scheduled access, VSA group). Results: In the first experiment, only the 5 mg/kg dose of MDL decreased responding for ethanol (approximately 20%) during the first 30 min of the 4-hr session. This dose also reduced total 4-hr responding for ethanol and water. In the second experiment, the 1 mg/kg dose of MDL had no effect on operant responding by the FSA group, but significantly reduced ethanol responding by the VSA group. Conclusions: These results suggest that 5-HT3 receptors may be involved in mediating the reinforcing effects of ethanol, and that temporal-environmental cues associated with the presentation of ethanol may be one factor involved in reducing the effectiveness of 5-HT3 antagonists to attenuate ethanol intake. [ABSTRACT FROM AUTHOR]

The novel mGlu2/3 receptor antagonist, LY3020371, has been shown to produce antidepressant-like effects comparable to that of the clinically-effective antidepressant ketamine. In the present study, we investigated whether LY3020371 would be predicted to be free of the side-effects and safety pharmacology issues associated with ketamine. In contrast to ketamine, LY3020371 produced small increases in locomotion and did not impair motor performance on an inverted screen. Ketamine, but not LY3020371, increased dopamine efflux in the nucleus accumbens of rats. Ketamine also produced cognitively-impairing effects in rats in a T-maze and in a psychomotor vigilance task and altered theta synchrony between the hippocampus and mPFC, whereas LY3020371 had either no significant impact or lesser effects in these assays. In mice, ketamine, but not LY3020371, negatively affected spontaneous alternation in a Y-maze. Rats were trained to discriminate LY3020371 from vehicle where 30mg/kg produced 100% drug-appropriate responding and the ED 50 for LY3020371 was 9.4mg/kg, i.p. In rats discriminating LY3020371, neither d-amphetamine nor phencyclidine fully substituted for LY3020371 (35-45%) and the mGlu2/3 receptor agonist LY354740 partially attenuated the discriminative stimulus effects of LY3020371. These are the first data to demonstrate the discriminative stimulus effects of an mGlu2/3 receptor antagonist. Some alterations were suggested to occur in the density of mGlu2/3 receptor binding sites in the drug discrimination rats relative to their age-matched non-drug-exposed controls. In preclinical toxicology studies of 14day dosing of doses up to 1000mg/kg, i.v. in rats and up to 500m/kg, i.v. in Cynomologous monkeys, LY3020371 produced uM plasma exposures without producing critical toxicological findings. It is concluded that LY3020371 does not recapitulate the motor, cognitive, subjective, neurochemical, electrophysiological, or toxicological findings reported with ketamine. Thus, LY3020371 possesses both the efficacy signatures of a rapidly-acting antidepressant and a safety profile enabling proof of concept studies in patients., (Copyright © 2017. Published by Elsevier Inc.)

The objective of this study was to determine whether functional differences exist in amphetamine-induced locomotor activity between alcohol-naive alcohol-preferring (P) and -nonpreferring (NP) rats during postnatal development and during adulthood. Using a between-subjects design, 20- and 28-day-old P and NP rats (male and female counterbalanced, n=11-16/line) were habituated for 30 min in a photocell activity field. Each rat received subcutaneous injections of saline or 0.3, 0.6 or 1.2 mg/kg d-amphetamine (AMPH) and were then tested for an additional 30 min. Because of age and line differences in basal locomotor activity, total activity counts during the 30-min postdrug period were standardized using Z-score transformations. In the 20- and 28-day-old rats, dose-dependent locomotor activity increases after AMPH injections were obtained at both ages, although activity levels were greater in the 20-day-old pups. The 20-day-old female NP rats showed greater AMPH-induced increases in locomotor activity than P rats, whereas at 28 days of age, male NP rats showed greater activity levels than P rats to AMPH. For the adult P and NP rats (n=8/line/gender), a within-subject design was used. In the adults, the NP line had higher locomotor activity than the P line following AMPH injection, and male rats were activated more by AMPH than female rats. The results suggest that functioning of the DA system in the adult P line is reduced compared to the adult NP line, and this line difference is also observed to some degree at an early postweaning developmental period.

