The γ-Benzylidene Digoxin Derivative BD-15 Increases the α3-Na, K-ATPase Activity in Rat Hippocampus and Prefrontal Cortex and no Change on Heart.

Our study aimed to investigate the effects of the new cardiotonic steroid BD-15 (γ-benzylidene derivatives) in the behavioral parameters, oxidative stress and the Na, K-ATPase activity in the hippocampus, prefrontal cortex and heart from rats to verify the safety and possible utilization in brain disorders. For this study, groups of male Wistar rats were used after intraperitoneal injection of 20, 100 and 200 µg/Kg with BD-15. The groups were treated for three consecutive days and the control group received 0.9% saline. BD-15 did not alter behavior of rats treated with different doses. An increase in the specific α2,3-Na, K-ATPase activity was observed for all doses of BD-15 tested in the hippocampus. However, in the prefrontal cortex, only the dose of 100 µg/Kg increased the activity of all Na, K-ATPase isoforms. BD-15 did not cause alteration in the lipid peroxidation levels in the hippocampus, but in the prefrontal cortex, a decrease of lipid peroxidation (~ 25%) was observed. In the hippocampus, GSH levels increased with all doses tested, while in the prefrontal cortex no changes were found. Subsequently, when the effect of BD-15 on cardiac tissue was analyzed, no changes were observed in the tested parameters. BD-15 at a dosage of 100 µg/Kg proved to be promising because it is considered therapeutic for brain disorders, since it increases the activity of the α3-Na, K-ATPase in the hippocampus and prefrontal cortex, as well as decreasing the oxidative stress in these brain regions. In addition, this drug did not cause changes in the tissues of the heart and kidneys, preferentially demonstrating specificity for the brain.

Dysregulation of Protein Kinase C in Adult Depression and Suicide: Evidence From Postmortem Brain Studies.

BACKGROUND: Several lines of evidence suggest the abnormalities of protein kinase C (PKC) signaling system in mood disorders and suicide based primarily on the studies of PKC and its isozymes in the platelets and postmortem brain of depressed and suicidal subjects. In this study, we examined the role of PKC isozymes in depression and suicide. METHODS: We determined the protein and mRNA expression of various PKC isozymes in the prefrontal cortical region (Brodmann area 9) in 24 normal control subjects, 24 depressed suicide (DS) subjects, and 12 depressed nonsuicide (DNS) subjects. The levels of mRNA in the prefrontal cortex were determined by quantitative real-time reverse transcription PCR, and the protein expression was determined by western blotting. RESULTS: We observed a significant decrease in mRNA expression of PKCα, PKCßI, PKCδ, and PKCε and decreased protein expression in either the membrane or the cytosol fraction of PKC isozymes PKCα, PKCßI, PKCßII, and PKCδ in DS and DNS subjects compared with normal control subjects. CONCLUSIONS: The current study provides detailed evidence of specific dysregulation of certain PKC isozymes in the postmortem brain of DS and DNS subjects and further supports earlier evidence for the role of PKC in the platelets and brain of the adult and teenage depressed and suicidal population. This comprehensive study may lead to further knowledge of the involvement of PKC in the pathophysiology of depression and suicide.

Phosphodiesterase type 1 inhibition alters medial prefrontal cortical activity during goal-driven behaviour and partially reverses neurophysiological deficits in the rat phencyclidine model of schizophrenia.

