Fatty acid binding proteins are novel modulators of synaptic epoxyeicosatrienoic acid signaling in the brain.

Fatty acid binding proteins (FABPs) govern intracellular lipid transport to cytosolic organelles and nuclear receptors. More recently, FABP5 has emerged as a key regulator of synaptic endocannabinoid signaling, suggesting that FABPs may broadly regulate the signaling of neuroactive lipids in the brain. Herein, we demonstrate that brain-expressed FABPs (FABP3, FABP5, and FABP7) interact with epoxyeicosatrienoic acids (EETs) and the peroxisome proliferator-activated receptor gamma agonist 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2). Among these lipids, EETs displayed highest affinities for FABP3 and FABP5, and 11,12-EET was identified as the preferred FABP ligand. Similarly, 15d-PGJ2 interacted with FABP3 and FABP5 while binding to FABP7 was markedly lower. Molecular modeling revealed unique binding interactions of the ligands within the FABP binding pockets and highlighted major contributions of van der Waals clashes and acyl chain solvent exposure in dictating FABP affinity and specificity. Functional studies demonstrated that endogenous EETs gate the strength of CA1 hippocampal glutamate synapses and that this function was impaired following FABP inhibition. As such, the present study reveals that FABPs control EET-mediated synaptic gating, thereby expanding the functional roles of this protein family in regulating neuronal lipid signaling.

Oxytocin excites dorsal raphe serotonin neurons and bidirectionally gates their glutamate synapses.

Oxytocin (OXT) modulates wide spectrum of social and emotional behaviors via modulation of numerous neurotransmitter systems, including serotonin (5-HT). However, how OXT controls the function of dorsal raphe nucleus (DRN) 5-HT neurons remains unknown. Here, we reveal that OXT excites and alters the firing pattern of 5-HT neurons via activation of postsynaptic OXT receptors (OXTRs). In addition, OXT induces cell-type-specific depression and potentiation of DRN glutamate synapses by two retrograde lipid messengers, 2-arachidonoylglycerol (2-AG) and arachidonic acid (AA), respectively. Neuronal mapping demonstrates that OXT preferentially potentiates glutamate synapses of 5-HT neurons projecting to medial prefrontal cortex (mPFC) and depresses glutamatergic inputs to 5-HT neurons projecting to lateral habenula (LHb) and central amygdala (CeA). Thus, by engaging distinct retrograde lipid messengers, OXT exerts a target-specific gating of glutamate synapses on the DRN. As such, our data uncovers the neuronal mechanisms by which OXT modulates the function of DRN 5-HT neurons.

Prenatal ethanol exposure leads to persistent anxiety-like behavior during adulthood indicated by reduced horizontal and vertical exploratory behaviors.

Background: Fetal alcohol spectrum disorders (FASD) caused by prenatal ethanol exposure (PE) consist of many cognitive/behavioral deficits. Studies have reported that PE leads to impairments of learning and memory, attention, executive function, and anxiety. Open field (OF) is a common behavioral model which offers comprehensive ethological information. Here, we analyzed multiple parameters of OF to examine anxiety behavior and habituation after PE. Material and Methods: Pregnant Sprague Dawley rats were gavaged twice/day with 0 or 3 g/kg/treatment ethanol (15% w/v) during gestational day (GD) 8-20, mimicking second-trimester heavy PE in humans. The control and PE adult offspring were subjected to OF task in different ambient light levels with or without acute stress. Results: Prenatal ethanol exposure did not influence the overall locomotor activities or habituation in the OF. In lower ambient light, no PE effects could be detected. In higher ambient light, female PE rats showed less activities in the center zone, indicative of increased anxiety. Males show lower activities in the center zone only after acute stress. Rats spent <2% of the time in the center zone compared to >75% of the time in the corner zone where they engaged in frequent rearing activities (vertical exploration; exploratory rearing). Prenatal ethanol exposure led to lower rearing activities in the corner in both males and females. Acute stress masks the PE effects in males but not in females. Discussion: The results support that heavy PE leads to persistent anxiety-like behavior during adulthood in both sexes. This conclusion is supported by using multiple parameters of exploratory behavior in the OF, including the rearing activities in the corner to reach reliable quantification of anxiety-like behavior.

