The fragile X mental retardation protein promotes adjustments in cocaine self-administration that preserve reinforcement level.

The fragile X mental retardation protein (FMRP), an RNA-binding protein, regulates cocaine-induced neuronal plasticity and is critical for the normal development of drug-induced locomotor sensitization, as well as reward-related learning in the conditioned place preference assay. However, it is unknown whether FMRP impacts behaviors that are used to more closely model substance use disorders. Utilizing a cocaine intravenous self-administration (IVSA) assay in Fmr1 knockout (KO) and wild-type (WT) littermate mice, we find that, despite normal acquisition and extinction learning, Fmr1 KO mice fail to make a normal upward shift in responding during dose-response testing. Later, when given access to the original acquisition dose under increasing fixed ratio (FR) schedules of reinforcement (FR1, FR3, and FR5), Fmr1 KO mice earn significantly fewer cocaine infusions than WT mice. Importantly, similar deficits are not present in operant conditioning using a palatable food reinforcer, indicating that our results do not represent broad learning or reward-related deficits in Fmr1 KO mice. Additionally, we find an FMRP target, the activity-regulated cytoskeleton-associated protein (Arc), to be significantly reduced in synaptic cellular fractions prepared from the nucleus accumbens of Fmr1 KO, compared with WT, mice following operant tasks reinforced with cocaine but not food. Overall, our findings suggest that FMRP facilitates adjustments in drug self-administration behavior that generally serve to preserve reinforcement level, and combined with our similar IVSA findings in Arc KO mice may implicate Arc, along with FMRP, in behavioral shifts that occur in drug taking when drug availability is altered.

The activity-regulated cytoskeleton-associated protein (Arc) functions in a cell type- and sex-specific manner in the adult nucleus accumbens to regulate non-contingent cocaine behaviors.

Repeated cocaine use produces adaptations in brain function that contribute to long-lasting behaviors associated with cocaine use disorder (CUD). In rodents, the activity-regulated cytoskeleton-associated protein (Arc) can regulate glutamatergic synaptic transmission, and cocaine regulates Arc expression and subcellular localization in multiple brain regions, including the nucleus accumbens (NAc)-a brain region linked to CUD-related behavior. We show here that repeated, non-contingent cocaine administration in global Arc KO male mice produced a dramatic hypersensitization of cocaine locomotor responses and drug experience-dependent sensitization of conditioned place preference (CPP). In contrast to the global Arc KO mice, viral-mediated reduction of Arc in the adult male, but not female, NAc (shArcNAc) reduced both CPP and cocaine-induced locomotor activity, but without altering basal miniature or evoked glutamatergic synaptic transmission. Interestingly, cell type-specific knockdown of Arc in D1 dopamine receptor-expressing NAc neurons reduced cocaine-induced locomotor sensitization, but not cocaine CPP; whereas, Arc knockdown in D2 dopamine receptor-expressing NAc neurons reduced cocaine CPP, but not cocaine-induced locomotion. Taken together, our findings reveal that global, developmental loss of Arc produces hypersensitized cocaine responses; however, these effects cannot be explained by Arc's function in the adult mouse NAc since Arc is required in a cell type- and sex-specific manner to support cocaine-context associations and locomotor responses.

Drug reinforcement impairs cognitive flexibility by inhibiting striatal cholinergic neurons.

Addictive substance use impairs cognitive flexibility, with unclear underlying mechanisms. The reinforcement of substance use is mediated by the striatal direct-pathway medium spiny neurons (dMSNs) that project to the substantia nigra pars reticulata (SNr). Cognitive flexibility is mediated by striatal cholinergic interneurons (CINs), which receive extensive striatal inhibition. Here, we hypothesized that increased dMSN activity induced by substance use inhibits CINs, reducing cognitive flexibility. We found that cocaine administration in rodents caused long-lasting potentiation of local inhibitory dMSN-to-CIN transmission and decreased CIN firing in the dorsomedial striatum (DMS), a brain region critical for cognitive flexibility. Moreover, chemogenetic and time-locked optogenetic inhibition of DMS CINs suppressed flexibility of goal-directed behavior in instrumental reversal learning tasks. Notably, rabies-mediated tracing and physiological studies showed that SNr-projecting dMSNs, which mediate reinforcement, sent axonal collaterals to inhibit DMS CINs, which mediate flexibility. Our findings demonstrate that the local inhibitory dMSN-to-CIN circuit mediates the reinforcement-induced deficits in cognitive flexibility.

What's Right and Wrong in Preclinical Science: A Matter of Principled Investigation.

