Adolescent nicotine exposure induces long-term, sex-specific disturbances in mood and anxiety-related behavioral, neuronal and molecular phenotypes in the mesocorticolimbic system.

The majority of lifetime smokers begin using nicotine during adolescence, a critical period of brain development wherein neural circuits critical for mood, affect and cognition are vulnerable to drug-related insults. Specifically, brain regions such as the medial prefrontal cortex (mPFC), the ventral tegmental area (VTA), nucleus accumbens (NAc) and hippocampus, are implicated in both nicotine dependence and pathological phenotypes linked to mood and anxiety disorders. Clinical studies report that females experience higher rates of mood/anxiety disorders and are more resistant to smoking cessation therapies, suggesting potential sex-specific responses to nicotine exposure and later-life neuropsychiatric risk. However, the potential neural and molecular mechanisms underlying such sex differences are not clear. In the present study, we compared the impacts of adolescent nicotine exposure in male vs. female rat cohorts. We performed a combination of behavioral, electrophysiological and targeted protein expression analyses along with matrix assisted laser deionization imaging (MALDI) immediately post-adolescent exposure and later in early adulthood. We report that adolescent nicotine exposure induced long-lasting anxiety/depressive-like behaviors, disrupted neuronal activity patterns in the mPFC-VTA network and molecular alterations in various neural regions linked to affect, anxiety and cognition. Remarkably, these phenotypes were only observed in males and/or were expressed in the opposite direction in females. These findings identify a series of novel, sex-selective biomarkers for adolescent nicotine-induced neuropsychiatric risk, persisting into adulthood.

Inhibition of fatty acid binding protein-5 in the basolateral amygdala induces anxiolytic effects and accelerates fear memory extinction.

RATIONALE: The endocannabinoid (eCB) system critically controls anxiety and fear-related behaviours. Anandamide (AEA), a prominent eCB ligand, is a hydrophobic lipid that requires chaperone proteins such as Fatty Acid Binding Proteins (FABPs) for intracellular transport. Intracellular AEA transport is necessary for degradation, so blocking FABP activity increases AEA neurotransmission. OBJECTIVE: To investigate the effects of a novel FABP5 inhibitor (SBFI-103) in the basolateral amygdala (BLA) on anxiety and fear memory. METHODS: We infused SBFI-103 (0.5 µg-5 µg) to the BLA of adult male Sprague Dawley rats and ran various anxiety and fear memory behavioural assays, neurophysiological recordings, and localized molecular signaling analyses. We also co-infused SBFI-103 with the AEA inhibitor, LEI-401 (3 µg and 10 µg) to investigate the potential role of AEA in these phenomena. RESULTS: Acute intra-BLA administration of SBFI-103 produced strong anxiolytic effects across multiple behavioural tests. Furthermore, animals exhibited acute and long-term accelerated associative fear memory extinction following intra-BLA FABP5 inhibition. In addition, BLA FABP5 inhibition induced strong modulatory effects on putative PFC pyramidal neurons along with significantly increased gamma oscillation power. Finally, we observed local BLA changes in the phosphorylation activity of various anxiety- and fear memory-related molecular biomarkers in the PI3K/Akt and MAPK/Erk signaling pathways. At all three levels of analyses, we found the functional effects of SBFI-103 depend on availability of the AEA ligand. CONCLUSIONS: These findings demonstrate a novel intra-BLA FABP5 signaling mechanism regulating anxiety and fear memory behaviours, neuronal activity states, local anxiety-related molecular pathways, and functional AEA modulation.

Identification of a novel fatty acid binding protein-5-CB2 receptor-dependent mechanism regulating anxiety behaviors in the prefrontal cortex.

The endocannabinoid (eCB) system represents a promising neurobiological target for novel anxiolytic pharmacotherapies. Previous clinical and preclinical evidence has revealed that genetic and/or pharmacological manipulations altering eCB signaling modulate fear and anxiety behaviors. Water-insoluble eCB lipid anandamide requires chaperone proteins for its intracellular transport to degradation, a process that requires fatty acid-binding proteins (FABPs). Here, we investigated the effects of a novel FABP-5 inhibitor, SBFI-103, on fear and anxiety-related behaviors using rats. Acute intra-prelimbic cortex administration of SBFI-103 induced a dose-dependent anxiolytic response and reduced contextual fear expression. Surprisingly, both effects were reversed when a cannabinoid-2 receptor (CB2R) antagonist, AM630, was co-infused with SBFI-103. Co-infusion of the cannabinoid-1 receptor antagonist Rimonabant with SBFI-103 reversed the contextual fear response yet showed no reversal effect on anxiety. Furthermore, in vivo neuronal recordings revealed that intra-prelimbic region SBFI-103 infusion altered the activity of putative pyramidal neurons in the basolateral amygdala and ventral hippocampus, as well as oscillatory patterns within these regions in a CB2R-dependent fashion. Our findings identify a promising role for FABP5 inhibition as a potential target for anxiolytic pharmacotherapy. Furthermore, we identify a novel, CB2R-dependent FABP-5 signaling pathway in the PFC capable of strongly modulating anxiety-related behaviors and anxiety-related neuronal transmission patterns.