Adolescent chronic unpredictable stress leads to increased anxiety and attention deficit/hyperactivity-like symptoms in adulthood.

BACKGROUND: Early-life adversities during development (e.g., child abuse and neglect) are linked to multiple behavioral and cognitive dysfunctions, such as attention deficit/hyperactivity disorder (ADHD) and anxiety disorders, which have high comorbidity. However, the impact of adversities during adolescence, a crucial period in early life for these disorders, is understudied. Using a chronic unpredictable stress (CUS) model in rats, we investigated whether adversities in adolescence could lead to increased anxiety and ADHD-like symptoms in adulthood. METHODS: Mid- to late-adolescent (5-7-week-old) male and female Sprague-Dawley rats underwent a mild CUS procedure for 2 weeks. Various stressors were applied in an unpredictable way. Rats of both sexes were then trained with a 2-choice reaction time (2-CRT) task during adulthood, which are designed to detect ADHD-like symptoms, including increased impulsivity and lapse of attention. In addition, an open field test was conducted to examine if CUS resulted in a persistent increase in anxiety-like behavior during adulthood. RESULTS: Both male and female rats with CUS exposure travelled shorter distances in the open field and spent less time in the center zone, indicating increased anxiety. In the 2-CRT task, rats of both sexes with CUS exposure showed increased impulsivity. Augmented lapses of attention were observed in female but not male rats. CONCLUSION: Chronic unpredictable stress during adolescence increases anxiety and leads to ADHD-like symptoms in both male and female rats in adulthood. The deficits are more severe in females than in males. These observations support that adversities during adolescence persistently increase anxiety, which is comorbid with attention deficits.

Prenatal ethanol exposure causes anxiety-like phenotype and alters synaptic nitric oxide and endocannabinoid signaling in dorsal raphe nucleus of adult male rats.

Mood disorders, including anxiety and depression caused by prenatal ethanol exposure (PE) are prevalent conditions in fetal alcohol spectrum disorders (FASDs). Prenatal ethanol exposure is associated with persistent dysfunctions of several neurotransmitter systems, including the serotonin (5-HT) system, which plays a major role in mood regulation and stress homeostasis. While PE is known to disrupt the development of the 5-HT system, the cellular mechanisms by which it alters the function of dorsal raphe nucleus (DRn) 5-HT neurons and their synaptic inputs remain unknown. Here, we used a second-trimester binge-drinking pattern PE (two daily gavages of 15% w/v ethanol at 3 g/kg, 5-6 h apart) during gestational days 8 - 20 and measured anxiety-like behaviors of adult male rats using the elevated plus (EPM) and zero (ZM) mazes. We also employed ex-vivo electrophysiological and pharmacological approaches to unravel the mechanisms by which PE alters the excitability and synaptic transmission onto DRn 5-HT neurons. We found that PE enhanced anxiety-like behaviors in adult male rats and induced a persistent activation of DRn 5-HT neurons. The PE-induced activation of DRn 5-HT neurons was largely mediated by potentiation of DRn glutamate synapses, which was caused by activation of the nitrergic system and impaired endocannabinoid signaling. As such, the present study reveals "push-pull" effects of PE on nitrergic and eCB signaling, respectively, which mediate the enhanced activity of DRn 5-HT neurons and could contribute to anxiety-like behaviors observed in animal model of FASD.

Fatty Acid-Binding Protein 5 Modulates Brain Endocannabinoid Tone and Retrograde Signaling in the Striatum.

