Opposing reduced VPAC1 and enhanced VPAC2 VIP receptors in the hippocampus of the Li2+-pilocarpine rat model of temporal lobe epilepsy.

VIP binding sites are upregulated in mesial temporal lobe epilepsy (MTLE) patients, also suffering from severe cognitive deficits. Although altered VIP and VIP receptor levels were described in rodent models of epilepsy, the VIP receptor subtype(s) were never identified. We now investigated how VPAC1 and VPAC2 receptor levels change in the Li2+-pilocarpine rat model of MTLE. Cognitive decline and altered synaptic plasticity as estimated from phosphorylation of AMPA GluA1 subunit on Ser831 and Ser845 and AMPA GluA1/GluA2 ratio was also probed. Animals showing spontaneous recurrent seizures (SRSs) for at least 4 weeks showed impaired learning in the radial arm maze (RAM) and presented decreased VPAC1 and increased VPAC2 receptor levels. In addition, SRSs rats showed increased AMPA GluA1 phosphorylation in Ser831 and Ser845, marked decrease in GluA1 levels and a milder decrease in GluA2 levels. Consequently, the GluA1/GluA2 ratio was also decreased in SRSs rats. Altered VIP receptor levels may differentially prevent or contribute to MTLE pathology, since VPAC1 receptors promote the endogenous control of LTP, mediate endogenous VIP neuroprotection against altered synaptic plasticity following epileptiform activity, and mediate anti-inflammatory actions in microglia, while VPAC2 receptors mediate VIP endogenous neuroprotection against neonatal excitotoxicity and prevent reactive astrogliosis. This discovery imposes a different mindset for considering VIP receptors as therapeutic targets in MTLE, allowing a differential targeting of the cellular events contributing to epileptogenesis.

The Role of cGMP on Adenosine A 1 Receptor-mediated Inhibition of Synaptic Transmission at the Hippocampus.

Both adenosine A1 receptor and cGMP inhibit synaptic transmission at the hippocampus and recently it was found that A1 receptor increased cGMP levels in hippocampus, but the role of cGMP on A1 receptor-mediated inhibition of synaptic transmission remains to be established. In the present work we investigated if blocking the NOS/sGC/cGMP/PKG pathway using nitric oxide synthase (NOS), protein kinase G (PKG), and soluble guanylyl cyclase (sGC) inhibitors modify the A1 receptor effect on synaptic transmission. Neurotransmission was evaluated by measuring the slope of field excitatory postsynaptic potentials (fEPSPs) evoked by electrical stimulation at hippocampal slices. N6-cyclopentyladenosine (CPA, 15 nM), a selective A1 receptor agonist, reversibly decreased the fEPSPs by 54 ± 5%. Incubation of the slices with an inhibitor of NOS (L-NAME, 200 µM) decreased the CPA effect on fEPSPs by 57 ± 9% in female rats. In males, ODQ (10 µM), an sGC inhibitor, decreased the CPA inhibitory effect on fEPSPs by 23 ± 6%, but only when adenosine deaminase (ADA,1 U/ml) was present; similar results were found in females, where ODQ decreased CPA-induced inhibition of fEPSP slope by 23 ± 7%. In male rats, the presence of the PKG inhibitor (KT5823, 1 nM) decreased the CPA effect by 45.0 ± 9%; similar results were obtained in females, where KT5823 caused a 32 ± 9% decrease on the CPA effect. In conclusion, the results suggest that the inhibitory action of adenosine A1 receptors on synaptic transmission at hippocampus is, in part, mediated by the NOS/sGC/cGMP/PKG pathway.

Combined neuroprotective action of adenosine A1 and cannabinoid CB1 receptors against NMDA-induced excitotoxicity in the hippocampus.

