P11 deficiency increases stress reactivity along with HPA axis and autonomic hyperresponsiveness.

Patients suffering from mood disorders and anxiety commonly exhibit hypothalamic-pituitary-adrenocortical (HPA) axis and autonomic hyperresponsiveness. A wealth of data using preclinical animal models and human patient samples indicate that p11 deficiency is implicated in depression-like phenotypes. In the present study, we used p11-deficient (p11KO) mice to study potential roles of p11 in stress responsiveness. We measured stress response using behavioral, endocrine, and physiological readouts across early postnatal and adult life. Our data show that p11KO pups respond more strongly to maternal separation than wild-type pups, even though their mothers show no deficits in maternal behavior. Adult p11KO mice display hyperactivity of the HPA axis, which is paralleled by depression- and anxiety-like behaviors. p11 was found to be highly enriched in vasopressinergic cells of the paraventricular nucleus and regulates HPA hyperactivity in a V1B receptor-dependent manner. Moreover, p11KO mice display sympathetic-adrenal-medullary (SAM) axis hyperactivity, with elevated adrenal norepinephrine and epinephrine levels. Using conditional p11KO mice, we demonstrate that this SAM hyperactivity is partially regulated by the loss of p11 in serotonergic neurons of the raphe nuclei. Telemetric electrocardiogram measurements show delayed heart rate recovery in p11KO mice in response to novelty exposure and during expression of fear following auditory trace fear conditioning. Furthermore, p11KO mice have elevated basal heart rate in fear conditioning tests indicating increased autonomic responsiveness. This set of experiments provide strong and versatile evidence that p11 deficiency leads to HPA and SAM axes hyperresponsiveness along with increased stress reactivity.

The Effect of Early Life Stress on Emotional Behaviors in GPR37KO Mice.

GPR37 is an orphan G-protein-coupled receptor, a substrate of parkin which is linked to Parkinson's disease (PD) and affective disorders. In this study, we sought to address the effects of early life stress (ELS) by employing the paradigm of limited nesting material on emotional behaviors in adult GPR37 knockout (KO) mice. Our results showed that, while there was an adverse effect of ELS on various domains of emotional behaviors in wild type (WT) mice in a sex specific manner (anxiety in females, depression and context-dependent fear memory in males), GPR37KO mice subjected to ELS exhibited less deteriorated emotional behaviors. GPR37KO female mice under ELS conditions displayed reduced anxiety compared to WT mice. This was paralleled by lower plasma corticosterone in GPR37KO females and a lower increase in P-T286-CaMKII by ELS in the amygdala. GPR37KO male mice, under ELS conditions, showed better retention of hippocampal-dependent emotional processing in the passive avoidance behavioral task. GPR37KO male mice showed increased immobility in the forced swim task and increased P-T286-CaMKII in the ventral hippocampus under baseline conditions. Taken together, our data showed overall long-term effects of ELS-deleterious or beneficial depending on the genotype, sex of the mice and the emotional context.

Effects of a Novel Psychomotor Stabilizer, IRL790, on Biochemical Measures of Synaptic Markers and Neurotransmission.

The novel small-molecule psychomotor stabilizer, IRL790, is currently in clinical trial for treatment of levodopa-induced dyskinesia and psychosis in patients with Parkinson disease. Here, we used naïve mice to investigate the effects of acute systemic administration of IRL790 on protein levels and phosphorylation states of proteins relevant for synaptic plasticity and transmission. IRL790 increased pro-brain-derived neurotrophic factor protein levels and phosphorylation at Ser1303 of the N-methyl-D-aspartate (NMDA) subtype 2B glutamate receptor (NR2B) in prefrontal cortex. IRL790 also increased the phosphorylation states at Ser19, Ser31, and Ser40, respectively, of tyrosine hydroxylase in striatum. IRL790 reduced protein levels of the NR2B receptor in striatum but not in prefrontal cortex. Taken together, we report that systemically administered IRL790 rapidly elicits changes in protein level and phosphorylation state of proteins associated with a beneficial effect on synaptic markers and neurotransmission. SIGNIFICANCE STATEMENT: The novel small-molecule psychomotor stabilizer, IRL790, is currently in clinical trial for treatment of levodopa-induced dyskinesia and psychosis in patients with Parkinson disease. In this study, we report that systemically administered IRL790 rapidly elicits changes in protein level and phosphorylation state of proteins associated with a beneficial effect on synaptic markers and neurotransmission.