The endocannabinoid (eCB) anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are endogenous lipid neurotransmitters that regulate an array of physiological functions, including pain, stress homeostasis, and reward. Fatty acid-binding protein 5 (FABP5) is a key modulator of intracellular eCB transport and inactivation. Recent evidence suggests that FABP5 controls synaptic 2-AG signaling at excitatory synapses in the dorsal raphe nucleus. However, it is currently not known whether this function extends to other brain areas. To address this, we first profiled eCB levels across several brain areas in FABP5 knockout mice and wild-type controls and report that FABP5 deletion elevates AEA levels in the striatum, prefrontal cortex, midbrain, and thalamus, as well as midbrain 2-AG levels. The expression of eCB biosynthetic and catabolic enzymes was largely unaltered in these regions, although minor sex and region-specific changes in the expression of 2-AG catabolic enzymes were observed in female FABP5 KO mice. Robust FABP5 expression was observed in the striatum, a region where both AEA and 2-AG control synaptic transmission. Deletion of FABP5 impaired tonic 2-AG and AEA signaling at striatal GABA synapses of medium spiny neurons, and blunted phasic 2-AG mediated short-term synaptic plasticity without altering CB1R expression or function. Collectively, these results support the role of FABP5 as a key regulator of eCB signaling at excitatory and inhibitory synapses in the brain.

Prenatal ethanol exposure impairs sensory processing and habituation to visual stimuli, effects normalized by enrichment of postnatal environmental.

BACKGROUND: Individuals with fetal alcohol spectrum disorders (FASD) often show processing deficits in all sensory modalities. Using an operant light reinforcement model, we tested whether prenatal ethanol exposure (PE) alters operant responding to elicit a contingent sensory stimulus-light onset (turning on the light) and habituation to this behavior in rats. We also explored whether postnatal environmental enrichment could ameliorate PE-induced deficits. METHODS: Pregnant Sprague Dawley rats were gavaged twice/day with 0 or 3 g/kg/treatment ethanol (15% w/v) during gestational days 8-20, mimicking second-trimester heavy PE in humans. The offspring were reared in a standard housing condition or an enriched condition. Adult male and female offspring underwent an operant light reinforcement experiment with either a short-access or a long-access procedure. A dishabituation test was also conducted to characterize the habituation process. RESULTS: In the short-access procedure, PE led to increased operant responding to the contingent light onset in both sexes reared in the standard housing condition. Such an effect was not observed in rats reared in enriched conditions due to an overall decrease in responding. Moreover, rats reared in enriched conditions showed greater short-term habituation. In the long access procedure, PE rats showed increased responding and impaired long-term habituation. The long-access procedure facilitated both short-term and long-term habituation in control and PE rats. CONCLUSION: Prenatal ethanol exposure increases responding to contingent light onset and impairs the long-term habituation process. The PE-induced deficits were ameliorated by rearing in the enriched environment and increasing the duration and frequency of exposure to light onset. The PE-induced effects are like increased sensation-seeking, a subtype of sensory-processing deficit that is often observed in individuals with FASD. Our findings could inform a suitable animal model for investigating the underlying mechanisms and possible intervention strategies for sensory deficits in FASD.

Tonic Endocannabinoid Signaling Gates Synaptic Plasticity in Dorsal Raphe Nucleus Serotonin Neurons Through Peroxisome Proliferator-Activated Receptors.

Endocannabinoids (eCBs), which include 2-arachidonoylglycerol (2-AG) and anandamide (AEA) are lipid signaling molecules involved in the regulation of an array of behavioral and physiological functions. Released by postsynaptic neurons, eCBs mediate both phasic and tonic signaling at central synapses. While the roles of phasic eCB signaling in modulating synaptic functions and plasticity are well characterized, very little is known regarding the physiological roles and mechanisms regulating tonic eCB signaling at central synapses. In this study, we show that both 2-AG and AEA are constitutively released in the dorsal raphe nucleus (DRN), where they exert tonic control of glutamatergic synaptic transmission onto serotonin (5-HT) neurons. The magnitude of this tonic eCB signaling is tightly regulated by the overall activity of neuronal network. Thus, short term in vitro neuronal silencing or blockade of excitatory synaptic transmission abolishes tonic eCB signaling in the DRn. Importantly, in addition to controlling basal synaptic transmission, this study reveals that tonic 2-AG, but not AEA signaling, modulates synaptic plasticity. Indeed, short-term increase in tonic 2-AG signaling impairs spike-timing dependent potentiation (tLTP) of glutamate synapses. This tonic 2-AG-mediated homeostatic control of DRN glutamate synapses is not signaled by canonical cannabinoid receptors, but by intracellular peroxisome proliferator-activated receptor gamma (PPARγ). Further examination reveals that 2-AG mediated activation of PPARγ blocks tLTP by inhibiting nitric oxide (NO), soluble guanylate cyclase, and protein kinase G (NO/sGC/PKG) signaling pathway. Collectively, these results unravel novel mechanisms by which tonic 2-AG signaling integrates network activities and controls the synaptic plasticity in the brain.

