Adenosine A2A receptors control generalization of contextual fear in rats.

Fear learning is essential to survival, but traumatic events may lead to abnormal fear consolidation and overgeneralization, triggering fear responses in safe environments, as occurs in post-traumatic stress disorder (PTSD). Adenosine A2A receptors (A2AR) control emotional memory and fear conditioning, but it is not known if they affect the consolidation and generalization of fear, which was now investigated. We now report that A2AR blockade through systemic administration of the A2AR antagonist SCH58261 immediately after contextual fear conditioning (within the consolidation window), accelerated fear generalization. Conversely, A2AR activation with CGS21680 decreased fear generalization. Ex vivo electrophysiological recordings of field excitatory post-synaptic potentials (fEPSPs) in CA3-CA1 synapses and of population spikes in the lateral amygdala (LA), showed that the effect of SCH58261 is associated with a reversion of fear conditioning-induced decrease of long-term potentiation (LTP) in the dorsal hippocampus (DH) and with increased amplitude of LA LTP in conditioned animals. These data suggest that A2AR are engaged during contextual fear consolidation, controlling long-term potentiation mechanisms in both DH and LA during fear consolidation, impacting on fear generalization; this supports targeting A2AR during fear consolidation to control aberrant fear processing in PTSD and other fear-related disorders.

An optimized spectrophotometric assay reveals increased activity of enzymes involved in 2-arachidonoyl glycerol turnover in the cerebral cortex of a rat model of Alzheimer's disease.

The endocannabinoid system is implicated in a plethora of neuropsychiatric disorders. However, it is technically challenging to assess the turnover of 2-arachidonoyl glycerol (2-AG), the principal endocannabinoid molecule in the brain. Two recent studies showed that diacylglycerol lipase α (DAGLα), an enzyme chiefly responsible for the cerebral production of 2-AG, also accepts the surrogate chromogenic substrate 4-nitrophenyl butyrate (4-NPB). Here, we aimed to optimize this spectrophotometric assay for ex vivo brain tissue, in particular, rat cerebrocortical homogenates, to measure the activity of the major enzymes responsible for the production and degradation of 2-AG. The initial velocity of 4-NPB hydrolysis was dependent on protein, substrate, and Ca2+ concentrations, and was sensitive to the non-selective serine hydrolase inhibitor, methoxy arachidonyl fluorophosphonate, the DAGLα inhibitors, OMDM188, tetrahydrolipstatin, and RHC80267, as well as the monoacylglycerol lipase (MAGL) inhibitor, JZL184, respectively. Next, we tested the usefulness of this assay in ex vivo brain tissue of rat models of human health conditions known to affect cerebrocortical 2-AG production, i.e. pathological stress and sporadic Alzheimer's disease (AD). In rats submitted to chronic restraint stress, cortical CB1 R density was significantly decreased, as assessed with radioligand binding. Nevertheless, 4-NPB hydrolysis remained at control levels. However, in rats 4 weeks after intracerebroventricular injection with streptozotocin - an established model of sporadic AD -, both CB1 R levels and 4-NPB hydrolysis and its DAGL- and MAGL-dependent fractions were significantly increased. Altogether, we optimized a simple complementary ex vivo technique for the quantification of DAGL and MAGL activity in brain samples.

The physiological effects of caffeine on synaptic transmission and plasticity in the mouse hippocampus selectively depend on adenosine A1 and A2A receptors.

Caffeine is the most consumed psychoactive drug worldwide and its intake in moderate amounts prevents neurodegenerative disorders. However, the molecular targets of caffeine to modulate activity in brain circuits are ill-defined. By electrophysiologically recording synaptic transmission and plasticity in Schaffer fibers-CA1 pyramid synapses of mouse hippocampal slices, we characterized the impact of caffeine using a concentration reached in the brain parenchyma upon moderate caffeine consumption. Caffeine (50 µM) facilitated synaptic transmission by 40%, while decreasing paired-pulse facilitation, and also decreased by 35% the amplitude of long-term potentiation (LTP). Clearance of extracellular adenosine with adenosine deaminase (2 U/mL) blunted all the effects of caffeine on synaptic transmission and plasticity. The A1R antagonist DPCPX (100 nM) only eliminated caffeine-induced facilitation of synaptic transmission while not affecting caffeine-induced depression of LTP; conversely, the genetic (using A2AR knockout mice) or the pharmacological blockade (with SCH58261, 50 nM) of A2AR eliminated the effect of caffeine on LTP while not affecting caffeine-induced facilitation of synaptic transmission. Finally, blockade of GABAA or of ryanodine receptors with bicuculline (10 µM) or dantrolene (10 µM), respectively, did not affect the ability of caffeine to alter synaptic transmission or plasticity. These results show that the effects of caffeine on synaptic transmission and plasticity in the hippocampus are selectively mediated by antagonizing adenosine receptors, where A1R are responsible for the impact of caffeine on synaptic transmission and A2AR regulate the impact of caffeine on LTP.