Positive modulation of cAMP signalling by phosphodiesterase (PDE) inhibitors has recently been explored as a potential target for the reversal of cognitive and behavioural deficits implicating the corticoaccumbal circuit. Previous studies show that PDE type 1 isoform B (PDE1B) inhibition may improve memory function in rodent models; however, the contribution of PDE1B inhibition to impulsivity, attentional and motivational functions as well as its neurophysiological effects have not been investigated. To address this, we recorded single unit activity in medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) in Lister Hooded rats treated with the PDE1B inhibitor Lu AF64386 and tested in the 5-choice serial reaction time task (5-CSRTT). We also asked whether PDE1B inhibition modulates neurophysiological deficits produced by subchronic phencyclidine (PCP) treatment, a rat pharmacological model of schizophrenia. Lu AF64386 significantly affected behavioural parameters consistent with a reduction in goal-directed behaviour, however without affecting accuracy. Additionally, it reduced mPFC neuronal activity. Pre-treatment with PCP did not affect behavioural parameters, however it significantly disrupted overall neuronal firing while increasing phasic responses to reward-predicting cues and disrupting mPFC-NAc cross-talk. The latter two effects were reversed by Lu AF64386. These findings suggest PDE1B inhibition may be beneficial in disorders implicating a dysfunction of the mPFC-NAc network.

Treatment with the calcineurin inhibitor and immunosuppressant cyclosporine A impairs sensorimotor gating in Dark Agouti rats.

RATIONALE: Calcineurin is a protein regulating cytokine expression in T lymphocytes and calcineurin inhibitors such as cyclosporine A (CsA) are widely used for immunosuppressive therapy. It also plays a functional role in distinct neuronal processes in the central nervous system. Disturbed information processing as seen in neuropsychiatric disorders is reflected by deficient sensorimotor gating, assessed as prepulse inhibition (PPI) of the acoustic startle response (ASR). OBJECTIVE: Patients who require treatment with immunosuppressive drugs frequently display neuropsychiatric alterations during treatment with calcineurin inhibitors. Importantly, knockout of calcineurin in the forebrain of mice is associated with cognitive impairments and symptoms of schizophrenia-like psychosis as seen after treatment with stimulants. METHODS: The present study investigated in rats effects of systemic acute and subchronic administration of CsA on sensorimotor gating. Following a single injection with effective doses of CsA, adult healthy male Dark Agouti rats were tested for PPI. For subchronic treatment, rats were injected daily with the same doses of CsA for 1 week before PPI was assessed. Since calcineurin works as a modulator of the dopamine pathway, activity of the enzyme tyrosine hydroxylase was measured in the prefrontal cortex and striatum after accomplishment of the study. RESULTS: Acute and subchronic treatment with the calcineurin inhibitor CsA disrupted PPI at a dose of 20 mg/kg. Concomitantly, following acute CsA treatment, tyrosine hydroxylase activity was reduced in the prefrontal cortex, which suggests that dopamine synthesis was downregulated, potentially reflecting a stimulatory impact of CsA on this neurotransmitter system. CONCLUSIONS: The results support experimental and clinical evidence linking impaired calcineurin signaling in the central nervous system to the pathophysiology of neuropsychiatric symptoms. Moreover, these findings suggest that therapy with calcineurin inhibitors may be a risk factor for developing neurobehavioral alterations as observed after the abuse of psychomotor stimulant drugs.

Active Transition of Fear Memory Phase from Reconsolidation to Extinction through ERK-Mediated Prevention of Reconsolidation.

