Cyclic AMP-regulated GABA release at inhibitory synapses in rat cerebellar slices

We have investigated the effects of agents interfering with the cAMP pathway on the rate of miniature IPSCs in cerebellar slices. Noradrenaline and group II glutamate metabotropic receptor agonists respectively enhance and reduce the rate of miniature IPSCs, presumably because they respectively increase and decrease the presynaptic concentration of cAMP.     

Regulation of AMPA Receptor Activity, Synaptic Targeting and Recycling: Role in Synaptic Plasticity

The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors for the neurotransmitter glutamate are oligomeric structures responsible for most fast excitatory responses in the central nervous system. The activity of AMPA receptors can be directly regulated by protein phosphorylation, which may also affect the interaction with intracellular proteins and, consequently, their recycling and localization to defined postsynaptic sites. This review focuses on recent advances in understanding the dynamic regulation of AMPA receptors, on a short- and long-term basis, and its implications in synaptic plasticity.     

Functional assembly of AMPA and kainate receptors is mediated by several discrete protein-protein interactions

Functional heterogeneity of ionotropic glutamate receptors arises not only from the existence of many subunits and isoforms, but also from combinatorial assembly creating channels with distinct properties. This heteromerization is subtype restricted and thought to be determined exclusively by the proximal extracellular N-terminal domain of the subunits. However, using functional assays for heteromer formation, we show that, besides the N-terminal domain, the membrane sector and the C-terminal part of S2 are critical determinants for the formation of functional channels. Our results are compatible with a model where the N-terminal domain only mediates the initial subunit associations into dimers, whereas for the assembly of the full functional tetramer, compatibility of the other regions is required.     

Modeling of slow glutamate diffusion and AMPA receptor activation in the cerebellar glomerulus

Synaptic conductances are influenced markedly by the geometry of the space surrounding the synapse since the transient glutamate concentration in the synaptic cleft is determined by this geometry. Our paper is an attempt to understand the reasons for slow glutamate diffusion in the cerebellar glomerulus, a structure situated around the enlarged mossy fiber terminal in the cerebellum and surrounded by a glial sheath. For this purpose, analytical expressions for glutamate diffusion in the glomerulus were considered in models with two-, three-, and fractional two-three-dimensional (2D-3D) geometry with an absorbing boundary. The time course of average glutamate concentration in the synaptic cleft of the mossy fiber-granule cell connection was calculated for both direct release of glutamate from the same synaptic unit, and for cumulative spillover of glutamate from neighboring release sites. Several kinetic schemes were examined, and the parameters of the diffusion models were estimated by identifying theoretical activation of AMPA receptors with direct release and spillover components of published experimental AMPA receptor-mediated EPSCs. For model selection, the correspondence of simulated paired-pulse ratio and EPSC increase after prevention of desensitization to experimental values were also taken into consideration. Our results suggest at least a 7- to 10-fold lower apparent diffusion coefficient of glutamate in the porous medium of the glomerulus than in water. The modeling of glutamate diffusion in the 2D-3D geometry gives the best fit of experimental EPSCs. We show that it could be only partly explained by normal diffusion of glutamate in the complex geometry of the glomerulus. We assume that anomalous diffusion of glutamate occurs in the glomerulus. A good match of experimental estimations and theoretical parameters, obtained in the simulations that use an approximation of anomalous diffusion by a solution for fractional Brownian motion, confirms our assumption.     

AMPA and NMDA glutamate receptors are found in both peptidergic and non-peptidergic primary afferent neurons in the rat

Two distinct classes of nociceptive primary afferents, peptidergic and non-peptidergic, respond similarly to acute noxious stimulation; however the peptidergic afferents are more likely to play a role in inflammatory pain, while the non-peptidergic afferents may be more characteristically involved in neuropathic pain. Using multiple immunofluorescence, we determined the proportions of neurons in the rat L4 dorsal root ganglion (DRG) that co-express AMPA or NMDA glutamate receptors and markers for the peptidergic and non-peptidergic classes of primary afferents, substance P and P2X(3), respectively. The fraction of DRG neurons immunostained for the NR1 subunit of the NMDA receptor (40%) was significantly higher than that of DRG neurons immunostained for the GluR2/3 (27%) or the GluR4 (34%) subunits of the AMPA receptor. Of all DRG neurons double-immunostained for glutamate receptor subunits and either marker for peptidergic and non-peptidergic afferents, a significantly larger proportion expressed GluR4 than GluR2/3 or NR1 and in a significantly larger proportion of P2X(3)- than SP-positive DRG neurons. These observations support the idea that nociceptors, involved primarily in the mediation of neuropathic pain, may be presynaptically modulated by GluR4-containing AMPA receptors.     