Moderate prenatal ethanol exposure leads to attention deficits in both male and female rats.

BACKGROUND: Attention deficits caused by prenatal ethanol (EtOH) exposure (PE) are a prevalent condition in fetal alcohol spectrum disorders (FASDs). Importantly, the deficits are observed in individuals with FASD who have normal IQs and show no dysmorphic facial features caused by heavy PE. These observations suggest that even moderate PE could lead to attention deficits. This possibility was investigated in the present study using a rat model. METHODS: Pregnant Sprague Dawley rats were administered EtOH (3 g/kg/day) or vehicle via intragastric gavage on gestational days 8 to 20. The blood EtOH concentration (BEC) in EtOH-treated rats was 87.7 ± 1.2 mg/dl (1 h after the gavage), similar to the BECs reported in other moderate PE studies in rodents. Moderate PE did not produce teratogenic effects on birthweight or litter size. The adult offspring underwent a 2-choice reaction time task. RESULTS: Moderate PE led to augmented action impulsivity in both sexes, indicated by more rapid response initiation and more premature responses. Deficits were more marked in males than in females. No greater lapses of attention, assessed by incorrect or relatively slow responses, were observed in rats of either sex with moderate PE. In addition, no deficits in learning or motor function were detected after moderate PE. Interestingly, rats with moderate PE completed more trials than controls. CONCLUSIONS: Our results confirm that moderate PE leads to attention deficits in both sexes, which is demonstrated by greater action impulsivity, but not more lapses of attention. This effect differs from that of heavy PE, as shown in our previous study, which is manifested as impaired action impulsivity and lapses of attention in both sexes.

Cocaine self-administration abolishes endocannabinoid-mediated long-term depression of glutamatergic synapses in the ventral tegmental area.

Drugs of abuse, including cocaine, alter the mechanisms underpinning synaptic plasticity, including long-term potentiation of glutamatergic synapses in the mesolimbic system. These effects are thought to underlie addictive behaviors. In the ventral tegmental area (VTA), glutamatergic synapses also exhibit long-term depression (LTD), a type of plasticity that weakens synaptic strength. This form of synaptic plasticity is induced by low-frequency stimulation and mediated by endocannabinoid (eCB) signaling, which also modulates addictive behaviors. However, it remains unknown whether eCB-LTD in the VTA could be altered by cocaine use. Therefore, the goal of the present study was to examine the impact of cocaine self-administration on eCB-LTD of glutamatergic synapses onto VTA dopaminergic (DA) neurons. To that end, male rats underwent cocaine (0.75 mg/kg/infusion) or saline self-administration under the fixed ratio 1 schedule for 6-9 days. One day after the last self-administration session, the magnitude of eCB-LTD was examined using ex vivo whole-cell recordings of putative VTA DA neurons from naïve rats and rats with saline or cocaine self-administration. The results revealed that cocaine self-administration abolished eCB-LTD. The cocaine-induced blockade of eCB-LTD in the VTA was mediated by an impaired function of presynaptic CB1 receptors. Collectively, these findings indicate that cocaine exposure blunts eCB-mediated synaptic plasticity in midbrain DA neurons. This effect could be one of the cellular mechanisms that mediate, at least in part, addictive behaviors.