The retrieval of fear memory induces two opposite memory process, i.e., reconsolidation and extinction. Brief retrieval induces reconsolidation to maintain or enhance fear memory, while prolonged retrieval extinguishes this memory. Although the mechanisms of reconsolidation and extinction have been investigated, it remains unknown how fear memory phases are switched from reconsolidation to extinction during memory retrieval. Here, we show that an extracellular signal-regulated kinase (ERK)-dependent memory transition process after retrieval regulates the switch of memory phases from reconsolidation to extinction by preventing induction of reconsolidation in an inhibitory avoidance (IA) task in male mice. First, the transition memory phase, which cancels the induction of reconsolidation, but is insufficient for the acquisition of extinction, was identified after reconsolidation, but before extinction phases. Second, the reconsolidation, transition, and extinction phases after memory retrieval showed distinct molecular and cellular signatures through cAMP responsive element binding protein (CREB) and ERK phosphorylation in the amygdala, hippocampus, and medial prefrontal cortex (mPFC). The reconsolidation phase showed increased CREB phosphorylation, while the extinction phase displayed several neural populations with various combinations of CREB and/or ERK phosphorylation, in these brain regions. Interestingly, the three memory phases, including the transition phase, showed transient ERK activation immediately after retrieval. Most importantly, the blockade of ERK in the amygdala, hippocampus, or mPFC at the transition memory phase disinhibited reconsolidation-induced enhancement of IA memory. These observations suggest that the ERK-signaling pathway actively regulates the transition of memory phase from reconsolidation to extinction and this process functions as a switch that cancels reconsolidation of fear memory.SIGNIFICANCE STATEMENT Retrieval of fear memory induces two opposite memory process; reconsolidation and extinction. Reconsolidation maintains/enhances fear memory, while extinction weakens fear memory. It remains unknown how memory phases are switched from reconsolidation to extinction during retrieval. Here, we identified an active memory transition process functioning as a switch that inhibits reconsolidation. This memory transition phase showed a transient increase of extracellular signal-regulated kinase (ERK) phosphorylation in the amygdala, hippocampus and medial prefrontal cortex (mPFC). Interestingly, inhibition of ERK in these regions at the transition phase disinhibited the reconsolidation-mediated enhancement of inhibitory avoidance (IA) memory. These findings suggest that the transition memory process actively regulates the switch of fear memory phases of fear memory by preventing induction of reconsolidation through the activation of the ERK-signaling pathway.

DNA Methyltransferase 3A Is Involved in the Sustained Effects of Chronic Stress on Synaptic Functions and Behaviors.

Emerging evidence suggests that epigenetic mechanisms regulate aberrant gene transcription in stress-associated mental disorders. However, it remains to be elucidated about the role of DNA methylation and its catalyzing enzymes, DNA methyltransferases (DNMTs), in this process. Here, we found that male rats exposed to chronic (2-week) unpredictable stress exhibited a substantial reduction of Dnmt3a after stress cessation in the prefrontal cortex (PFC), a key target region of stress. Treatment of unstressed control rats with DNMT inhibitors recapitulated the effect of chronic unpredictable stress on decreased AMPAR expression and function in PFC. In contrast, overexpression of Dnmt3a in PFC of stressed animals prevented the loss of glutamatergic responses. Moreover, the stress-induced behavioral abnormalities, including the impaired recognition memory, heightened aggression, and hyperlocomotion, were partially attenuated by Dnmt3a expression in PFC of stressed animals. Finally, we found that there were genome-wide DNA methylation changes and transcriptome alterations in PFC of stressed rats, both of which were enriched at several neural pathways, including glutamatergic synapse and microtubule-associated protein kinase signaling. These results have therefore recognized the potential role of DNA epigenetic modification in stress-induced disturbance of synaptic functions and cognitive and emotional processes.

Evidences for agmatine alterations in Aß1-42induced memory impairment in mice.

Alzheimer's disease is an age related progressive neurodegenerative disorder characterized by decline in cognitive functions, such as memory loss and behavioural abnormalities. The present study sought to assess alterations in agmatine metabolism in the beta-amyloid (Aß1-42) Alzheimer's disease mouse model. Aß1-42 injected mice showed impairment of cognitive functioning as evidenced by increased working and reference memory errors in radial arm maze (RAM). This cognitive impairment was associated with a reduction in the agmatine levels and elevation in its degrading enzyme, agmatinase, whereas reduced immunocontent was observed in its synthesizing enzyme arginine decarboxylase expression within hippocampus and prefrontal cortex. Chronic agmatine treatment and its endogenous modulation by l-arginine, or arcaine or aminoguanidine prevented the learning and memory impairment induced by single intracranial Aß1-42 peptide injection. In conclusion, the present study suggests the importance of the endogenous agmatinergic system in ß-amyloid induced memory impairment in mice.

Involvement of kynurenine pathway in depressive-like behaviour induced by nandrolone decanoate in mice.