Adolescent Δ-9-tetrahydrocannabinol exposure induces differential acute and long-term neuronal and molecular disturbances in dorsal vs. ventral hippocampal subregions.

Chronic exposure to Δ-9-tetrahydrocannabinol (THC) during adolescence is associated with long-lasting cognitive impairments and enhanced susceptibility to anxiety and mood disorders. Previous evidence has revealed functional and anatomical dissociations between the posterior vs. anterior portions of the hippocampal formation, which are classified as the dorsal and ventral regions in rodents, respectively. Notably, the dorsal hippocampus is critical for cognitive and contextual processing, whereas the ventral region is critical for affective and emotional processing. While adolescent THC exposure can induce significant morphological disturbances and glutamatergic signaling abnormalities in the hippocampus, it is not currently understood how the dorsal vs. ventral hippocampal regions are affected by THC during neurodevelopment. In the present study, we used an integrative combination of behavioral, molecular, and neural assays in a neurodevelopmental rodent model of adolescent THC exposure. We report that adolescent THC exposure induces long-lasting memory deficits and anxiety like-behaviors concomitant with a wide range of differential molecular and neuronal abnormalities in dorsal vs. ventral hippocampal regions. In addition, using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS), we show for the first time that adolescent THC exposure induces significant and enduring dysregulation of GABA and glutamate levels in dorsal vs. ventral hippocampus. Finally, adolescent THC exposure induced dissociable dysregulations of hippocampal glutamatergic signaling, characterized by differential glutamatergic receptor expression markers, profound alterations in pyramidal neuronal activity and associated oscillatory patterns in dorsal vs. ventral hippocampal subregions.

Anxiety and cognitive-related effects of Δ 9-tetrahydrocannabinol (THC) are differentially mediated through distinct GSK-3 vs. Akt-mTOR pathways in the nucleus accumbens of male rats.

RATIONALE: Δ9-tetrahydrocannabinol (THC) is the primary psychoactive compound in cannabis and is responsible for cannabis-related neuropsychiatric side effects, including abnormal affective processing, cognitive and sensory filtering deficits and memory impairments. A critical neural region linked to the psychotropic effects of THC is the nucleus accumbens shell (NASh), an integrative mesocorticolimbic structure that sends and receives inputs from multiple brain areas known to be dysregulated in various disorders, including schizophrenia and anxiety-related disorders. Considerable evidence demonstrates functional differences between posterior vs. anterior NASh sub-regions in the processing of affective and cognitive behaviours influenced by THC. Nevertheless, the neuroanatomical regions and local molecular pathways responsible for these psychotropic effects are not currently understood. OBJECTIVES: The objectives of this study were to characterize the effects of intra-accumbens THC in the anterior vs. posterior regions of the NASh during emotional memory formation, sensorimotor gating and anxiety-related behaviours. METHODS: We performed an integrative series of translational behavioural pharmacological studies examining anxiety, sensorimotor gating and fear-related associative memory formation combined with regionally specific molecular signalling analyses in male Sprague Dawley rats. RESULTS: We report that THC in the posterior NASh causes distortions in emotional salience attribution, impaired sensory filtering and memory retention and heightened anxiety, through a glycogen-synthase-kinase-3 (GSK-3)-ß-catenin dependent signalling pathway. In contrast, THC in the anterior NASh produces anxiolytic effects via modulation of protein kinase B (Akt) phosphorylation states. CONCLUSIONS: These findings reveal critical new insights into the neuroanatomical and molecular mechanisms associated with the differential neuropsychiatric side effects of THC in dissociable nucleus accumbens sub-regions.

l-Theanine Prevents Long-Term Affective and Cognitive Side Effects of Adolescent Δ-9-Tetrahydrocannabinol Exposure and Blocks Associated Molecular and Neuronal Abnormalities in the Mesocorticolimbic Circuitry.