The M(5) muscarinic receptor is the most recent member of the muscarinic acetylcholine receptor family (M(1)-M(5)) to be cloned. At present, the physiological relevance of this receptor subtype remains unknown, primarily because of its low expression levels and the lack of M(5) receptor-selective ligands. To circumvent these difficulties, we used gene targeting technology to generate M(5) receptor-deficient mice (M5R(-/-) mice). M5R(-/-) mice did not differ from their wild-type littermates in various behavioral and pharmacologic tests. However, in vitro neurotransmitter release experiments showed that M(5) receptors play a role in facilitating muscarinic agonist-induced dopamine release in the striatum. Because M(5) receptor mRNA has been detected in several blood vessels, we also investigated whether the lack of M(5) receptors led to changes in vascular tone by using several in vivo and in vitro vascular preparations. Strikingly, acetylcholine, a powerful dilator of most vascular beds, virtually lost the ability to dilate cerebral arteries and arterioles in M5R(-/-) mice. This effect was specific for cerebral blood vessels, because acetylcholine-mediated dilation of extra-cerebral arteries remained fully intact in M5R(-/-) mice. Our findings provide direct evidence that M(5) muscarinic receptors are physiologically relevant. Because it has been suggested that impaired cholinergic dilation of cerebral blood vessels may play a role in the pathophysiology of Alzheimer's disease and focal cerebral ischemia, cerebrovascular M(5) receptors may represent an attractive therapeutic target.

Background: The alcohol deprivation effect (ADE) is a temporary increase in the ratio of alcohol/total fluid intake and voluntary intake of ethanol (EtOH) solutions over baseline drinking conditions when EtOH access is reinstated after a period of alcohol deprivation. The ADE has been posited to be an animal model for alcohol craving. In the current study, we examined the effects of initial deprivation length and number of deprivation exposures on the ADE in the replicate lines of the high-alcohol-drinking (HAD) rats., Methods: Adult male HAD-1 and HAD-2 rats received 24 hr free-choice access to 10% (v/v) EtOH and water for 6 weeks. Rats were then assigned to groups deprived of EtOH for 0 (control), or 2 to 8 weeks. All deprived groups were then given 24 hr access to EtOH for 2 weeks before being deprived of EtOH for another 2 weeks. This cycle of 2 weeks of access and 2 weeks of deprivation was carried out for a total of four deprivations., Results: After the initial EtOH deprivation period, EtOH consumption in HAD-1 and HAD-2 rats returned to baseline levels but failed to exhibit either an early onset ADE (initial 2 hr) or prolonged ADE (24 hr). An ADE was observed in two of the four deprived groups for the HAD-1 rats (2 week and 6 week groups) and in all deprived groups for the HAD-2 rats after a second deprivation, and in all deprived groups of both lines after a third deprivation. In the HAD-2 line, but not in the HAD-1 line, the duration of the ADE was prolonged into the second reinstatement day after the fourth deprivation., Conclusions: The expression of an ADE was observed only after repeated deprivation periods in the HAD lines. The duration of the ADE was prolonged in the HAD-2 line, but not in the HAD-1 line, with repeated deprivations, which suggests a dissociation between selection for alcohol preference and the effects of repeated deprivations on the duration of the ADE.

Rationale: Because current findings indicate that the selectively bred alcohol-preferring P line of rats self-administers 50-200 mg% ethanol (EtOH) directly into the ventral tegmental area (VTA), whereas the alcohol-nonpreferring NP line does not, it is important to determine whether unselected, common stock rats would self-administer EtOH directly into the VTA. In addition, because neuroanatomical and self-administration studies indicate that the VTA may be functionally heterogeneous, the present study was designed to determine whether there were subregional differences within the VTA for the intracranial self-administration (ICSA) of EtOH., Objectives: The objective of this study was to employ the ICSA technique to determine whether adult female Wistar rats would self-administer EtOH directly into the VTA, and whether regional heterogeneity existed for EtOH self-infusion within the VTA., Methods: Following surgery to implant guide cannulae aimed at either the posterior or anterior VTA, subjects were placed in standard experimental chambers equipped with an 'active lever' [fixed ratio (FR)1 schedule of reinforcement], which caused the delivery of the infusate, and an 'inactive lever', which had no programmed consequence. Subjects were assigned to groups that self-administered either artificial cerebrospinal fluid (aCSF) throughout, or 100-400 mg% EtOH for the first four sessions (acquisition), aCSF in sessions 5 and 6 (extinction), and EtOH again during session 7 (reinstatement)., Results: During the four acquisition sessions, rats with posterior VTA placements readily self-administered 200 mg% and 250 mg% EtOH and discriminated between the active and inactive levers. These subjects also demonstrated extinction, when aCSF was substituted for EtOH, and reinstatement when EtOH was reintroduced. Rats with posterior VTA placements self-infused 300 mg% and 400 mg% EtOH, and demonstrated lever discrimination only during the initial acquisition sessions. In contrast, rats with anterior VTA placements did not self-administer EtOH., Conclusions: The findings suggest that EtOH is reinforcing within the posterior VTA of Wistar rats, and the VTA is a functionally heterogeneous structure with regard to EtOH reinforcement.