Nandrolone decanoate (ND) belongs to the class II of anabolic-androgenic steroids (AAS), which is composed of 19-nor-testosterone-derivatives. AAS represent a group of synthetic testosterone that is used in clinical treatment. However, these drugs are widely abused among individuals as a means of promoting muscle growth or enhancing athletic performance. AAS in general and ND in particular have been associated with several behavioral disturbances, such as anxiety, aggressiveness and depression. A factor that contributes to the development of depression is the brain activation of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme of kynurenine pathway (KP). In the present study, we examined the involvement of KP in depressive phenotype induced by a ND treatment (10 mg/kg/day/s.c., for 28 days) that mimics human abuse system (e.g. supraphysiological doses) in C57B/6J mice. Our results showed that ND caused depressive like-behavior in the tail suspension test and anhedonic-like state measured in the sucrose preference test. ND administration decreased the levels of brain-derived neurotrophic factor and neurotrophin-3 and reduced Na+,K+-ATPase activity in the hippocampus, striatum and prefrontal cortex. We also found that ND elicited KP activation, as reflected by the increase of IDO activity and kynurenine levels in these brain regions. Moreover, ND decreased serotonin levels and increased 5-hydroxyindoleacetic acid levels in the brain. Treatment with IDO inhibitor 1-methyl-dl-trypthophan (1 mg/kg/i.p.) reversed the behavioral and neurochemical alterations induced by ND. These results indicate for the first time that KP plays a key role in depressive-like behavior and neurotoxicity induced by supraphysiologicaldoses of ND in mice.

Galantamine improves enhanced impulsivity, impairments of attention and long-term potentiation induced by prenatal nicotine exposure to mice.

Prenatal nicotine exposure (PNE) causes behavioral abnormalities in offspring, such as an enhancement of impulsivity and decrease in attention at adolescence. Here we examined the effects of galantamine (GAL) on the behavioral and electrophysiological changes induced by PNE in mice. Pregnant C57BL/6J mice were exposed to nicotine (0.2 mg/mL) dissolved in sweetened (2% saccharin) drinking water during gestational day 14 and perinatal day 0 (P0). At the ages of postnatal days 42-49 (P42-P49), female offspring displayed impulsivity in the cliff avoidance test and impairment of visual attention in the object-based attention test. Decrease of long-term potentiation (LTP) and extracellular glutamate levels were observed in the prefrontal cortex of PNE mice. Systemic treatment with GAL (1 mg/kg, s.c.), an allosteric potentiating ligand for the nicotinic acetylcholine receptor (nAChR) and a weak cholinesterase inhibitor, attenuated the enhancement of impulsivity and impairment of attention induced by PNE in mice. Further, GAL reversed the impairment of LTP induced by PNE in the prefrontal cortex of mice, although it failed to attenuate the decrease of extracellular glutamate levels. The effects of GAL were blocked by an α 7 nAChR antagonist, methyllycaconitine (1 mg/kg, i.p.). These results suggest that PNE during cortex development affects nicotinic cholinergic-dependent plasticity and formation of impulsivity and attention. Furthermore, GAL could be a useful drug for cognitive impairments-related to attention deficit hyperactivity disorder.

The impact of sleep deprivation on sexual behaviors and FAAH expression in the prefrontal cortex of male rats.

Sleep deprivation (SD) causes alterations in the function of the endocannabinoid (EC) system and also results in alteration in many behaviors such as increased anxiety, deteriorated alertness, memory deficits, as well as sexual behaviors. Controversial data about the effects of SD on sexual response are provided. Fatty acid amide hydrolase (FAAH), the enzymes involved in the degradation of the EC system play an important role in the function of the EC system. This study aimed to investigate the effect of REM SD (RSD) and total SD (TSD) on the sexual behaviors and FAAH expression in the prefrontal cortex (PFC) of male rats. RSD was carried out through the flower pot technique for 24 h and 48 h, and TSD also was induced by keeping awake the rats by gentle handling for 6 h. Immediately after RSD and TSD, sexual behaviors were recorded for 45 min. Sexual behaviors were reduced by both types of RSD and TSD. The deleterious effects of 24 h RSD were more severe compared with 6 h of TSD. Serum testosterone concentration was significantly higher after TSD but not RSD compared to the normal sleep (NS) group. FAAH expression in the PFC was significantly reduced after both RSD and TSD compared to the NS group. Given that the function of the EC system has been previously shown to change different behaviors such as sexual activity, our results could suggest that behavioral effects of both types of SD on sexual behavior may partially result from activation of this signaling pathway by the reduction of FAAH in the PFC.