Chronic adolescent exposure to Δ-9-tetrahydrocannabinol (THC) is linked to elevated neuropsychiatric risk and induces neuronal, molecular and behavioral abnormalities resembling neuropsychiatric endophenotypes. Previous evidence has revealed that the mesocorticolimbic circuitry, including the prefrontal cortex (PFC) and mesolimbic dopamine (DA) pathway are particularly susceptible to THC-induced pathologic alterations, including dysregulation of DAergic activity states, loss of PFC GABAergic inhibitory control and affective and cognitive abnormalities. There are currently limited pharmacological intervention strategies capable of preventing THC-induced neuropathological adaptations. l-Theanine is an amino acid analog of l-glutamate and l-glutamine derived from various plant sources, including green tea leaves. l-Theanine has previously been shown to modulate levels of GABA, DA, and glutamate in various neural regions and to possess neuroprotective properties. Using a preclinical model of adolescent THC exposure in male rats, we report that l-theanine pretreatment before adolescent THC exposure is capable of preventing long-term, THC-induced dysregulation of both PFC and VTA DAergic activity states, a neuroprotective effect that persists into adulthood. In addition, pretreatment with l-theanine blocked THC-induced downregulation of local GSK-3 (glycogen synthase kinase 3) and Akt signaling pathways directly in the PFC, two biomarkers previously associated with cannabis-related psychiatric risk and subcortical DAergic dysregulation. Finally, l-theanine powerfully blocked the development of both affective and cognitive abnormalities commonly associated with adolescent THC exposure, further demonstrating functional and long-term neuroprotective effects of l-theanine in the mesocorticolimbic system.SIGNIFICANCE STATEMENT With the increasing trend of cannabis legalization and consumption during adolescence, it is essential to expand knowledge on the potential effects of adolescent cannabis exposure on brain development and identify potential pharmacological strategies to minimize Δ-9-tetrahydrocannabinol (THC)-induced neuropathology. Previous evidence demonstrates that adolescent THC exposure induces long-lasting affective and cognitive abnormalities, mesocorticolimbic dysregulation, and schizophrenia-like molecular biomarkers that persist into adulthood. We demonstrate for the first time that l-theanine, an amino acid analog of l-glutamate and l-glutamine, is capable of preventing long-term THC side effects. l-Theanine prevented the development of THC-induced behavioral aberrations, blocked cortical downregulation of local GSK-3 (glycogen synthase kinase 3) and Akt signaling pathways, and normalized dysregulation of both PFC and VTA DAergic activity, demonstrating powerful and functional neuroprotective effects against THC-induced developmental neuropathology.

THC and CBD produce divergent effects on perception and panic behaviours via distinct cortical molecular pathways.

Clinical and pre-clinical evidence demonstrates divergent psychotropic effects of THC vs. CBD. While THC can induce perceptual distortions and anxiogenic effects, CBD displays antipsychotic and anxiolytic properties. A key brain region responsible for regulation of cognition and affect, the medial prefrontal cortex (PFC), is strongly modulated by cannabinoids, suggesting that these dissociable THC/CBD-dependent effects may involve functional and molecular interplay within the PFC. The primary aim of this study was to investigate potential interactions and molecular substrates involved in PFC-mediated effects of THC and CBD on differential cognitive and affective behavioural processing. Male Sprague Dawley rats received intra-PFC microinfusions of THC, CBD or their combination, and tested in the latent inhibition paradigm, spontaneous oddity discrimination test, elevated T-maze and open field. To identify local, drug-induced molecular modulation in the PFC, PFC samples were collected and processed with Western Blotting. Intra-PFC THC induced strong panic-like responses that were counteracted with CBD. In contrast, CBD did not affect panic-like behaviours but blocked formation of associative fear memories and impaired latent inhibition and oddity discrimination performance. Interestingly, these CBD effects were dependent upon 5-HT1A receptor transmission but not influenced by THC co-administration. Moreover, THC induced robust phosphorylation of ERK1/2 that was prevented by CBD, while CBD decreased phosphorylation of p70S6K, independently of THC. These results suggest that intra-PFC infusion of THC promotes panic-like behaviour associated with increased ERK1/2 phosphorylation. In contrast, CBD impairs perceptive functions and latent inhibition via activation of 5-HT1A receptors and reduced phosphorylation of p70S6K.