The discovery of efficacious treatment options for neuropsychiatric conditions is a process that remains in jeopardy. Contributing to the failure of clinical trials, a strong positive bias exists in the reported results of preclinical studies, including in the field of neuroscience. However, despite clear recognition of major factors that lead to bias, efforts to address them have not made much meaningful change, receiving inadequate attention from the scientific community. In truth, little real-world value is currently attached to efforts made to oppose positive bias, and instead-partially driven by competitive conditions-the opposite has become true. Since pressures throughout our system of scientific discovery, particularly those tied to definitions of individual success, hold these damaging practices firmly in place, we urgently need to make changes to the system itself. Such a transformation should include a pivot away from explicit or tacit requirements for statistical significance and clean narratives, particularly in publishing, and should promote a priori power calculations as the determinant of final sample size. These systemic changes must be reinforced and upheld in responsible decisions made by individual scientists concerning the planning, analysis, and presentation of their own research.

Jugular Vein Catheter Design and Cocaine Self-Administration Using Mice: A Comprehensive Method.

Intravenous self-administration (IVSA) is a behavioral method of voluntary drug intake in animal models which is used to study the reinforcing effects of drugs of abuse. It is considered to have greater face validity in the study of substance use and abuse than other assays, and thus, allows for valuable insight into the neurobiological basis of addiction, and the development of substance abuse disorders. The technique typically involves surgically inserting a catheter into the jugular vein, which enables the infusion of drug solution after the performance of a desired operant behavior. Two nose- poke ports or levers are offered as manipulanda and are randomly assigned as active (reinforced) or inactive (non-reinforced) to allow for the examination of discrimination in the assessment of learning. Here, we describe our methodological approach to this assay in a mouse model, including construction and surgical implantation of a jugular vein catheter, set up of operant chambers, and considerations during each phase of the operant task.

The role of the dorsal striatum in a mouse model for fragile X syndrome: Behavioral and dendritic spine assessment.

Fragile X syndrome (FXS), a leading monogenic cause of autism spectrum disorders (ASDs), typically occurs as the result of a mutation silencing the Fmr1 gene, preventing production of the fragile X messenger ribonucleoprotein (FMRP). FXS is characterized, in part, by hyperactivity, impaired behavioral flexibility, and the development of repetitive, or stereotyped, behaviors. While these phenotypes are influenced by striatal activity, few studies have examined FXS or FMRP in the context of striatal function. Here, we report enhanced repetitive behaviors in Fmr1 knockout (KO) compared to wild type (WT) mice according to multiple measures, including quantity and intensity of stereotypic behaviors in an open field and nose poking activity in an unbaited hole board test. However, using a baited version of the hole board assay, we see that KO mice do show some behavioral flexibility in that they make changes in their nose poking behavior following familiarization with an appetitive bait. By contrast, repeated exposure to cocaine (15 mg/kg) promotes repetitive behavior in both WT and KO mice, in a manner mostly independent of genotype. Branch length alterations in medium spiny neurons (MSNs) of the dorsolateral striatum (DLS) are similar between WT cocaine-treated and KO saline-treated mice, possibly suggesting shared synaptic mechanisms. Overall, we suggest that scoring open field behavior is a sensitive measure for repetitive sensory-motor behaviors in Fmr1 KO mice. In addition, our findings show that synaptic contacts onto MSNs in the DLS should be examined in conjunction with measures of stereotypical behavior.

Chronic alcohol drinking persistently suppresses thalamostriatal excitation of cholinergic neurons to impair cognitive flexibility.

Exposure to addictive substances impairs flexible decision making. Cognitive flexibility is mediated by striatal cholinergic interneurons (CINs). However, how chronic alcohol drinking alters cognitive flexibility through CINs remains unclear. Here, we report that chronic alcohol consumption and withdrawal impaired reversal of instrumental learning. Chronic alcohol consumption and withdrawal also caused a long-lasting (21 days) reduction of excitatory thalamic inputs onto CINs and reduced pause responses of CINs in the dorsomedial striatum (DMS). CINs are known to inhibit glutamatergic transmission in dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) but facilitate this transmission in D2-MSNs, which may contribute to flexible behavior. We discovered that chronic alcohol drinking impaired CIN-mediated inhibition in D1-MSNs and facilitation in D2-MSNs. Importantly, in vivo optogenetic induction of long-term potentiation of thalamostriatal transmission in DMS CINs rescued alcohol-induced reversal learning deficits. These results demonstrate that chronic alcohol drinking reduces thalamic excitation of DMS CINs, compromising their regulation of glutamatergic transmission in MSNs, which may contribute to alcohol-induced impairment of cognitive flexibility. These findings provide a neural mechanism underlying inflexible drinking in alcohol use disorder.