Background: The alcohol deprivation effect (ADE) is a temporary increase in the ratio of ethanol/total fluid intake and the voluntary intake of ethanol solutions over baseline drinking conditions when ethanol access is reinstated after a period of alcohol deprivation. The ADE has been posited to be an animal model for alcohol craving. The current study examined the effects of initial deprivation length and number of deprivation exposures on the ADE in alcohol-preferring (P) rats., Methods: Adult female P rats received 24-hr free-choice access to 10% (v/v) ethanol and water for 6 weeks. Rats were then randomly assigned to five groups deprived of ethanol for 0 (control), 2, 4, 6, or 8 weeks (W). All deprived groups were then given 24-hr access to ethanol for 2 weeks before being deprived of ethanol for another 2 weeks., Results: After the initial ethanol deprivation period, the deprived groups displayed a similar 2-fold ADE (e.g., 4-W group; 4.6 +/- 0.5 for baseline vs. 10.5 +/- 0.3 g/kg/day for the 1st reinstatement day) during the initial 24-hr period. Ethanol consumption began to return to control levels 48 (7.1 +/- 0.4 g/kg/day) and 72 (6.4 +/- 0.4 g/kg/day) hrs later. In addition, each deprived group showed increases in the ratio of ethanol/total fluid intake upon reinstatement, and there was a tendency for sustained higher ethanol intake ratios during the first 3 postexposure days for the 4-, 6-, and 8-W groups, but only during the first 2 reinstatement days for the 2-W group. The second deprivation did not increase the magnitude of the ADE over that observed in the first deprivation during the initial 24-hr period of re-exposure, but it did prolong the duration of the ADE into the 2nd and 3rd reinstatement day for the 2-, 4-, and 6-W groups and into the 5th reinstatement day for the 8-W group., Conclusions: Equivalent robust ADEs can be seen in P rats with deprivation periods of 2-8 W, which suggests that the ADE has a rapid onset and is not affected by the durations of deprivation that were tested. The duration of the ADE was prolonged in P rats exposed to a second deprivation period, suggesting that factors associated with the ADE phenomenon could be strengthened by repeated deprivations.

Background: Using a procedure first developed by Hall (1979), we examined ethanol self-administration in preweanling pups from Wistar rats and in lines of rats selectively bred for divergent ethanol preference (alcohol-preferring P, alcohol-nonpreferring NP, high-alcohol-drinking HAD-1 and -2, and low-alcohol-drinking LAD-2) to determine if factors contributing to high and low alcohol intakes are present early in development., Methods: From postnatal days 5 to 20, nondeprived male and female rat pups received 30 min daily access to either water or a 15% (v/v) ethanol solution. In each daily session, pups were placed in a heated chamber containing Kimwipes soaked with a water or ethanol solution. Pups were weighed before and after each session, and intake levels were calculated as a percentage of body weight change., Results: Similar to previous reports, Wistar pups exhibited over a 2-fold higher level of ethanol ingestion than water on postnatal days 9 through 14, with ethanol intakes approaching 3 g/kg body weight. When the drinking patterns of the selected lines were examined, only the HAD replicate lines showed a comparable preference for ethanol versus water during the preweanling period. The ethanol consumption of P, NP, and LAD lines was not consistently distinguishable from water intake levels. To reveal whether early ethanol exposure would influence later ethanol drinking behavior, a subset of HAD and LAD rat pups received free-choice ethanol access upon weaning. Although the divergent ethanol preference between high- and low-alcohol-drinking lines was evident within the first 4 days of access (>4 g/kg/day for HAD; <2 g/kg/day for LAD), preweanling ethanol exposure did not alter the acquisition or maintenance of ethanol drinking in either line., Conclusions: Overall, these results suggest that (a) the enhanced ethanol ingestion observed during the middle portion of the preweanling period is a robust phenomenon and generalizes across nonselected strains of rats, (b) selective breeding for divergent alcohol preference has arrested this age-specific effect in all but the HAD lines of rats, and (c) early ethanol exposure does not alter genetic dispositions for later high or low alcohol preference.