Cannabidiol Counteracts the Psychotropic Side-Effects of Δ-9-Tetrahydrocannabinol in the Ventral Hippocampus through Bidirectional Control of ERK1-2 Phosphorylation.

Evidence suggests that the phytocannabinoids Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) differentially regulate salience attribution and psychiatric risk. The ventral hippocampus (vHipp) relays emotional salience via control of dopamine (DA) neuronal activity states, which are dysregulated in psychosis and schizophrenia. Using in vivo electrophysiology in male Sprague Dawley rats, we demonstrate that intra-vHipp THC strongly increases ventral tegmental area (VTA) DA neuronal frequency and bursting rates, decreases GABA frequency, and amplifies VTA beta, gamma and ε oscillatory magnitudes via modulation of local extracellular signal-regulated kinase phosphorylation (pERK1-2). Remarkably, whereas intra-vHipp THC also potentiates salience attribution in morphine place-preference and fear conditioning assays, CBD coadministration reverses these changes by downregulating pERK1-2 signaling, as pharmacological reactivation of pERK1-2 blocked the inhibitory properties of CBD. These results identify vHipp pERK1-2 signaling as a critical neural nexus point mediating THC-induced affective disturbances and suggest a potential mechanism by which CBD may counteract the psychotomimetic and psychotropic side effects of THC.SIGNIFICANCE STATEMENT Strains of marijuana with high levels of delta-9-tetrahydrocannabinol (THC) and low levels of cannabidiol (CBD) have been shown to underlie neuropsychiatric risks associated with high-potency cannabis use. However, the mechanisms by which CBD mitigates the side effects of THC have not been identified. We demonstrate that THC induces cognitive and affective abnormalities resembling neuropsychiatric symptoms directly in the hippocampus, while dysregulating dopamine activity states and amplifying oscillatory frequencies in the ventral tegmental area via modulation of the extracellular signal-regulated kinase (ERK) signaling pathway. In contrast, CBD coadministration blocked THC-induced ERK phosphorylation, and prevented THC-induced behavioral and neural abnormalities. These findings identify a novel molecular mechanism that may account for how CBD functionally mitigates the neuropsychiatric side effects of THC.

Effects of Adolescent THC Exposure on the Prefrontal GABAergic System: Implications for Schizophrenia-Related Psychopathology.

Marijuana is the most commonly used drug of abuse among adolescents. Considerable clinical evidence supports the hypothesis that adolescent neurodevelopmental exposure to high levels of the principal psychoactive component in marijuana, -delta-9-tetrahydrocanabinol (THC), is associated with a high risk of developing psychiatric diseases, such as schizophrenia later in life. This marijuana-associated risk is believed to be related to increasing levels of THC found within commonly used marijuana strains. Adolescence is a highly vulnerable period for the development of the brain, where the inhibitory GABAergic system plays a pivotal role in the maturation of regulatory control mechanisms in the central nervous system (CNS). Specifically, adolescent neurodevelopment represents a critical period wherein regulatory connectivity between higher-order cortical regions and sub-cortical emotional processing circuits such as the mesolimbic dopamine (DA) system is established. Emerging preclinical evidence demonstrates that adolescent exposure to THC selectively targets schizophrenia-related molecular and neuropharmacological signaling pathways in both cortical and sub-cortical regions, including the prefrontal cortex (PFC) and mesolimbic DA pathway, comprising the ventral tegmental area (VTA) and nucleus accumbens (NAc). Prefrontal cortical GABAergic hypofunction is a key feature of schizophrenia-like neuropsychopathology. This GABAergic hypofunction may lead to the loss of control of the PFC to regulate proper sub-cortical DA neurotransmission, thereby leading to schizophrenia-like symptoms. This review summarizes preclinical evidence demonstrating that reduced prefrontal cortical GABAergic neurotransmission has a critical role in the sub-cortical DAergic dysregulation and schizophrenia-like behaviors observed following adolescent THC exposure.

Adolescent THC Exposure Causes Enduring Prefrontal Cortical Disruption of GABAergic Inhibition and Dysregulation of Sub-Cortical Dopamine Function.