The Fragile X Mental Retardation Protein Regulates Striatal Medium Spiny Neuron Synapse Density and Dendritic Spine Morphology.

The fragile X mental retardation protein (FMRP), an RNA-binding protein that mediates the transport, stability, and translation of hundreds of brain RNAs, is critically involved in regulating synaptic function. Loss of FMRP, as in fragile X syndrome (FXS), is a leading monogenic cause of autism and results in altered structural and functional synaptic plasticity, widely described in the hippocampus and cortex. Though FXS is associated with hyperactivity, impaired social interaction, and the development of repetitive or stereotyped behaviors, all of which are influenced by striatal activity, few studies have investigated the function of FMRP here. Utilizing a cortical-striatal co-culture model, we find that striatal medium spiny neurons (MSNs) lacking FMRP fail to make normal increases in PSD95 expression over a short time period and have significant deficits in dendritic spine density and colocalized synaptic puncta at the later measured time point compared to wildtype (WT) MSNs. Acute expression of wtFMRP plasmid in Fmr1 KO co-cultures results in contrasting outcomes for these measures on MSNs at the more mature time point, reducing spine density across multiple spine types but making no significant changes in colocalized puncta. FMRP's KH2 and RGG RNA-binding domains are required for normal elimination of PSD95, and interruption of these domains slightly favors elimination of immature spine types. Further, KH2 is required for normal levels of colocalized puncta. Our data are largely consistent with a basal role for FMRP and its RNA-binding domains in striatal synapse stabilization on developing MSNs, and in light of previous findings, suggest distinct regional and/or cell type-specific roles for FMRP in regulating synapse structure.

The activity-regulated cytoskeleton-associated protein, Arc/Arg3.1, influences mouse cocaine self-administration.

The activity-regulated cytoskeleton-associated protein (Arc, also known as Arg3.1), an immediate early gene and synaptic regulator, is upregulated following a single cocaine exposure. However, there is not much known regarding Arc/Arg3.1's potential contribution to addiction-relevant behaviors. Despite known learning and memory deficits in contextual fear and water-maze reversal learning tasks, we find that mice lacking Arc/Arg3.1 perform conditioned place preference and operant conditioning involving positive reinforcers (food and cocaine) with little-to-no impairment. However, following normal saline-extinction, wild type (WT) mice show a classic inverted-U dose-response function, while Arc/Arg3.1 knockout (KO) mice fail to adjust their intake across multiple doses. Importantly, Arc/Arg3.1 KO and WT mice behave comparably on an increasing cost task (FR1-FR3; acquisition dose), providing evidence that both groups find cocaine reinforcing. Differences in individuals that drive variations in use patterns and particularly, drug intake levels, are critical as they influence the likelihood of developing dependence. Our data suggest that Arc/Arg3.1 may contribute to addiction as a regulator of drug-taking vulnerability under different drug availability conditions.

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) regulates anxiety- and novelty-related behaviors.

The activity-regulated cytoskeleton-associated protein (Arc, also known as Arg3.1) regulates glutamatergic synapse plasticity and has been linked to neuropsychiatric illness; however, its role in behaviors associated with mood and anxiety disorders remains unclear. We find that stress upregulates Arc expression in the adult mouse nucleus accumbens (NAc)-a brain region implicated in mood and anxiety behaviors. Global Arc knockout mice have altered AMPAR-subunit surface levels in the adult NAc, and the Arc-deficient mice show reductions in anxiety-like behavior, deficits in social novelty preference, and antidepressive-like behavior. Viral-mediated expression of Arc in the adult NAc of male, global Arc KO mice restores normal levels of anxiety-like behavior in the elevated plus maze (EPM). Consistent with this finding, viral-mediated reduction of Arc in the adult NAc reduces anxiety-like behavior in male, but not female, mice in the EPM. NAc-specific reduction of Arc also produced significant deficits in both object and social novelty preference tasks. Together our findings indicate that Arc is essential for regulating normal mood- and anxiety-related behaviors and novelty discrimination, and that Arc's function within the adult NAc contributes to these behavioral effects.

A novel method for oral stimulant administration in the neonate rat and similar species.

Sixty male and female Long-Evans hooded rats were administered 1, 2, or 5mg/kg methylphenidate (MPH) suspended in apple juice on postnatal day (P)15 or P40 using a novel, non-invasive oral administration technique. Plasma was collected 15 min after ingestion and analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS) to confirm appropriate concentrations. HPLC-MS plasma analysis showed levels comparable to previous gavage studies using MPH. We have used this method successfully in subsequent behavioral studies as well. Since therapeutic MPH in humans is typically administered orally, oral dosing methods that have been verified in the rodent model are of value. We recommend employment of this alternative oral dosing technique as it is minimally invasive, can be used anytime during postnatal development, and does not depend upon voluntary consumption.