nNOS-expressing neurons in the vmPFC transform pPVT-derived chronic pain signals into anxiety behaviors.

Anxiety is common in patients suffering from chronic pain. Here, we report anxiety-like behaviors in mouse models of chronic pain and reveal that nNOS-expressing neurons in ventromedial prefrontal cortex (vmPFC) are essential for pain-induced anxiety but not algesia, using optogenetic and chemogenetic strategies. Additionally, we determined that excitatory projections from the posterior subregion of paraventricular thalamic nucleus (pPVT) provide a neuronal input that drives the activation of vmPFC nNOS-expressing neurons in our chronic pain models. Our results suggest that the pain signal becomes an anxiety signal after activation of vmPFC nNOS-expressing neurons, which causes subsequent release of nitric oxide (NO). Finally, we show that the downstream molecular mechanisms of NO likely involve enhanced glutamate transmission in vmPFC CaMKIIα-expressing neurons through S-nitrosylation-induced AMPAR trafficking. Overall, our data suggest that pPVT excitatory neurons drive chronic pain-induced anxiety through activation of vmPFC nNOS-expressing neurons, resulting in NO-mediated AMPAR trafficking in vmPFC pyramidal neurons.

Adenosine Kinase Expression in the Frontal Cortex in Schizophrenia.

The adenosine hypothesis of schizophrenia posits that reduced availability of the neuromodulator adenosine contributes to dysregulation of dopamine and glutamate transmission and the symptoms associated with schizophrenia. It has been proposed that increased expression of the enzyme adenosine kinase (ADK) may drive hypofunction of the adenosine system. While animal models of ADK overexpression support such a role for altered ADK, the expression of ADK in schizophrenia has yet to be examined. In this study, we assayed ADK gene and protein expression in frontocortical tissue from schizophrenia subjects. In the dorsolateral prefrontal cortex (DLPFC), ADK-long and -short splice variant expression was not significantly altered in schizophrenia compared to controls. There was also no significant difference in ADK splice variant expression in the frontal cortex of rats treated chronically with haloperidol-decanoate, in a study to identify the effect of antipsychotics on ADK gene expression. ADK protein expression was not significantly altered in the DLPFC or anterior cingulate cortex (ACC). There was no significant effect of antipsychotic medication on ADK protein expression in the DLPFC or ACC. Overall, our results suggest that increased ADK expression does not contribute to hypofunction of the adenosine system in schizophrenia and that alternative mechanisms are involved in dysregulation of this system in schizophrenia.

Dysregulation of histone acetylation pathways in hippocampus and frontal cortex of Alzheimer's disease patients.

Memory impairment is the main feature of Alzheimer's disease (AD). Initial impairments originate in the temporal lobe area and propagate throughout the brain in a sequential manner. Epigenetic mechanisms, especially histone acetylation, regulate plasticity and memory processes. These may be dismantled during the disease. The aim of this work was to establish changes in the acetylation-associated pathway in two key brain regions affected in AD: the hippocampus and the F2 area of frontal cortex in end-stage AD patients and age-matched controls. We found that the F2 area was more affected than the hippocampus. Indeed, CREB-Binding Protein (CBP), P300/CBP-associated protein (PCAF), Histone Deacetylase 1 (HDAC1) and HDAC2 (but not HDAC3) levels were strongly decreased in F2 area of AD compared to controls patients, whereas only HDAC1 was decreased and CBP showed a downward trend in the hippocampus. At the histone level, we detected a substantial increase in total (H3 and H2B) histone levels in the frontal cortex, but these were decreased in nuclear extracts, pointing to a dysregulation in histone trafficking/catabolism in this brain region. Histone H3 acetylation levels were increased in cell nuclei mainly in the frontal cortex. These findings provide evidence for acetylation dysfunctions at the level of associated enzymes and of histones in AD brains, which may underlie transcriptional dysregulations and AD-related cognitive impairments. They further point to stronger dysregulations in the F2 area of the frontal cortex than in the hippocampus at an end-stage of the disease, suggesting a differential vulnerability and/or compensatory mechanisms efficiency towards epigenetic alterations.