Both adenosine A1 and cannabinoid CB1 receptors trigger similar transduction pathways and protect against neurotoxic insults at the hippocampus, but their combined neuroprotective potential has not been investigated. We set forth to assess the combined action of A1 and CB1 receptors against glutamate NMDA receptor-mediated excitotoxicity at the hippocampus. Cell damage was assessed by measuring propidium iodide (PI) uptake and lactate dehydrogenase (LDH) activity released from cultured hippocampal slices exposed to NMDA. Exposure to NMDA (50 µM) for 1 h increased LDH activity by 92% ± 16%. WIN55212-2 (30 µM), a cannabinoid CB1 receptor agonist, decreased NMDA-induced LDH activity by 53% ± 10% while N6-cyclopentyladenosine (CPA, 100 nM), an adenosine A1 receptor selective agonist, decreased it by 37% ± 11%. The combined inhibitory effect of WIN55212-2 and CPA (88% ± 12%) did not differ from the sum of the individual inhibitory effects of each agonist (90% ± 15%) but was different from the effects of CPA or WIN55212-2 alone. An additive inhibitory effect of co-application of WIN55212-2 (30 µM) and CPA (100 nM) on NMDA (50 µM)-induced PI uptake was also observed in CA3 but not in CA1 area of the hippocampal slice. The results suggest that both CB1 and A1 receptors produce additive cumulative neuroprotection against NMDA-induced excitotoxicity in the hippocampus.

The combined inhibitory effect of the adenosine A1 and cannabinoid CB1 receptors on cAMP accumulation in the hippocampus is additive and independent of A1 receptor desensitization.

Adenosine A1 and cannabinoid CB1 receptors are highly expressed in hippocampus where they trigger similar transduction pathways. We investigated how the combined acute activation of A1 and CB1 receptors modulates cAMP accumulation in rat hippocampal slices. The CB1 agonist WIN55212-2 (0.3-30 µM) decreased forskolin-stimulated cAMP accumulation with an EC50 of 6.6±2.7 µM and an Emax of 31%±2%, whereas for the A1 agonist, N6-cyclopentyladenosine (CPA, 10-150 nM), an EC50 of 35±19 nM, and an Emax of 29%±5 were obtained. The combined inhibitory effect of WIN55212-2 (30 µM) and CPA (100 nM) on cAMP accumulation was 41%±6% (n=4), which did not differ (P>0.7) from the sum of the individual effects of each agonist (43%±8%) but was different (P<0.05) from the effects of CPA or WIN55212-2 alone. Preincubation with CPA (100 nM) for 95 min caused desensitization of adenosine A1 activity, which did not modify the effect of WIN55212-2 (30 µM) on cAMP accumulation. In conclusion, the combined effect of CB1 and A1 receptors on cAMP formation is additive and CB1 receptor activity is not affected by short-term A1 receptor desensitization.

Association of the transcobalamin II gene 776C → G polymorphism with Alzheimer's type dementia: dependence on the 5, 10-methylenetetrahydrofolate reductase 1298A → C polymorphism genotype.

BACKGROUND: Decreased serum concentrations of vitamin B12 are associated with Alzheimer's type dementia. The transcobalamin II gene (TCN2) 776C → G polymorphism affects transcobalamin II function as a carrier of vitamin B12 and might modify its availability. The association of the TCN2 776C → G polymorphism with Alzheimer's type dementia is unclear and was investigated in the present study. METHODS: Case-control study including 27 individuals diagnosed with Alzheimer's type dementia and 28 healthy controls. Serum concentrations of vitamin B12, homocysteine and other analytes were determined and the presence of TCN2 776C → G and 5, 10-methylenetetrahydrofolate reductase 1298A → C polymorphisms genotypes was ascertained by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: Serum concentrations of vitamin B12 were lower while those of homocysteine were higher in patients than in controls (P < 0.05). The frequency of individuals carrying at least one 5, 10-methylenetetrahydrofolate reductase 1298C allele was higher (59% versus 32%) while frequency of individuals harbouring at least one TCN2 776G allele was lower (58% versus 86%) in patients than in controls (P < 0.05). Univariate logistic regression showed negative association of TCN2 776CG genotype with Alzheimer's type dementia (OR = 0.17 versus CC genotype, P < 0.02). Multivariate logistic regression identified TCN2 776C → G polymorphism as independent predictor of Alzheimer's type dementia together with higher concentrations of homocysteine, cholesterol and uric acid and lower concentrations of oestradiol. Association of TCN2 776C → G polymorphism with Alzheimer's type dementia was observed for individuals carrying the 5,10-methylenetetrahydrofolate reductase 1298AA genotype but not the AC or CC genotypes, indicating interaction between the two polymorphisms. CONCLUSIONS: The TCN2 776C → G polymorphism is negatively associated with Alzheimer's type dementia, suggesting a protective role against the disease in subjects with the 5, 10-methylenetetrahydrofolate reductase 1298AA genotype.