Patterns of drinking and responding for ethanol (EtOH) and saccharin (SACC) were examined in the alcohol-preferring P rat using various limited-access paradigms. Adult female P rats (n = 10-20) were given 2-h access to EtOH (10-13% v/v) and SACC (0.0125% g/v) concurrently each day, or each solution individually on alternate days. Total 2-h SACC intake was significantly greater than EtOH under both concurrent (12+/-2 vs. 7+/-0.8 ml, p<0.05) and alternate-day access (18+/-1.6 vs. 10+/-0.5 ml) conditions. Under both conditions, however, EtOH intake (over 55% of the total) in the first 15 min was significantly greater than that of SACC (<25% of total). In an operant paradigm, total responding for EtOH (124+/-29) and SACC (114+/-7) under 2-h alternate-day conditions did not differ, but 65% of total EtOH responding occurred during the first 20 min versus less than 45% for SACC (p<0.05). Increasing response requirements (FR-1 to FR-5) did not significantly alter the total number of EtOH reinforcements, but decreased the total number of SACC reinforcements by approximately 50% (p<0.05). Increasing the EtOH concentration from 15% to 35% decreased the number of reinforcements approximately 50% but did not decrease the estimated g/kg EtOH intake. Increasing the SACC concentration from 0.0125% to 0.05%, however, nearly doubled the number of reinforcements. The greater preference for EtOH versus SACC during the initial part of the access period, together with the maintenance of EtOH intake in g/kg when the response requirements and the EtOH concentration were increased, suggests that EtOH intake is motivated by pharmacological consequences. Therefore, different motivational factors appear to underlie EtOH and SACC intake of the P rat. Furthermore, the pattern of EtOH intake and responding displayed by the P rat may be the result of a "bout-" or "binge-" like loss of control under restricted EtOH access conditions.

Preweanling 17-day-old rats were tested for retention of the conditioned emotional response after a 5-min or 24-hr retention interval. For a variety of conditioning parameters (i.e., variation in conditioned stimulus modality, unconditioned stimulus intensity, number of training trials), conditioned responding was consistently weaker after 5 min than after 24 hr. This apparent "incubation," or "hypermnesic," effect was not found in adult rats, even when comparable conditioning levels were indicated on the 24-hr test. The transient short-term retention deficit observed in 17-day-old preweanlings was alleviated by placing the pup in its home cage during the 5-min retention interval or by extending the conditioning session. Fifteen-day-old rat pups did not benefit from home cage exposure or extended training and displayed the transient short-term retention deficit regardless. The results are discussed in terms of age-related effects on time-dependent memory consolidation.

We found previously that alcohol-preferring (P) rats have fewer serotonin (5-HT) neurons and fibers in key brain regions than alcohol-nonpreferring (NP) rats. Because 5-HT uptake blockers increase synaptic 5-HT content and 5-HT1A receptor antagonists increase 5-HT release by disinhibiting 5-HT autoinnervation, in the present study, our intent was to determine whether increased synaptic 5-HT content and/or 5-HT release in P rats would effectively reduce alcohol consumption. In experiment 1, the 5-HT antagonist WAY 100635 (WAY) was tested on adult female P rats maintained on 24-hr free-choice access to ethanol (10% v/v) and water. Twice daily doses of WAY (0.05, 0.1, 0.5, and 1.0 mg/kg, subcutaneously) were administered to each rat in a counterbalanced order. Baseline ethanol intake, derived from the mean ethanol intakes of the three previous non-drug days, was approximately 8 g/kg/day. Results indicated that 0.05, 0.1, and 0.5 mg/kg doses of WAY reduced 24-hr ethanol drinking by 25-30% (p < 0.01) without affecting 24-hr water intake or body weight. In the second experiment, the effects of WAY (0.5 mg/kg), fluoxetine (1.0 mg/kg), or a combination of both were tested in another group of female P rats. WAY and fluoxetine, each alone, reduced ethanol drinking by around 20% and, when combined, decreased ethanol intake by 50%, whereas the body weight and the total fluid intake were not significantly affected. Taken together, these results indicate that both fluoxetine and WAY preferentially reduce ethanol drinking in the P line of rats and, when administered together, reduce ethanol intake in an additive manner. It is proposed that coadministration of these two compounds with distinct mechanisms of action may be a new strategy for reducing alcohol intake.

Three experiments were conducted to assess the plasticity of ethanol-mediated conditioned aversions to a tactile stimulus in infant rats. Ten- and 11-day-old rats first acquired an aversion to a texture, by virtue of its pairing with alcohol-induced intoxication. This first conditioning phase was followed by an associative devaluation procedure, a second phase in which sucrose was intraorally infused during alcohol-induced intoxication. Pups were then tested for their texture preference. Results indicated that infant rats readily express conditioned aversion to a tactile cue as a result of tactile-alcohol pairings and that this associative learning was not state dependent. When alcohol-texture conditioning was followed by sucrose-alcohol pairings, the magnitude of the texture aversion was dramatically reduced (Experiments 1 and 2). In Experiment 3 citric acid rather than sucrose was paired with alcohol intoxication following texture-alcohol pairings. The results indicated that this procedure strengthened texture conditioned aversions in terms of increased resistance to extinction. Taken as a whole these studies indicate that infants rapidly acquire alcohol-mediated texture aversions and that this memory is malleable and can be reduced or potentiated through manipulation of the representation of alcohol's unconditioned properties.