Chronic adolescent marijuana use has been linked to the later development of psychiatric diseases such as schizophrenia. GABAergic hypofunction in the prefrontal cortex (PFC) is a cardinal pathological feature of schizophrenia and may be a mechanism by which the PFC loses its ability to regulate sub-cortical dopamine (DA) resulting in schizophrenia-like neuropsychopathology. In the present study, we exposed adolescent rats to Δ-9-tetra-hydrocannabinol (THC), the psychoactive component in marijuana. At adulthood, we characterized the functionality of PFC GABAergic neurotransmission and its regulation of sub-cortical DA function using molecular, behavioral and in-vivo electrophysiological analyses. Our findings revealed a persistent attenuation of PFC GABAergic function combined with a hyperactive neuronal state in PFC neurons and associated disruptions in cortical gamma oscillatory activity. These PFC abnormalities were accompanied by hyperactive DAergic neuronal activity in the ventral tegmental area (VTA) and behavioral and cognitive abnormalities similar to those observed in psychiatric disorders. Remarkably, these neuronal and behavioral effects were reversed by pharmacological activation of GABAA receptors in the PFC. Together, these results identify a mechanistic link between dysregulated frontal cortical GABAergic inhibition and sub-cortical DAergic dysregulation, characteristic of well-established neuropsychiatric endophenotypes.

Opiate exposure state controls dopamine D3 receptor and cdk5/calcineurin signaling in the basolateral amygdala during reward and withdrawal aversion memory formation.

The dopamine (DA) D3 receptor (D3R) is highly expressed in the basolateral nucleus of the amygdala (BLA), a neural region critical for processing opiate-related reward and withdrawal aversion-related memories. Functionally, D3R transmission is linked to downstream Cdk5 and calcineurin signaling, both of which regulate D3R activity states and play critical roles in memory-related synaptic plasticity. Previous evidence links D3R transmission to opiate-related memory processing, however little is known regarding how chronic opiate exposure may alter D3R-dependent memory mechanisms. Using conditioned place preference (CPP) and withdrawal aversion (conditioned place aversion; CPA) procedures in rats, combined with molecular analyses of BLA protein expression, we examined the effects of chronic opiate exposure on the functional role of intra-BLA D3R transmission during the acquisition of opiate reward or withdrawal aversion memories. Remarkably, we report that the state of opiate exposure during behavioural conditioning (opiate-naïve/non-dependent vs. chronically exposed and in withdrawal) controlled the functional role of intra-BLA D3R transmission during the acquisition of both opiate reward memories and withdrawal-aversion associative memories. Thus, whereas intra-BLA D3R blockade had no effect on opiate reward memory formation in the non-dependent state, blockade of intra-BLA D3R transmission prevented the formation of opiate reward and withdrawal aversion memory in the chronically exposed state. This switch in the functional role of D3R transmission corresponded to significant increases in Cdk5 phosphorylation and total expression levels of calcineurin, and a corresponding decrease in intra-BLA D3R expression. Inhibition of either intra-BLA Cdk5 or calcineurin reversed these effects, switching intra-BLA associative memory formation back to a D3R-independent mechanism.

Adolescent Cannabinoid Exposure Induces a Persistent Sub-Cortical Hyper-Dopaminergic State and Associated Molecular Adaptations in the Prefrontal Cortex.

Considerable evidence suggests that adolescent exposure to delta-9-tetrahydrocanabinol (THC), the psychoactive component in marijuana, increases the risk of developing schizophrenia-related symptoms in early adulthood. In the present study, we used a combination of behavioral and molecular analyses with in vivo neuronal electrophysiology to compare the long-term effects of adolescent versus adulthood THC exposure in rats. We report that adolescent, but not adult, THC exposure induces long-term neuropsychiatric-like phenotypes similar to those observed in clinical populations. Thus, adolescent THC exposure induced behavioral abnormalities resembling positive and negative schizophrenia-related endophenotypes and a state of neuronal hyperactivity in the mesocorticolimbic dopamine (DA) pathway. Furthermore, we observed profound alterations in several prefrontal cortical molecular pathways consistent with sub-cortical DAergic dysregulation. Our findings demonstrate a profound dissociation in relative risk profiles for adolescent versus adulthood exposure to THC in terms of neuronal, behavioral, and molecular markers resembling neuropsychiatric pathology.

What Can Rats Tell Us about Adolescent Cannabis Exposure? Insights from Preclinical Research.