Blockade of adenosine A2A receptors recovers early deficits of memory and plasticity in the triple transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) begins with a deficit of synaptic function and adenosine A2A receptors (A2AR) are mostly located in synapses controlling synaptic plasticity. The over-activation of adenosine A2A receptors (A2AR) causes memory deficits and the blockade of A2AR prevents memory damage in AD models. We now enquired if this prophylactic role of A2AR might be extended to a therapeutic potential. We used the triple transgenic model of AD (3xTg-AD) and defined that the onset of memory dysfunction occurred at 4 months of age in the absence of locomotor or emotional alterations. At the onset of memory deficits, 3xTg mice displayed a decreased density of markers of excitatory synapses (10.6 ±â€¯3.8% decrease of vGluT1) without neuronal or glial overt damage and an increase of synaptic A2AR in the hippocampus (130 ±â€¯22%). After the onset of memory deficits in 3xTg-AD mice, a three weeks treatment with the selective A2AR antagonist normalized the up-regulation of hippocampal A2AR and restored hippocampal-dependent reference memory, as well as the decrease of hippocampal synaptic plasticity (60.0 ±â€¯3.7% decrease of long-term potentiation amplitude) and the decrease of global (syntaxin-I) and glutamatergic synaptic markers (vGluT1). These findings show a therapeutic-like ability of A2AR antagonists to recover synaptic and memory dysfunction in early AD.

Age-Related Changes in the Synaptic Density of Amyloid-ß Protein Precursor and Secretases in the Human Cerebral Cortex.

Amyloid-ß protein precursor (AßPP) is involved in synaptic formation and function. In the human cingulate cortex, AßPP was preferentially located in the presynaptic active zone as in rodents, indicating a preserved subsynaptic AßPP distribution across species and brain regions. Synaptic AßPP immunoreactivity was decreased with aging in cortical samples collected from autopsies of males (20-80 years), whereas the synaptic levels of α-secretase (ADAM10) and ß-secretase (BACE1) did not significantly change. Decreased AßPP levels may be related to lower allostasis of synapses in the aged brain and their greater susceptibility to dysfunction characteristic of the onset of neurodegenerative disorders.

The Association of Amyloid-ß Protein Precursor With α- and ß-Secretases in Mouse Cerebral Cortex Synapses Is Altered in Early Alzheimer's Disease.

Amyloid-ß peptides (Aß), the proposed triggers of synaptic dysfunction in early Alzheimer's disease (AD), derive from the endoproteolytic cleavage of amyloid-ß precursor protein (APP) by ß-secretases (BACE1), whereas APP cleavage by α-secretases (ADAM10) abrogates Aß formation. We now mapped the synaptic localization of APP, ADAM10, and BACE1 in the mouse cerebral cortex. All three proteins were present in cortical synapses and subsynaptic fractionation revealed that APP was located mainly in the pre-synaptic active zone (53 %) and in the post-synaptic density (37 %), whereas ADAM10 was enriched in the post-synaptic density (61 %) and BACE1 was concentrated in extra-synaptic regions (72 %). Immunocytochemistry analysis further showed that APP and BACE1 were co-localized in about 30 % of both glutamatergic and GABAergic terminals, whereas few terminals were endowed with ADAM10. This distribution is modified in a mouse model of early AD based on Aß1-42-intracerebroventricular injection, where the synaptic levels of APP and ADAM10 increased by 30 %, whereas BACE1 levels were reduced. This suggests that, in early AD, there are compensatory mechanisms to avoid Aß overload in cortical synapses favoring the non-amyloidogenic processing of APP.

Adenosine A2b receptors control A1 receptor-mediated inhibition of synaptic transmission in the mouse hippocampus.