Nicotine causes age-dependent changes in gene expression in the adolescent female rat brain.

Humans often start smoking during adolescence. Recent results suggest that rodents may also be particularly vulnerable to nicotine dependence during adolescence. We examined the effect of chronic nicotine exposure on gene expression profiles during adolescence in female rats, who were dosed with nicotine (and control animals were dosed with saline) via subcutaneously implanted osmotic minipumps. Brain samples were collected at four ages: before puberty (postnatal day 25), at about the time of puberty in females (postnatal day 35), and after puberty (postnatal days 45 and 55). The expression of 7931 genes in three brain areas was measured using DNA microarrays. Quantitative RT-PCR was also employed to confirm the expression patterns of selected genes. We used a novel clustering technique (principal cluster analysis) to classify 162 nicotine-regulated genes into five clusters, of which only one (cluster A) showed similar patterns of gene expression across all three brain areas (ventral striatum, prefrontal cortex, and hippocampus). Three clusters of genes (A, B, and C) showed dramatic peaks in their nicotine responses at the same age (p35). The other two clusters (D1 and D2) showed smaller peaks and/or valleys in their nicotine responses at p35 and p45. Thus, the age of maximal gene expression response to nicotine in female rats corresponds approximately to the age of maximal behavioral response and the age of puberty.

HDAC5 and Its Target Gene, Npas4, Function in the Nucleus Accumbens to Regulate Cocaine-Conditioned Behaviors.

Individuals suffering from substance-use disorders develop strong associations between the drug's rewarding effects and environmental cues, creating powerful, enduring triggers for relapse. We found that dephosphorylated, nuclear histone deacetylase 5 (HDAC5) in the nucleus accumbens (NAc) reduced cocaine reward-context associations and relapse-like behaviors in a cocaine self-administration model. We also discovered that HDAC5 associates with an activity-sensitive enhancer of the Npas4 gene and negatively regulates NPAS4 expression. Exposure to cocaine and the test chamber induced rapid and transient NPAS4 expression in a small subpopulation of FOS-positive neurons in the NAc. Conditional deletion of Npas4 in the NAc significantly reduced cocaine conditioned place preference and delayed learning of the drug-reinforced action during cocaine self-administration, without affecting cue-induced reinstatement of drug seeking. These data suggest that HDAC5 and NPAS4 in the NAc are critically involved in reward-relevant learning and memory processes and that nuclear HDAC5 limits reinstatement of drug seeking independent of NPAS4.

Long-term changes in fear conditioning and anxiety-like behavior following nicotine exposure in adult versus adolescent rats.

Adolescent nicotine exposure is associated with long-term use, and it has been suggested that this vulnerability to addiction may relate to lasting anxiogenic effects of the drug. However, few studies have addressed long-term effects of adolescent nicotine, and fewer yet have compared adolescent to adult exposure. Male and female Long-Evans rats continuously received nicotine bitartrate or sodium tartrate via osmotic mini-pumps over 15 days either during adolescence (p28-42) or adulthood (p85-99). Initial nicotine dose (free base) was either low (1 mg/kg/day) or high (2 mg/kg/day). Open field behavior and fear conditioning were assessed in adulthood, 1 month post-dosing. Animals pretreated with nicotine during adolescence showed less center time in a novel open field than sham controls. Conversely, the two nicotine doses differentially affected fear conditioning. Animals pretreated with low nicotine during adolescence demonstrated superior acquisition of the task compared to sham control animals; however, unlike either high nicotine-pretreated or sham control animals, they failed to extinguish the learned behavior. In contrast, animals pretreated during adulthood did not behave significantly different from sham controls on either task. Overall, nicotine-pretreatment during adolescence induced effects on behaviors related to fear and anxiety in adulthood, while comparable pretreatment during adulthood failed to produce significant residual effects.

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice.

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence(2). In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

Periadolescent nicotine administration produces enduring changes in dendritic morphology of medium spiny neurons from nucleus accumbens.