Monoacylglycerol lipase alpha inhibition alters prefrontal cortex excitability and blunts the consequences of traumatic stress in rat.

Neural activity within the ventromedial prefrontal cortex (vmPFC) is a critical determinant of stressor-induced anxiety. Pharmacological activation of the vmPFC during stress protects against stress-induced social anxiety suggesting that altering the excitatory/inhibitory (E/I) tone in the vmPFC may promote stress resilience. E/I balance is maintained, in part, by endogenous cannabinoid (eCB) signaling with the calcium dependent retrograde release of 2-arachidonoylglycerol (2-AG) suppressing presynaptic neurotransmitter release. We hypothesized that raising 2-AG levels, via inhibition of its degradation enzyme monoacylglycerol lipase (MAGL) with KML29, would shift vmPFC E/I balance and promote resilience. In acute slice experiments, bath application of KML29 (100 nM) augmented evoked excitatory neurotransmission as evidenced by a left-shift in fEPSP I/O curve, and decreased sIPSC amplitude. In whole-cell recordings, KML29 increased resting membrane potential but reduced the after depolarization, bursting rate, membrane time constant and slow after hyperpolarization. Intra-vmPFC administration of KML29 (200ng/0.5µL/hemisphere) prior to inescapable stress (IS) exposure (25, 5s tail shocks) prevented stress induced anxiety as measured by juvenile social exploration 24 h after stressor exposure. Conversely, systemic administration of KML29 (40 mg/kg, i.p.) 2 h before IS exacerbated stress induced anxiety. MAGL inhibition in the vmPFC may promote resilience by augmenting the output of neurons that project to brainstem and limbic structures that mediate stress responses.

Reversal of peripheral nerve injury-induced neuropathic pain and cognitive dysfunction via genetic and tomivosertib targeting of MNK.

Neuropathic pain caused by nerve injury presents with severe spontaneous pain and a variety of comorbidities, including deficits in higher executive functions. None of these clinical problems are adequately treated with current analgesics. Targeting of the mitogen-activated protein kinase-interacting kinase (MNK1/2) and its phosphorylation target, the mRNA cap binding protein eIF4E, attenuates many types of nociceptive plasticity induced by inflammatory mediators and chemotherapeutic drugs but inhibiting this pathway does not alter nerve injury-induced mechanical allodynia. We used genetic manipulations and pharmacology to inhibit MNK-eIF4E activity in animals with spared nerve injury, a model of peripheral nerve injury (PNI)-induced neuropathic pain. We assessed the presence of spontaneous pain using conditioned place preference. We also tested performance in a medial prefrontal cortex (mPFC)-dependent rule-shifting task. WT neuropathic animals showed signs of spontaneous pain and were significantly impaired in the rule-shifting task while genetic and pharmacological inhibition of the MNK-eIF4E signaling axis protected against and reversed spontaneous pain and PNI-mediated cognitive impairment. Additionally, pharmacological and genetic inhibition of MNK-eIF4E signaling completely blocked and reversed maladaptive shortening in the length of axon initial segments (AIS) in the mPFC of PNI mice. Surprisingly, these striking positive outcomes on neuropathic pain occurred in the absence of any effect on mechanical allodynia, a standard test for neuropathic pain efficacy. Our results illustrate new testing paradigms for determining preclinical neuropathic pain efficacy and point to the MNK inhibitor tomivosertib (eFT508) as an important drug candidate for neuropathic pain treatment.