Epigenetics and energetics in ventral hippocampus mediate rapid antidepressant action: Implications for treatment resistance.

Although regulation of energy metabolism has been linked with multiple disorders, its role in depression and responsiveness to antidepressants is less known. We found that an epigenetic and energetic agent, acetyl-l-carnitine (LAC, oral administration), rapidly rescued the depressive- and central and systemic metabolic-like phenotype of LAC-deficient Flinders Sensitive Line rats (FSL). After acute stress during LAC treatment, a subset of FSL continued to respond to LAC (rFSL), whereas the other subset did not (nrFSL). RNA sequencing of the ventral dentate gyrus, a mood-regulatory region, identified metabolic factors as key markers predisposing to depression (insulin receptors Insr, glucose transporters Glut-4 and Glut-12, and the regulator of appetite Cartpt) and to LAC responsiveness (leptin receptors Lepr, metabotropic glutamate receptors-2 mGlu2, neuropeptide-Y NPY, and mineralocorticoid receptors MR). Furthermore, we found that stress-induced treatment resistance in nrFSL shows a new gene profile, including the metabolic regulator factors elongation of long chain fatty acids 7 (Elovl7) and cytochrome B5 reductase 2 (Cyb5r2) and the synaptic regulator NPAS4. Finally, while improving central energy regulation and exerting rapid antidepressant-like effects, LAC corrected a systemic hyperinsulinemia and hyperglicemia in rFSL and failed to do that in nrFSL. These findings establish CNS energy regulation as a factor to be considered for the development of better therapeutics. Agents such as LAC that regulate metabolic factors and reduce glutamate overflow could rapidly ameliorate depression and could also be considered for treatment of insulin resistance in depressed subjects. The approach here serves as a model for identifying markers and underlying mechanisms of predisposition to diseases and treatment responsiveness that may be useful in translation to human behavior and psychopathology.

Lurasidone and fluoxetine reduce novelty-induced hypophagia and NMDA receptor subunit and PSD-95 expression in mouse brain.

Lurasidone, a novel second-generation antipsychotic agent, exerts antidepressant actions in patients suffering from bipolar type I disorder. Lurasidone acts as a high affinity antagonist at multiple monoamine receptors, particularly 5-HT2A, 5-HT7, D2 and α2 receptors, and as a partial agonist at 5-HT1A receptors. Accumulating evidence indicates therapeutic actions by monoaminergic antidepressants are mediated via alterations of glutamate receptor-mediated neurotransmission. Here, we used mice and investigated the effects of chronic oral administration of vehicle, lurasidone (3 or 10mg/kg) or fluoxetine (20mg/kg) in the novelty induced hypophagia test, a behavioral test sensitive to chronic antidepressant treatment. We subsequently performed biochemical analyses on NMDA receptor subunits and associated proteins. Both lurasidone and fluoxetine reduced the latency to feed in the novelty-induced hypophagia test. Western blotting experiments showed that both lurasidone and fluoxetine decreased the total levels of NR1, NR2A and NR2B subunits of NMDA receptors and PSD-95 (PostSynaptic Density-95) in hippocampus and prefrontal cortex. Taken together, these data indicate that antidepressant/anxiolytic-like effects of lurasidone, as well as fluoxetine, could involve reduced NMDA receptor-mediated signal transduction, particularly in pathways regulated by PSD-95, in hippocampus and prefrontal cortex.

Maternal separation impairs long term-potentiation in CA1-CA3 synapses and hippocampal-dependent memory in old rats.