GABA release by basket cells onto Purkinje cells, in rat cerebellar slices, is directly controlled by presynaptic purinergic receptors, modulating Ca2+ influx

In many brain regions, Ca(2+) influx through presynaptic P2X receptors influences GABA release from interneurones. In patch-clamp recordings of Purkinje cells (PCs) in rat cerebellar slices, broad spectrum P2 receptor antagonists, PPADS (30microM) or suramin (12microM), result in a decreased amplitude and increased failure rate of minimal evoked GABAergic synaptic currents from basket cells. The effect is mimicked by desensitizing P2X1/3-containing receptors with alpha,beta-methylene ATP. This suggests presynaptic facilitation of GABA release via P2XR-mediated Ca(2+) influx activated by endogenously released ATP. In contrast, activation of P2Y4 receptors (using UTP, 30microM, but not P2Y1 or P2Y6 receptor ligands) results in inhibition of GABA release. Immunological studies reveal the presence of most known P2Rs in >or=20% of GABAergic terminals in the cerebellum. P2X3 receptors and P2Y4 receptors occur in approximately 60% and 50% of GABAergic synaptosomes respectively and are localized presynaptically. Previous studies report that PC output is also influenced by postsynaptic purinergic receptors located on both PCs and interneurones. The high Ca(2+) permeability of the P2X receptor and the ability of ATP to influence intracellular Ca(2+) levels via P2Y receptor-mediated intracellular pathways make ATP the ideal transmitter for the multisite bidirectional modulation of the cerebellar cortical neuronal network.     

Modulation of potassium evoked secretory function in rat cerebellar slices measured by real time monitoring: Evidence of a possible role for methylfolate in cerebral tissue

The real time dynamics of K⁺ evoked neurosecretion in cerebellar slices has been monitored electrochemically. In the presence of 5-methyltetrahydrofolate a statistically significant diminution in secretory response occurs. Agonists to probe the pharmacological basis for this indicate it is not due to voltage sensitive Ca²⁺ channel blockade, nor does it show any similarity of effect with kainate, whose receptor is a putative binding site for 5-methyltetrahydrofolate. The method is fully validated, although no account is taken of individual molecular species. High performance liquid chromatography combined with off line microbiological assay could only detect 5-methyltetrahydrofolate in cerebrospinal fluid. We therefore discuss our findings in relation to possible cerebral roles for cerebrospinal fluid 5-methyltetrahydrofolate in the context of both membrane and transmitter related interactions.     

Macrophage-induced neurotoxicity is mediated by glutamate and attenuated by glutaminase inhibitors and gap junction inhibitors

We have shown previously, that the most neurotoxic factor from activated microglia is glutamate that is produced by glutaminase utilizing extracellular glutamine as a substrate. Drugs that inhibit glutaminase or gap junction through which the glutamate is released were effective in reducing neurotoxic activity of microglia. In this study, to elucidate whether or not a similar mechanism is operating in macrophages infiltrating into the central nervous system during inflammatory, demyelinating, and ischemic brain diseases, we examined the neurotoxicity induced by macrophages, in comparison with microglia in vitro. LPS- or TNF-alpha-stimulated macrophage-conditioned media induced robust neurotoxicity, which was completely inhibited by the NMDA receptor antagonist MK801. Both the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine (DON), and the gap junction inhibitor carbenoxolone (CBX), effectively suppressed glutamate production and subsequent neurotoxicity by activated macrophages. These results revealed that macrophages produce glutamate via glutaminase from extracelluar glutamine, and release it through gap junctions. This study demonstrated that a similar machinery is operating in macrophages as well, and DON and CBX that prevent microglia-mediated neurotoxicity should be effective for preventing macrophage-mediated neurotoxicity. Thus, these drugs may be effective therapeutic reagents for inflammatory, demyelinating, and ischemic brain diseases.     

Methotrexate induces seizure and decreases glutamate uptake in brain slices: prevention by ionotropic glutamate receptors antagonists and adenosine

Methotrexate (MTX)-induced neurotoxicity may occur after intrathecal or systemic administration at low, intermediate and high doses for the treatment of malignant or inflammatory diseases. The mechanisms of MTX neurotoxicity are not totally understood, and appear to be multifactorial. In this study we characterized a model of MTX-induced seizures in mice to evaluate the convulsive and toxic MTX properties. Additionally, the effect of MTX-induced seizures on the activity of glutamate transporters, as well as the anticonvulsant role of MK-801, DNQX and adenosine on glutamate uptake in brain slices was investigated . MTX induced tonic-clonic seizures in approximately 95% of animals and pre-treatment with MK-801, DNQX and adenosine prevented seizure in 80%, 62% and 50% of animals, respectively. Moreover, MTX leads 59% of mice to death, which was prevented in 100% and 94% when animals received MK-801 and DNQX, respectively. Glutamate uptake decreased by 20% to 30% in cortical slices after MTX-induced seizures. Interestingly, when seizures were prevented by MK-801, DNQX or adenosine, glutamate uptake activity remained at the same level as the control group. Thus, our results demonstrate the involvement of the glutamatergic system in MTX-induced seizures.     