Vitamin D3 favorable outcome on recognition memory and prefrontal cortex expression of choline acetyltransferase and acetylcholinesterase in experimental model of chronic high-fat feeding.

Aim of the study: High-fat diet (HFD) consumption and insufficient vitamin D levels are globally increasing phenomena. The present study assessed the effect of chronic HFD feeding with and without vitamin D supplementation on recognition memory and prefrontal cortex expression of choline acetyltransferase (CAT) and acetylcholinesterase (Achase).Materials and methods: Forty male Wistar rats were subjected to four dietary regimens (n = 10); control diet (10% fat), control + vitamin D3, high-fat diet (HFD 45% fat) and HFD + vitamin D3 for 6 months. Rats were tested for the novel object recognition test, and their prefrontal cortices were assessed for expression of CAT and Achase.Results: Recognition memory was impaired in HFD-fed rats compared to control rats as evidenced by significantly decreased discrimination index in the novel object recognition test. Moreover, CAT expression was significantly decreased while Achase expression was significantly increased in the prefrontal cortex of HFD-fed rats. Vitamin D3 supplementation with HFD significantly increased the exploration of the novel object and the discrimination index and attenuated the alterations in the prefrontal cortex CAT and Achase expression.Conclusions: The present findings support the potential effect of vitamin D on recognition memory and cholinergic transmission in the prefrontal cortex and add to the pathophysiology of HFD consumption.

Lithium attenuates d-amphetamine-induced hyperlocomotor activity in mice via inhibition of interaction between cyclooxygenase-2 and indoleamine-2,3-dioxygenase.

In the present study, we investigated whether mood stabilizer lithium (Li) protects against d-amphetamine (AMP)-induced mania-like behaviours via modulating the novel proinflammatory potential. Repeated treatment with AMP resulted in significant increases in proinflammatory cyclooxygenase-2 (COX-2) and indolemaine-2,3-dioxygenase-1 (IDO)-1 expression in the prefrontal cortex (PFC) of mice. However, AMP treatment did not significantly change IDO-2 and 5-lipoxygenase (5-LOX) expression, suggesting that proinflammatory parameters such as COX-2 and IDO-1 are specific for AMP-induced behaviours. AMP-induced initial expression of COX-2 (15 minutes post-AMP) was earlier than that of IDO-1 (1 hour post-AMP). Mood stabilizer Li and COX-2 inhibitor meloxicam significantly attenuated COX-2 expression 15 minutes post-AMP, whereas IDO-1 inhibitor 1-methyl-DL-tryptophan (1-MT) did not affect COX-2 expression. However, AMP-induced IDO-1 expression was significantly attenuated by Li, meloxicam or 1-MT, suggesting that COX-2 is an upstream molecule for the induction of IDO-1 caused by AMP. Consistently, co-immunoprecipitation between COX-2 and IDO-1 was observed at 30 minutes, 1, 3, and 6 hours after the final AMP treatment. This interaction was also significantly inhibited by Li, meloxicam or 1-MT. Furthermore, AMP-induced hyperlocomotion was significantly attenuated by Li, meloxicam or 1-MT. We report, for the first time, that mood stabilizer Li attenuates AMP-induced mania-like behaviour via attenuation of interaction between COX-2 and IDO-1, and that the interaction of COX-2 and IDO-1 may be critical for the therapeutic intervention mediated by mood stabilizer.

Biased M1 receptor-positive allosteric modulators reveal role of phospholipase D in M1-dependent rodent cortical plasticity.