Adenosine is a neuromodulator mostly acting through A1 (inhibitory) and A2A (excitatory) receptors in the brain. A2B receptors (A(2B)R) are G(s/q)--protein-coupled receptors with low expression in the brain. As A(2B)R function is largely unknown, we have now explored their role in the mouse hippocampus. We performed electrophysiological extracellular recordings in mouse hippocampal slices, and immunological analysis of nerve terminals and glutamate release in hippocampal slices and synaptosomes. Additionally, A(2B)R-knockout (A(2B)R-KO) and C57/BL6 mice were submitted to a behavioural test battery (open field, elevated plus-maze, Y-maze). The A(2B)R agonist BAY60-6583 (300 nM) decreased the paired-pulse stimulation ratio, an effect prevented by the A(2B)R antagonist MRS 1754 (200 nM) and abrogated in A(2B)R-KO mice. Accordingly, A(2B)R immunoreactivity was present in 73 ± 5% of glutamatergic nerve terminals, i.e. those immunopositive for vesicular glutamate transporters. Furthermore, BAY 60-6583 attenuated the A(1)R control of synaptic transmission, both the A(1)R inhibition caused by 2-chloroadenosine (0.1-1 µM) and the disinhibition caused by the A(1)R antagonist DPCPX (100 nM), both effects prevented by MRS 1754 and abrogated in A(2B)R-KO mice. BAY 60-6583 decreased glutamate release in slices and also attenuated the A(1)R inhibition (CPA 100 nM). A(2B)R-KO mice displayed a modified exploratory behaviour with an increased time in the central areas of the open field, elevated plus-maze and the Y-maze and no alteration of locomotion, anxiety or working memory. We conclude that A(2B)R are present in hippocampal glutamatergic terminals where they counteract the predominant A(1)R-mediated inhibition of synaptic transmission, impacting on exploratory behaviour.

Localization and Trafficking of Amyloid-ß Protein Precursor and Secretases: Impact on Alzheimer's Disease.

Alzheimer's disease (AD) affects almost 35 million people worldwide. One of the neuropathological features of AD is the presence of extracellular amyloid plaques, which are mainly composed of amyloid-ß (Aß) peptides. These peptides derive from the amyloidogenic proteolytic processing of the amyloid-ß protein precursor (AßPP), through the sequential action of ß- and γ-secretases. However, AßPP can also be cleaved by a non-amyloidogenic pathway, involving an α-secretase, and in this case the Aß formation is precluded. The production of Aß and of other AßPP catabolites depends on the spatial and temporal co-localization of AßPP with α- or ß-secretases and γ-secretase, which traffic through the secretory pathway in a highly regulated manner. Disturbances on AßPP and secretases intracellular trafficking and, consequently, in their localization may affect dynamic interactions between these proteins with consequences in the AD pathogenesis. In this article, we critically review the recent knowledge about the trafficking and co-localization of AßPP and related secretases in the brain under physiological and AD conditions. A particular focus is given to data concerning the distribution of AßPP and secretases in different types of synapses relatively to other neuronal or glial localizations. Furthermore, we discuss some possible signals that govern the dynamic encounter of AßPP with each group of secretases, such as AßPP mutations, estrogen deprivation, chronic stress, metabolic impairment, and alterations in sleep pattern-associated with aging. The knowledge of key signals that are responsible for the shifting of AßPP processing away from α-secretases and toward the ß-secretases might be useful to develop AD therapeutic strategies.

Hyperactivation of D1 and A2A receptors contributes to cognitive dysfunction in Huntington's disease.

Stimulation of dopamine D1 receptor (D1R) and adenosine A2A receptor (A2AR) increases cAMP-dependent protein kinase (PKA) activity in the brain. In Huntington's disease, by essentially unknown mechanisms, PKA activity is increased in the hippocampus of mouse models and patients and contributes to hippocampal-dependent cognitive impairment in R6 mice. Here, we show for the first time that D1R and A2AR density and functional efficiency are increased in hippocampal nerve terminals from R6/1 mice, which accounts for increased cAMP levels and PKA signaling. In contrast, PKA signaling was not altered in the hippocampus of Hdh(Q7/Q111) mice, a full-length HD model. In line with these findings, chronic (but not acute) combined treatment with D1R plus A2AR antagonists (SCH23390 and SCH58261, respectively) normalizes PKA activity in the hippocampus, facilitates long-term potentiation in behaving R6/1 mice, and ameliorates cognitive dysfunction. By contrast, chronic treatment with either D1R or A2AR antagonist alone does not modify PKA activity or improve cognitive dysfunction in R6/1 mice. Hyperactivation of both D1R and A2AR occurs in HD striatum and chronic treatment with D1R plus A2AR antagonists normalizes striatal PKA activity but it does not affect motor dysfunction in R6/1 mice. In conclusion, we show that parallel alterations in dopaminergic and adenosinergic signaling in the hippocampus contribute to increase PKA activity, which in turn selectively participates in hippocampal-dependent learning and memory deficits in HD. In addition, our results point to the chronic inhibition of both D1R and A2AR as a novel therapeutic strategy to manage early cognitive impairment in this neurodegenerative disease.