Marijuana is the most widely used drug of abuse among adolescents. Adolescence is a vulnerable period for brain development, during which time various neurotransmitter systems such as the glutamatergic, GABAergic, dopaminergic, and endocannabinoid systems undergo extensive reorganization to support the maturation of the central nervous system (CNS). ▵-9-tetrahydrocannabinol (THC), the psychoactive component of marijuana, acts as a partial agonist of CB1 cannabinoid receptors (CB1Rs). CB1Rs are abundant in the CNS and are central components of the neurodevelopmental changes that occur during adolescence. Thus, overactivation of CB1Rs by cannabinoid exposure during adolescence has the ability to dramatically alter brain maturation, leading to persistent and enduring changes in adult cerebral function. Increasing preclinical evidence lends support to clinical evidence suggesting that chronic adolescent marijuana exposure may be associated with a higher risk for neuropsychiatric diseases, including schizophrenia. In this review, we present a broad overview of current neurobiological evidence regarding the long-term consequences of adolescent cannabinoid exposure on adult neuropsychiatric-like disorders.

Cannabidiol Counteracts Amphetamine-Induced Neuronal and Behavioral Sensitization of the Mesolimbic Dopamine Pathway through a Novel mTOR/p70S6 Kinase Signaling Pathway.

UNLABELLED: Schizophrenia-related psychosis is associated with disturbances in mesolimbic dopamine (DA) transmission, characterized by hyperdopaminergic activity in the mesolimbic pathway. Currently, the only clinically effective treatment for schizophrenia involves the use of antipsychotic medications that block DA receptor transmission. However, these medications produce serious side effects leading to poor compliance and treatment outcomes. Emerging evidence points to the involvement of a specific phytochemical component of marijuana called cannabidiol (CBD), which possesses promising therapeutic properties for the treatment of schizophrenia-related psychoses. However, the neuronal and molecular mechanisms through which CBD may exert these effects are entirely unknown. We used amphetamine (AMPH)-induced sensitization and sensorimotor gating in rats, two preclinical procedures relevant to schizophrenia-related psychopathology, combined with in vivo single-unit neuronal electrophysiology recordings in the ventral tegmental area, and molecular analyses to characterize the actions of CBD directly in the nucleus accumbens shell (NASh), a brain region that is the current target of most effective antipsychotics. We demonstrate that Intra-NASh CBD attenuates AMPH-induced sensitization, both in terms of DAergic neuronal activity measured in the ventral tegmental area and psychotomimetic behavioral analyses. We further report that CBD controls downstream phosphorylation of the mTOR/p70S6 kinase signaling pathways directly within the NASh. Our findings demonstrate a novel mechanism for the putative antipsychotic-like properties of CBD in the mesolimbic circuitry. We identify the molecular signaling pathways through which CBD may functionally reduce schizophrenia-like neuropsychopathology. SIGNIFICANCE STATEMENT: The cannabis-derived phytochemical, cannabidiol (CBD), has been shown to have pharmacotherapeutic efficacy for the treatment of schizophrenia. However, the mechanisms by which CBD may produce antipsychotic effects are entirely unknown. Using preclinical behavioral procedures combined with molecular analyses and in vivo neuronal electrophysiology, our findings identify a functional role for the nucleus accumbens as a critical brain region whereby CBD can produce effects similar to antipsychotic medications by triggering molecular signaling pathways associated with the effects of classic antipsychotic medications. Specifically, we report that CBD can attenuate both behavioral and dopaminergic neuronal correlates of mesolimbic dopaminergic sensitization, via a direct interaction with mTOR/p70S6 kinase signaling within the mesolimbic pathway.

NMDA receptor blockade in the prelimbic cortex activates the mesolimbic system and dopamine-dependent opiate reward signaling.

RATIONALE: N-Methyl-D-aspartate (NMDA) receptors in the medial prefrontal cortex (mPFC) are involved in opiate reward processing and modulate sub-cortical dopamine (DA) activity. NMDA receptor blockade in the prelimbic (PLC) division of the mPFC strongly potentiates the rewarding behavioural properties of normally sub-reward threshold doses of opiates. However, the possible functional interactions between cortical NMDA and sub-cortical DAergic motivational neural pathways underlying these effects are not understood. OBJECTIVE: This study examines how NMDA receptor modulation in the PLC influences opiate reward processing via interactions with sub-cortical DAergic transmission. We further examined whether direct intra-PLC NMDA receptor modulation may activate DA-dependent opiate reward signaling via interactions with the ventral tegmental area (VTA). METHODS: Using an unbiased place conditioning procedure (CPP) in rats, we performed bilateral intra-PLC microinfusions of the competitive NMDA receptor antagonist, (2R)-amino-5-phosphonovaleric acid (AP-5), prior to behavioural morphine place conditioning and challenged the rewarding effects of morphine with DA receptor blockade. We next examined the effects of intra-PLC NMDA receptor blockade on the spontaneous activity patterns of presumptive VTA DA or GABAergic neurons, using single-unit, extracellular in vivo neuronal recordings. RESULTS: We show that intra-PLC NMDA receptor blockade strongly activates sub-cortical DA neurons within the VTA while inhibiting presumptive non-DA GABAergic neurons. Behaviourally, NMDA receptor blockade activates a DA-dependent opiate reward system, as pharmacological blockade of DA transmission blocked morphine reward only in the presence of intra-PLC NMDA receptor antagonism. CONCLUSIONS: These findings demonstrate a cortical NMDA-mediated mechanism controlling mesolimbic DAergic modulation of opiate reward processing.