AMPA receptor-dependent clustering of synaptic NMDA receptors is mediated by Stargazin and NR2A/B in spinal neurons and hippocampal interneurons

Under standard conditions, cultured ventral spinal neurons cluster AMPA- but not NMDA-type glutamate receptors at excitatory synapses on their dendritic shafts in spite of abundant expression of the ubiquitous NMDA receptor subunit NR1. We demonstrate here that the NMDA receptor subunits NR2A and NR2B are not routinely expressed in cultured spinal neurons and that transfection with NR2A or NR2B reconstitutes the synaptic targeting of NMDA receptors and confers on exogenous application of the immediate early gene product Narp the ability to cluster both AMPA and NMDA receptors. The use of dominant-negative mutants of GluR2 further showed that the synaptic targeting of NMDA receptors is dependent on the presence of synaptic AMPA receptors and that synaptic AMPA and NMDA receptors are linked by Stargazin and a MAGUK protein. This system of AMPA receptor-dependent synaptic NMDA receptor localization was preserved in hippocampal interneurons but reversed in hippocampal pyramidal neurons.     

Exercise-induced apoptosis in rat kidney is mediated by both angiotensin II AT1 and AT2 receptors

Excessive physical exercise may lead to disturbance of the entire homeostasis in the body, including damage not only in skeletal muscles but also in many distant organs. The mechanisms responsible for the exercise-induced changes could include oxidative stress or angiotensin II. We previously showed that acute exercise led to apoptosis in kidney but not as a result of oxidative stress. In this study, we examined the role of angiotensin II and its AT1 and AT2 receptors in mediation of exercise-induced apoptosis in kidney. We clearly demonstrated that acute physical exercise induced apoptosis in renal cells of distal convoluted tubuli and cortical and medullary collecting ducts. Moreover, the cells displayed an increased expression of both AT1 and AT2 angiotensin II receptors and of p53 protein. The results suggest that angiotensin II could upregulate p53 expression in renal distal convoluted tubular cells and in the cells collecting ducts via both AT1 and AT2 receptors, which might be the crucial apoptosis-mediating mechanism in kidneys after excessive exercise.     

Functional and structural characterization of the secretin receptors in rat gastric glands: Desensitization and glycoprotein nature

We have documented and characterized the down-regulation of the¹²⁵I-secretin binding sites and the associated desensitization of the secretin receptor-cAMP system in rat gastric glands. Secretin induced a rapid decrease of the high-affinity¹²⁵I-secretin binding sites with t^1/2=30 min at 37°C. Half-maximal down-regulation and desensitization occurred at 10^–9 M secretin, a physiological concentration corresponding to the half-maximal activation of the secretin receptor. The Scatchard parameters of the low-affinity¹²⁵I-secretin binding sites were unaffected by the pretreatment. This desensitization is heterologous in view of the loss of responsiveness to the truncated glucagon-like peptide 1 (TGLP-1), and pharmacologically selective since the sectetin-related analogue VIP (10^–7 M) does not alter the secretin-induced cAMP generation in rat gastric glands. The glycoprotein nature of the secretin receptor has also been demonstrated using WGA-agarose affinity chromatography of the solubilized¹²⁵I-secretin receptor complex.     

Tianeptine potentiates AMPA receptors by activating CaMKII and PKA via the p38, p42/44 MAPK and JNK pathways

Impairments of cellular plasticity appear to underlie the pathophysiology of major depression. Recently, elevated levels of phosphorylated AMPA receptor were implicated in the antidepressant effect of various drugs. Here, we investigated the effects of an antidepressant, Tianeptine, on synaptic function and GluA1 phosphorylation using murine hippocampal slices and in vivo single-unit recordings. Tianeptine, but not imipramine, increased AMPA receptor-mediated neuronal responses both in vitro and in vivo, in a staurosporine-sensitive manner. Paired-pulse ratio was unaltered by Tianeptine, suggesting a postsynaptic site of action. Tianeptine, 10 μM, enhanced the GluA1-dependent initial phase of LTP, whereas 100 μM impaired the latter phases, indicating a critical role of GluA1 subunit phosphorylation in the excitation. Tianeptine rapidly increased the phosphorylation level of Ser⁸³¹-GluA1 and Ser⁸⁴⁵-GluA1. Using H-89 and KN-93, we show that the activation of both PKA and CaMKII is critical in the effect of Tianeptine on AMPA responses. Moreover, the phosphorylation states of Ser^217/221-MEK and Thr¹⁸³/Tyr¹⁸⁵-p42MAPK were increased by Tianeptine and specific kinase blockers of the MAPK pathways (PD 98095, SB 203580 and SP600125) prevented the effects of Tianeptine. Overall these data suggest that Tianeptine potentiates several signaling cascades associated with synaptic plasticity and provide further evidence that a major mechanism of action for Tianeptine is to act as an enhancer of glutamate neurotransmission via AMPA receptors.     