Synaptic and sub-synaptic localization of amyloid-ß protein precursor in the rat hippocampus.

Amyloid-ß protein precursor (AßPP) is a large transmembrane protein highly expressed in the central nervous system and cleavage of it can produce amyloid-ß peptides (Aß) involved in synaptic dysfunction and loss associated with cognitive impairment in Alzheimer's disease (AD). Surprisingly, little is known about the synaptic and sub-synaptic distribution of AßPP in different types of nerve terminals. We used total, synaptic, sub-synaptic, and astrocytic membrane preparations obtained from the hippocampus of adult rats to define the localization of AßPP, using two different antibodies against different AßPP epitopes. Western blot analysis revealed that AßPP was not significantly enriched in synaptosomal as compared to total membranes. Within synapses, AßPP immunoreactivity was more abundant in pre- (60 ± 4%) than post- (30 ± 5%) or extra-synaptic fractions (10 ± 2%). Immunocytochemical analysis of purified nerve terminals indicated that AßPP was more frequently associated with glutamatergic (present in 31 ± 4% of glutamatergic terminals) rather than with GABAergic (16 ± 3%) or cholinergic terminals (4 ± 1%, n = 4). We also observed a general lack of co-localization of AßPP and GFAP immunoreactivities in the hippocampus of sections of adult rat brain, albeit we could detect the presence of AßPP in gliosomes (vesicular specializations of astrocytic membranes), suggesting that AßPP has a heterogeneous localization restricted to certain regions of astrocytes. These results provide the first direct demonstration that AßPP is mostly distributed among glutamatergic rather than GABAergic or cholinergic terminals of the adult rat hippocampus, in remarkable agreement with the particular susceptibility to dysfunction and degeneration of glutamatergic synapses in early AD.

The P2X7 receptor antagonist Brilliant Blue G attenuates contralateral rotations in a rat model of Parkinsonism through a combined control of synaptotoxicity, neurotoxicity and gliosis.

Parkinson's disease (PD) involves an initial loss of striatal dopaminergic terminals evolving into a degeneration of dopaminergic neurons in the substantia nigra (SN), which can be modeled by 6-hydroxydopamine (6-OHDA) administration. Since ATP is a danger signal acting through its P2X7 receptors (P2X7R), we now tested if a blood-brain barrier-permeable P2X7R antagonist, Brilliant Blue G (BBG), controlled the 6-OHDA-induced PD-like features in rats. BBG (45 mg/kg) attenuated the 6-OHDA-induced: 1) increase of contralateral rotations in the apomorphine test, an effect mimicked by another P2X7R antagonist A438079 applied intra-cerebroventricularly; 2) short-term memory impairment in the passive avoidance and cued version of the Morris Water maze; 3) reduction of dopamine content in the striatum and SN; 4) microgliosis and astrogliosis in the striatum. To grasp the mechanism of action of BBG, we used in vitro models exploring synaptotoxicity (striatal synaptosomes) and neurotoxicity (dopamine-differentiated neuroblastoma SH-SY5Y cells). P2X7R were present in striatal dopaminergic terminals, and BBG (100 nM) prevented the 6-OHDA-induced synaptosomal dysfunction. P2X7R were also co-localized with tyrosine hydroxylase in SH-SY5Y cells, where BBG (100 nM) attenuated the 6-OHDA-induced neurotoxicity. This suggests that P2X7R contribute to PD pathogenesis through a triple impact on synaptotoxicity, gliosis and neurotoxicity, highlighting the therapeutic potential of P2X7R antagonists in PD.