Highly selective, positive allosteric modulators (PAMs) of the M1 subtype of muscarinic acetylcholine receptor have emerged as an exciting new approach to potentially improve cognitive function in patients suffering from Alzheimer's disease and schizophrenia. Discovery programs have produced a structurally diverse range of M1 receptor PAMs with distinct pharmacological properties, including different extents of agonist activity and differences in signal bias. This includes biased M1 receptor PAMs that can potentiate coupling of the receptor to activation of phospholipase C (PLC) but not phospholipase D (PLD). However, little is known about the role of PLD in M1 receptor signaling in native systems, and it is not clear whether biased M1 PAMs display differences in modulating M1-mediated responses in native tissue. Using PLD inhibitors and PLD knockout mice, we showed that PLD was necessary for the induction of M1-dependent long-term depression (LTD) in the prefrontal cortex (PFC). Furthermore, biased M1 PAMs that did not couple to PLD not only failed to potentiate orthosteric agonist-induced LTD but also blocked M1-dependent LTD in the PFC. In contrast, biased and nonbiased M1 PAMs acted similarly in potentiating M1-dependent electrophysiological responses that were PLD independent. These findings demonstrate that PLD plays a critical role in the ability of M1 PAMs to modulate certain central nervous system (CNS) functions and that biased M1 PAMs function differently in brain regions implicated in cognition.

mTORC1 in the orbitofrontal cortex promotes habitual alcohol seeking.

The mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in dendritic translation and in learning and memory. We previously showed that heavy alcohol use activates mTORC1 in the orbitofrontal cortex (OFC) of rodents (Laguesse et al., 2017a). Here, we set out to determine the consequences of alcohol-dependent mTORC1 activation in the OFC. We found that inhibition of mTORC1 activity in the OFC attenuates alcohol seeking and restores sensitivity to outcome devaluation in rats that habitually seek alcohol. In contrast, habitual responding for sucrose was unaltered by mTORC1 inhibition, suggesting that mTORC1's role in habitual behavior is specific to alcohol. We further show that inhibition of GluN2B in the OFC attenuates alcohol-dependent mTORC1 activation, alcohol seeking and habitual responding for alcohol. Together, these data suggest that the GluN2B/mTORC1 axis in the OFC drives alcohol seeking and habit.

Impaired ß-glucocerebrosidase activity and processing in frontotemporal dementia due to progranulin mutations.

Loss-of-function mutations in progranulin (GRN) are a major autosomal dominant cause of frontotemporal dementia. Most pathogenic GRN mutations result in progranulin haploinsufficiency, which is thought to cause frontotemporal dementia in GRN mutation carriers. Progranulin haploinsufficiency may drive frontotemporal dementia pathogenesis by disrupting lysosomal function, as patients with GRN mutations on both alleles develop the lysosomal storage disorder neuronal ceroid lipofuscinosis, and frontotemporal dementia patients with GRN mutations (FTD-GRN) also accumulate lipofuscin. The specific lysosomal deficits caused by progranulin insufficiency remain unclear, but emerging data indicate that progranulin insufficiency may impair lysosomal sphingolipid-metabolizing enzymes. We investigated the effects of progranulin insufficiency on sphingolipid-metabolizing enzymes in the inferior frontal gyrus of FTD-GRN patients using fluorogenic activity assays, biochemical profiling of enzyme levels and posttranslational modifications, and quantitative neuropathology. Of the enzymes studied, only ß-glucocerebrosidase exhibited impairment in FTD-GRN patients. Brains from FTD-GRN patients had lower activity than controls, which was associated with lower levels of mature ß-glucocerebrosidase protein and accumulation of insoluble, incompletely glycosylated ß-glucocerebrosidase. Immunostaining revealed loss of neuronal ß-glucocerebrosidase in FTD-GRN patients. To investigate the effects of progranulin insufficiency on ß-glucocerebrosidase outside of the context of neurodegeneration, we investigated ß-glucocerebrosidase activity in progranulin-insufficient mice. Brains from Grn-/- mice had lower ß-glucocerebrosidase activity than wild-type littermates, which was corrected by AAV-progranulin gene therapy. These data show that progranulin insufficiency impairs ß-glucocerebrosidase activity in the brain. This effect is strongest in neurons and may be caused by impaired ß-glucocerebrosidase processing.