The objective of the current study was to examine how periadolescent nicotine exposure affects dendritic morphology of medium spiny neurons from the nucleus accumbens shell. Male Long-Evans hooded rats were chronically administered nicotine or saline for a period extending from postnatal day 22 (p22) to p69. Nicotine and saline administration was via subcutaneously implanted osmotic pumps. At p144, 75 days after conclusion of nicotine administration, brains were processed for Golgi-Cox staining. Medium spiny neurons from the nucleus accumbens shell were digitally reconstructed. It was found that neurons from nicotine-treated animals possessed significantly longer dendrites and a greater number of dendritic segments than control animals. A branch order analysis indicated that differences in dendritic length and segment number were most pronounced in third and fourth order segments. A subsequent behavioral experiment suggests that the observed anatomical changes are associated with enduring psychomotor differences. These findings indicate that periadolescent exposure to nicotine can result in long-lasting structural changes in the nucleus accumbens shell and are consistent with behavioral data suggesting that adolescent nicotine exposure may result in vulnerability to nicotine addiction in adulthood.

Role of the D3 dopamine receptor in nicotine sensitization.

Adolescent cigarette use is associated with reduced quitting success and continued smoking in adulthood. Interestingly, polymorphisms of the dopamine D3 receptor (DRD3) gene have been associated with smoking behavior, and the receptor is expressed in an age- and brain region-dependent manner that suggests relevance to addiction. Here, we investigate the possible role of dopamine-related receptors, including DRD3 and an intriguing splice variant known as D3nf, in nicotine-induced sensitization. In adolescent and adult male rats, we examined (1) alterations occurring in dopamine receptor-related mRNAs (DRD1, DRD2, DRD3 and D3nf) at two time points during a sensitizing regimen of nicotine and (2) whether DRD3 antagonism either during the initial treatment (induction) or at a later challenge exposure (expression) is able to block nicotine sensitization. Nicotine-induced changes were seen for DRD3 and D3nf mRNAs in the nucleus accumbens shell early in repeated exposure in both age groups. DRD3 antagonism only blocked the induction of sensitization in adolescents and did not block the expression of sensitization in either age group. Adolescents and adults showed opposite DRD1 mRNA responses to nicotine treatment, while no age- and nicotine-related changes in DRD2 mRNA were observed. These data reveal important age-dependent regulation of DRD1- and DRD3-related mRNAs during the course of nicotine exposure. Furthermore, they highlight a requirement for DRD3 signaling in the development of adolescent nicotine sensitization, suggesting it may represent an appropriate target in the prevention of nicotine dependence initiated at this age.

Folic acid use by women receiving routine gynecologic care.

OBJECTIVE: Many health professional groups recommend folic acid supplementation for all women able to become pregnant. In this study, we document folic acid supplement use among a sample of women receiving routine gynecologic care. METHODS: A short questionnaire was administered to 322 women aged 18-45 years who were seeking routine gynecologic care at participating clinics in Little Rock, Arkansas. Questions covered knowledge and use of folic acid supplements, pregnancy intention, and demographic and socioeconomic characteristics. Primary study outcomes were self-reported folic acid awareness, daily or weekly use of folic acid supplements, and intention to begin taking folic acid. Factors affecting study outcomes were examined individually by computing crude odd ratios and adjusted for other covariates using unconditional logistic regression. RESULTS: Although 61.8% of women reported awareness of the association between folic acid and birth defects prevention, only 27.1% of these women, and 22.7% of all study participants, reported daily use of a folic acid supplement. Substantially more women (39.8%) were taking a folic acid supplement at least once per week. Age, race, educational level, folic acid awareness, marital status, pregnancy intent, and other preventive health behaviors were the most important predictors of compliance. CONCLUSION: The results indicate a need for targeted interventions directed toward minority women, young women, and those of lower socioeconomic and educational status. The routine gynecologic visit is an ideal opportunity to counsel women of reproductive age to take folic acid daily. LEVEL OF EVIDENCE: III

Fragile X mental retardation protein regulates synaptic and behavioral plasticity to repeated cocaine administration.

Repeated cocaine exposure causes persistent, maladaptive alterations in brain and behavior, and hope for effective therapeutics lies in understanding these processes. We describe here an essential role for fragile X mental retardation protein (FMRP), an RNA-binding protein and regulator of dendritic protein synthesis, in cocaine conditioned place preference, behavioral sensitization, and motor stereotypy. Cocaine reward deficits in FMRP-deficient mice stem from elevated mGluR5 (or GRM5) function, similar to a subset of fragile X symptoms, and do not extend to natural reward. We find that FMRP functions in the adult nucleus accumbens (NAc), a critical addiction-related brain region, to mediate behavioral sensitization but not cocaine reward. FMRP-deficient mice also exhibit several abnormalities in NAc medium spiny neurons, including reduced presynaptic function and premature changes in dendritic morphology and glutamatergic neurotransmission following repeated cocaine treatment. Together, our findings reveal FMRP as a critical mediator of cocaine-induced behavioral and synaptic plasticity.