The dopamine D2 receptor regulates Akt and GSK-3 via Dvl-3.

The dopamine D2 receptor (D2DR) regulates Akt and may also target the Wnt pathway, two signalling cascades that inhibit glycogen synthase kinase-3 (GSK-3). This study examined whether the Wnt pathway is regulated by D2DR and the role of Akt and dishevelled-3 (Dvl-3) in regulating GSK-3 and the transcription factor ß-catenin in the rat brain. Western blotting showed that subchronic treatment of raclopride (D2DR antagonist) increase phosphorylated Akt, Dvl-3, GSK-3, phosphorylated GSK-3 and ß-catenin, whereas subchronic treatment of quinpirole (D2DR agonist) induced the opposite response. Co-immunopreciptations revealed an association between GSK-3 and the D2DR complex that was altered following raclopride and quinpirole, albeit in opposite directions. SCH23390 (D1DR antagonist) and nafadotride (D3DR antagonist) were also used to determine if the response was specific to the D2DR. Neither subchronic treatment affected Dvl-3, GSK-3, Akt nor ß-catenin protein levels, although nafadotride altered the phosphorylation state of Akt and GSK-3. In addition, in-vitro experiments were conducted to manipulate Akt and Dvl-3 activity in SH-SY5Y cells to elucidate how the pattern of change observed following manipulation of D2DR developed. Results indicate that Akt affects the phosphorylation state of GSK-3 but has no effect on ß-catenin levels. However, altering Dvl-3 levels resulted in changes in Akt and the Wnt pathway similar to what was observed following raclopride or quinpirole treatment. Collectively, the data suggests that the D2DR very specifically regulates Wnt and Akt signalling via Dvl-3.

Regulation of Akt and Wnt signaling by the group II metabotropic glutamate receptor antagonist LY341495 and agonist LY379268.

Metabotropic glutamate receptors 2/3 (mGlu(2/3)) have been implicated in schizophrenia and as a novel treatment target for schizophrenia. The current study examined whether mGlu(2/3) regulates Akt (protein kinase B) and Wnt (Wingless/Int-1) signaling, two cascades associated with schizophrenia and modified by antipsychotics. Western blotting revealed increases in phosphorylated Akt (pAkt) and phosphorylated glycogen synthase kinase-3 (pGSK-3) following acute and repeated treatment of LY379268 (mGlu(2/3) agonist), whereas increases in dishevelled-2 (Dvl-2), dishevelled-3 (Dvl-3), GSK-3 and ß-catenin were only observed following repeated treatment. LY341495 (mGlu(2/3) antagonist) induced the opposite response compared with LY379268. Co-immunoprecipitation experiments showed an association between the mGlu(2/3) complex and Dvl-2 providing a possible mechanism to explain how the mGlu(2/3) can mediate changes in Wnt signaling. However, there was no association between the mGlu(2/3) complex and Akt suggesting that changes in Akt signaling following LY341495 and LY379268 treatments may not be directly mediated by the mGlu(2/3) . Finally, an increase in locomotor activity induced by LY341495 treatment correlated with increased pAkt and pGSK-3 levels and was attenuated by the administration of the GSK-3 inhibitor, SB216763. Overall, the results suggest that mGlu(2/3) regulates Akt and Wnt signaling and LY379268 treatment has overlapping effects with D(2) dopamine receptor antagonists (antipsychotic drugs).

PKC inhibition increases gap junction intercellular communication and cell adhesion in human neuroblastoma.