Modulation of cGMP accumulation by adenosine A1 receptors at the hippocampus: influence of cGMP levels and gender.

Adenosine A1 receptor is highly expressed in hippocampus where it inhibits neurotransmitter release and has neuroprotective activity. Similar actions are obtained by increasing cGMP concentration, but a clear link between adenosine A1 receptor and cGMP levels remains to be established. The present work aims to investigate if cGMP formation is modulated by adenosine A1 receptors at the hippocampus and if this effect is gender dependent. cGMP accumulation, induced by phosphodiesterases inhibitors Zaprinast (100 µM) and Bay 60-7550 (10 µM), and cAMP accumulation, induced by Forskolin (20 µM) and Rolipram (50 µM), were quantified in rat hippocampal slices using specific enzymatic immunoassays. N6-cyclopentyladenosine (CPA, 100 nM) alone failed to modify basal cGMP accumulation. However, the presence of adenosine deaminase (ADA, 2 U/ml) unmasked a CPA (0.03-300 nM) stimulatory effect on basal cGMP accumulation (EC50: 4.2±1.4 nM; Emax: 17±0.9%). ADA influence on CPA activity was specific for cGMP, since inhibition of cAMP accumulation by CPA was not affected by the presence of ADA, though ADA inhibited cAMP accumulation in the absence of CPA. Increasing cGMP accumulation, by about four-fold, with sodium nitroprusside (SNP, 100 µM) abolished the CPA (100 nM) effect on cGMP accumulation in males but did not modify the effect of CPA in female rats. This effect was reversed by 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 nM), indicating an adenosine A1 receptor mediated effect on cGMP accumulation. In conclusion, adenosine A1 receptors increase intracellular cGMP formation at hippocampus both in males and females under basal conditions, but only in females when cGMP levels are increased by SNP.

Cannabinoid CB(1) and adenosine A(1) receptors independently inhibit hippocampal synaptic transmission.

Adenosine A(1) and cannabinoid CB(1) receptors are affected by drugs widely consumed by humans, as it is the case for caffeine, an adenosine receptor antagonist, and tetrahydrocannabinol, a cannabinoid receptor agonist. These receptors are present in the hippocampus and inhibit neurotransmitter release by operating similar transduction mechanisms. We, therefore, evaluated if they cross-talk to modulate synaptic transmission in the hippocampus. To do so, field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 area of rat hippocampal slices and the consequences of activation or blockade of cannabinoid CB(1) or adenosine A(1) receptors upon neuromodulation exerted by the other receptor were assessed. The cannabinoid CB(1) receptor agonist, WIN55212-2 (300nM), slowly decreased ( congruent with40%) the fEPSP slope, while the adenosine A(1) receptor agonist, cyclopenthyladenosine (CPA, 15nM) rapidly decreased ( congruent with50%) it. Blockade of cannabinoid CB(1) receptors with AM251 (1microM) did not influence the adenosine A(1) receptor-mediated inhibition of synaptic transmission. Blockade of adenosine A(1) receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 50nM) did not affect the inhibition perpetrated by cannabinoid CB(1) receptor activation. When both receptors were simultaneously activated (5nM CPA plus 300nM WIN55212-2) the net inhibition of synaptic transmission was about the sum of the effect of each drug applied independently. These results indicate independent synaptic transmission modulation by adenosine A(1) and cannabinoid CB(1) receptors at the hippocampus, suggesting that availability of G-proteins coupled to each receptor and availability of other signalling molecules involved in their transducing pathways, are not limiting factors for their modulatory role.