Exposure to chronic stress during the neonatal period is known to induce permanent long-term changes in the central nervous system and hipothalamic-pituitary-adrenal axis reactivity that are associated with increased levels of depression, anxiety, and cognitive impairments. In rodents, a validated model of early life stress is the maternal separation (MS) paradigm, which has been shown to have long-term consequences for the pups that span to adulthood. We hypothesized that the early life stress-associated effects could be exacerbated with aging, because it is often accompanied by cognitive decline. Using a MS model in which rat pups were separated from their mothers for 3 hours daily, during postnatal days 2-14, we evaluated the long-term functional consequences to aged animals (70-week-old), by measuring synaptic plasticity and cognitive performance. The baseline behavioral deficits of aged control rats were further exacerbated in MS animals, indicating that early-life stress induces sustained changes in anxiety-like behavior and hippocampal-dependent memory that are maintained much later in life. We then investigated whether these differences are linked to impaired function of hippocampal neurons by recording hippocampal long-term potentiation from Schaffer collaterals/CA1 synapses. The magnitude of the hippocampal long-term potentiation induced by high-frequency stimulation was significantly lower in aged MS animals than in age-matched controls. These results substantiate the hypothesis that the neuronal and endocrine alterations induced by early-life stress are long lasting, and are able to exacerbate the mild age-associated deficits.

Regulation of TrkB receptor translocation to lipid rafts by adenosine A(2A) receptors and its functional implications for BDNF-induced regulation of synaptic plasticity.

Brain-derived neurotrophic factor (BDNF) signalling is critical for neuronal development and transmission. Recruitment of TrkB receptors to lipid rafts has been shown to be necessary for the activation of specific signalling pathways and modulation of neurotransmitter release by BDNF. Since TrkB receptors are known to be modulated by adenosine A(2A) receptor activation, we hypothesized that activation of A(2A) receptors could influence TrkB receptor localization among different membrane microdomains. We found that adenosine A(2A) receptor agonists increased the levels of TrkB receptors in the lipid raft fraction of cortical membranes and potentiated BDNF-induced augmentation of phosphorylated TrkB levels in lipid rafts. Blockade of the clathrin-mediated endocytosis with monodansyl cadaverine (100 µM) did not modify the effects of the A(2A) receptor agonists, but significantly impaired BDNF effects on TrkB recruitment to lipid rafts. The effect of A(2A) receptor activation in TrkB localization was mimicked by 5 µM forskolin, an adenylyl cyclase activator. Also, it was blocked by the PKA inhibitors RpcAMPs and PKI-(14-22) and by the Src-family kinase inhibitor PP2. Moreover, removal of endogenous adenosine or disruption of lipid rafts reduced BDNF stimulatory effects on glutamate release from cortical synaptosomes. Lipid raft integrity was also required for the effects of BDNF upon hippocampal long-term potentiation at CA1 synapses. Our data demonstrate, for the first time, a BDNF-independent recruitment of TrkB receptors to lipid rafts, induced by the activation of adenosine A(2A) receptors, with functional consequences for TrkB phosphorylation and BDNF-induced modulation of neurotransmitter release and hippocampal plasticity.

Enhancement of LTP in aged rats is dependent on endogenous BDNF.

Long-term potentiation (LTP), considered the neurophysiological basis for learning and memory, is facilitated by brain-derived neurotrophic factor (BDNF), an action more evident when LTP is evoked by weak θ-burst stimuli and dependent on co-activation of adenosine A(2A) receptors (A(2A)R), which are more expressed in aged rats. As θ-burst stimuli also favor LTP in aged animals, we hypothesized that enhanced LTP in aging could be related to changes in neuromodulation by BDNF. The magnitude of CA1 LTP induced by a weak θ-burst stimuli delivered to the Schaffer collaterals was significantly higher in hippocampal slices taken from 36 to 38 and from 70 to 80-week-old rats, when compared with LTP magnitude in slices from 4 or 10 to 15-week-old rats; this enhancement does not impact in cognitive improvement as aged rats revealed an impairment on hippocampal-dependent learning and memory performance, as assessed by the Morris water maze tests. The scavenger for BDNF, TrkB-Fc, and the inhibitor of Trk phosphorylation, K252a, attenuated LTP in slices from 70 to 80-week-old rats, but not from 10 to 15-week-old rats. When exogenously added, BDNF significantly increased LTP in slices from 4 and 10 to 15-week-old rats, but did not further increased LTP in 36 to 38 or 70 to 80-week-old rats. The effects of exogenous BDNF on LTP were prevented by the A(2A)R antagonist, SCH58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine). These results indicate that the higher LTP magnitude observed upon aging, which does not translate into improved spatial memory performance, is a consequence of an increase in the tonic action of endogenous BDNF.