Gap junction intercellular communication and cell-cell adhesion are essential for maintaining a normal cellular phenotype, including the control of growth and proliferation. Loss of either cell-cell adhesion or communication is common in cancers, while restoration of function is associated with tumor suppression. Protein kinase C (PKC) isozymes regulate a broad spectrum of cellular functions including growth and proliferation, and their overexpression has been correlated with carcinogenesis. Consequently, PKC inhibitors are currently undergoing clinical trials as an anti-cancer agents although the precise cellular alterations induced by PKC inhibitors remain to be elucidated. In the current study, the effects of PKC inhibitors on cell interactions were investigated using human neuroblastoma (IMR32, SKNMC, and SHSY-5Y) cell lines. An analysis of intercellular communication revealed an increase in gap junctional coupling with PKC inhibition. The observed increase in coupling was not associated with a change in Connexin 43 distribution or an alteration of phosphorylation status of the protein. There was also an increase in cell-cell adhesion with PKC inhibitor treatment as indicated by a cell aggregation assay. Therefore, the growth suppressive abilities of PKC inhibition on tumors may be due to the cancer suppressive effects of increased gap junction intercellular communication and cell-cell adhesion.

Prefrontal cortical D1 dopamine receptors modulate subcortical D2 dopamine receptor-mediated stress responsiveness.

Increased responsiveness to stress plays an important role in the manifestation of schizophrenia symptoms. Evidence indicates that the prefrontal cortex (PFC), and dopamine neurotransmission in the PFC in particular, is involved in the modulation of stress responsiveness. Decreased dopaminergic activity and loss of dopamine fibres have been reported in PFC in schizophrenia patients. Consequently, it was hypothesized that depletion of dopamine in PFC may facilitate increased stress responsiveness. Adult Sprague-Dawley rats received injections of 6-hydroxydopamine or saline bilaterally into the medial PFC (mPFC) following desipramine pretreatment to selectively deplete dopaminergic fibres. Following a 3-wk recovery period, the lesioned and control rats received injections of a D1 or D2 dopamine receptor agonist or vehicle into the mPFC and were immediately subjected to forced swimming as a stressor. Results showed that frequency of locomotion and rearing, behavioural measures indicative of increased dopaminergic activity in the nucleus accumbens (NAc), were significantly increased following stress in prefrontal cortical dopamine-depleted rats. This effect was significantly ameliorated by infusions of a D1 dopamine receptor agonist directly into the mPFC in a dose-dependent manner but not by infusion of a D2 dopamine receptor agonist. In addition, stress-induced behavioural changes in prefrontal cortical dopamine-depleted rats were significantly reduced following selective discrete infusions of a D2 dopamine receptor antagonist into the NAc shell. The results suggest that dopaminergic transmission via D1 receptors in the mPFC modulates D2 dopamine receptor-mediated stress responsiveness in the NAc, a feature that may be disrupted in schizophrenia patients.

Dose-dependent differential upregulation of CCN1/Cyr61 and CCN3/NOV by the gap junction protein Connexin43 in glioma cells.

Gap junctions form channels that allow exchange of materials between cells and are composed of transmembrane protein subunits called connexins. While connexins are believed to mediate cellular signaling by permitting intercellular communication to occur, there is also increasing evidence that suggest connexins may mediate growth control via a junction-independent mechanism. Connexin43 (Cx43) is the most abundant gap junction protein found in astrocytes, and gliomas exhibit reduced Cx43 expression. We have previously observed that restoration of Cx43 levels in glioma cells led to increased expression of CCN3 (NOV) proteins. We now report that overexpression of Cx43 in C6-glioma cells (C6-Cx43) also upregulates the expression of CCN1 (Cyr61). Both CCN1 and CCN3 belong to the Cyr61/Connective tissue growth factor/Nephroblastoma-overexpressed (CCN) family of secretory proteins. The CCN proteins are tightly associated with the extracellular matrix and have important roles in cell proliferation and migration. CCN1 promotes growth in glioma cells, as shown by the increased proliferation rate of CCN1-overexpressing C6 cells. In addition to its effect on cell growth, CCN1 also increased the motility of glioma cells in the presence of extracellular substrates such as fibronectin. Gliomas expressing high levels of Cx43 preferentially upregulated CCN3 which resulted in reduced growth rate. CCN3 could also be observed in Cx43 gap junction plaques in confluent C6-Cx43H culture at the stationary phase of their growth. Our results suggest that the dissimilar growth characteristics between high and low Cx43 expressors may be due to differential regulation of CCN3 by varying levels of Cx43.