Data from a variety of sources, including a number of tasks and dependent measures, suggests that a systematic inverse relationship exists between age and the amount of variability observed on simple measures of learning. Implications for general theoretical perspectives concerning ontogenetic differences in stimulus and response selection, acquired knowledge or experience, and general speed of information processing are discussed. It is argued that such a regularity offers possibilities for both methodological and theoretical explorations.

Depending on dose and task requirements, ethanol can have either a facilitative or an impairing influence on learning. Some explanations for this dichotomy have considered ethanol's suppression of behavioral variability and processing of incidental stimuli (e.g., context). The present study examined the effect of ethanol on context and conditioned stimulus (CS) learning in the preweanling rat. To assess state-dependent effects, a drug dissociation design was used. Learning to both context and CS were analyzed within each dose of ethanol (0, 1.2, 1.6, or 2.0 g/kg) and a trend analysis was conducted to determine dose-response relationships as a function of train-test state. The 1.2 g/kg dose of ethanol did not affect conditioning to either the context or the CS. A 1.6 g/kg dose tended to disrupt context, but not CS, conditioning. The influence of 2.0 g/kg ethanol depended on train-test conditions. Ethanol administration prior to training resulted in the stronger impairment of CS learning while context conditioning was most disrupted if ethanol was given only prior to testing. The results suggest that ethanol selectively attenuates processing of stimuli, possibly dependent on relative saliency at the time of testing.

Nonassociative learning and retention were assessed in terms of habituation of a heart-rate (HR) orienting response (bradycardia) to a tone in 16-day-old male and female rats prenatally exposed to cocaine and in control offspring. Offspring were derived from Sprague-Dawley dams given daily subcutaneous injections of 40 mg/kg/3 cc cocaine HCI (C40) from gestational days 8-20, pair-fed control dams given saline injections (PF), and nontreated control dams (LC). Each pup was adapted to the test apparatus for 15 min prior to being given 10 presentations of a pulsing tone, each separated by a 65-sec intertrial interval, with HR measured during a 5-sec pretone period and throughout the 10-sec tone for each trial. To assess retention, subjects were given 10 additional tone trials either 1, 2, 4, or 6 hr later. C40 male offspring displayed significantly lower basal HR following the initial 15-min isolation period than either LC or PF offspring, whereas prenatal treatment had no effect on basal HR among females. Although no differences were seen in rate of habituation, prenatal cocaine exposure was observed to affect retention of the habituated orienting response. Whereas LC and PF offspring retained habituation of the orienting response for less than 4 hr, C40 offspring exhibited no forgetting after a 4-hr interval, and showed significant loss of the habituation response only after a 6-hr interval.

The impact of rearing condition was assessed in Sprague-Dawley dams given 40 mg/kg cocaine (C40) or saline (LC control) subcutaneously (SC) from gestational days 8-20 and their offspring. Treated pups reared by their biological dams (LC/LC; C40/C40), treated pups reared by surrogate dams (FOS/LC; FOS/C40), and foster pups raised by treated dams (LC/FOS; C40/FOS) were examined. On postnatal day 7 (P7), pups received either 0 (unpaired) 2, 3, or 4 pairings of an odor and footshock and were tested for their aversion to this odor. Foster and LC pups, regardless of rearing condition, exhibited significant odor aversions following either 2, 3, or 4 training trials. In contrast, C40 pups reared by surrogate dams required 4 trials to acquire the aversion, and C40 pups reared by their own dams did not exhibit conditioning even after 4 trials. At P17, no differences were seen among the groups in the aversion formed to an auditory or an olfactory stimulus that was paired with footshock. At P60, shock-elicited aggression among pairs of siblings was examined. Regardless of prenatal exposure condition, offspring reared by dams given cocaine showed a decreased latency to the first aggressive contact, an effect that was evident without any alteration in shock sensitivity. Together these data suggest that being reared by a dam previously exposed to cocaine has an impact on offspring behavioral function apart from the effects of prenatal cocaine exposure per se. The implications of the data regarding the cognitive performance of pups exposed prenatally to cocaine are also discussed.