Regulation of hippocampal cannabinoid CB1 receptor actions by adenosine A1 receptors and chronic caffeine administration: implications for the effects of Δ9-tetrahydrocannabinol on spatial memory.

The cannabinoid CB(1) receptor-mediated modulation of γ-aminobutyric acid (GABA) release from inhibitory interneurons is important for the integrity of hippocampal-dependent spatial memory. Although adenosine A(1) receptors have a central role in fine-tuning excitatory transmission in the hippocampus, A(1) receptors localized in GABAergic cells do not directly influence GABA release. CB(1) and A(1) receptors are the main targets for the effects of two of the most heavily consumed psychoactive substances worldwide: Δ(9)-tetrahydrocannabinol (THC, a CB(1) receptor agonist) and caffeine (an adenosine receptor antagonist). We first tested the hypothesis that an A(1)-CB(1) interaction influences GABA and glutamate release in the hippocampus. We found that A(1) receptor activation attenuated the CB(1)-mediated inhibition of GABA and glutamate release and this interaction was manifested at the level of G-protein activation. Using in vivo and in vitro approaches, we then investigated the functional implications of the adenosine-cannabinoid interplay that may arise following chronic caffeine consumption. Chronic administration of caffeine in mice (intraperitoneally, 3 mg/kg/day, for 15 days, >12 h before trials) led to an A(1)-mediated enhancement of the CB(1)-dependent acute disruptive effects of THC on a short-term spatial memory task, despite inducing a reduction in cortical and hippocampal CB(1) receptor number and an attenuation of CB(1) coupling with G protein. A(1) receptor levels were increased following chronic caffeine administration. This study shows that A(1) receptors exert a negative modulatory effect on CB(1)-mediated inhibition of GABA and glutamate release, and provides the first evidence of chronic caffeine-induced alterations on the cannabinoid system in the cortex and hippocampus, with functional implications in spatial memory.

Activation of adenosine A2A receptors induces TrkB translocation and increases BDNF-mediated phospho-TrkB localization in lipid rafts: implications for neuromodulation.

Brain-derived neurotrophic factor (BDNF) signaling is critical for neuronal development and transmission. Recruitment of TrkB receptors to lipid rafts has been shown to be necessary for the activation of specific signaling pathways and modulation of neurotransmitter release by BDNF. Since TrkB receptors are known to be modulated by adenosine A(2A) receptor activation, we hypothesized that activation of A(2A) receptors could influence TrkB receptor localization among different membrane microdomains. We found that adenosine A(2A) receptor agonists increased the levels of TrkB receptors in the lipid raft fraction of cortical membranes and potentiated BDNF-induced augmentation of phosphorylated TrkB levels in lipid rafts. Blockade of the clathrin-mediated endocytosis with monodansylcadaverine (100 microm) did not modify the effects of the A(2A) receptor agonists but significantly impaired BDNF effects on TrkB recruitment to lipid rafts. The effect of A(2A) receptor activation in TrkB localization was mimicked by 5 microm forskolin, an adenylyl cyclase activator. Also, it was blocked by the PKA inhibitors Rp-cAMPs and PKI-(14-22), and by the Src-family kinase inhibitor PP2. Moreover, removal of endogenous adenosine or disruption of lipid rafts reduced BDNF stimulatory effects on glutamate release from cortical synaptosomes. Lipid raft integrity was also required for the effects of BDNF on hippocampal long-term potentiation at CA1 synapses. Our data demonstrate, for the first time, a BDNF-independent recruitment of TrkB receptors to lipid rafts induced by activation of adenosine A(2A) receptors, with functional consequences for TrkB phosphorylation and BDNF-induced modulation of neurotransmitter release and hippocampal plasticity.