Sequential Delivery of Synaptic GluA1- and GluA4-containing AMPA Receptors (AMPARs) by SAP97 Anchored Protein Complexes in Classical Conditioning

Multiple signaling pathways are involved in AMPAR trafficking to synapses during synaptic plasticity and learning. The mechanisms for how these pathways are coordinated in parallel but maintain their functional specificity involves subcellular compartmentalization of kinase function by scaffolding proteins, but how this is accomplished is not well understood. Here, we focused on characterizing the molecular machinery that functions in the sequential synaptic delivery of GluA1- and GluA4-containing AMPARs using an in vitro model of eyeblink classical conditioning. We show that conditioning induces the interaction of selective protein complexes with the key structural protein SAP97, which tightly regulates the synaptic delivery of GluA1 and GluA4 AMPAR subunits. The results demonstrate that in the early stages of conditioning the initial activation of PKA stimulates the formation of a SAP97-AKAP/PKA-GluA1 protein complex leading to synaptic delivery of GluA1-containing AMPARs through a SAP97-PSD95 interaction. This is followed shortly thereafter by generation of a SAP97-KSR1/PKC-GluA4 complex for GluA4 AMPAR subunit delivery again through a SAP97-PSD95 interaction. These data suggest that SAP97 forms the molecular backbone of a protein scaffold critical for delivery of AMPARs to the PSD during conditioning. Together, the findings reveal a cooperative interaction of multiple scaffolding proteins for appropriately timed delivery of subunit-specific AMPARs to synapses and support a sequential two-stage model of AMPAR synaptic delivery during classical conditioning.     

The characteristics of arginine transport by rat cerebellar and cortical synaptosomes

The uptake ofl-[³H]arginine into synaptosomes prepared from rat cerebellum and cortex occurred by a high-affinity carrier-mediated process. The uptake of arginine appeared to be potentiated by removal of extracellular Na⁺, inhibited by high levels of extracellular K⁺, but not by depolarization with veratridine or 4-amino pyridine. The effect of Na⁺ removal or K⁺ elevation did not seem to be due to changes in intracellular Ca²⁺ or pH. In both brain regions, uptake was significantly inhibited byl-arginine,l-lysine,l-ornithine, andl-homoarginine, but not byd-arginine norl-citrulline. Uptake was also inhibited by N^G-monomethyl-l-arginine acetate, but not by N^G-nitro-l-arginine methyl ester nor N^G-nitro-l-arginine except in the cortex at a concentration of 1 mM. The results indicate that the carrier system operating in synaptosomes showed many of the characteristics of the ubiquitous y⁺ system seen in many other tissues, although its apparent sensitivity to variations in extracellular Na⁺ was unusual.     

Nitric oxide-evoked glutamate release and cGMP production in cerebellar slices: control by presynaptic 5-HT1D receptors

We previously reported that pre- and postsynaptic 5-hydroxytryptamine (5-HT) receptors effectively control glutamatergic transmission in adult rat cerebellum. To investigate where 5-HT acts in the glutamate ionotropic receptors/nitric oxide/guanosine 3',5'-cyclic monophosphate (cGMP) pathway, in the present study 5-HT modulation of the cGMP response to the nitric oxide donor S-nitroso-penicillamine (SNAP) was studied in adult rat cerebellar slices. While cGMP elevation produced by high-micromolar SNAP was insensitive to 5-HT, 1 microM SNAP, expected to release nitric oxide in the low-nanomolar concentration range, elicited cGMP production and endogenous glutamate release both of which could be prevented by activating presynaptic 5-HT1D receptors. Released nitric oxide appeared responsible for cGMP production and glutamate release evoked by 1 microM SNAP, as both the effects were mimicked by the structurally unrelated nitric oxide donor 2-(N,N-diethylamino)-diazenolate-2-oxide (0.1 microM). Dependency of the 1 microM SNAP-evoked release of glutamate on external Ca2+, sensitivity to presynaptic release-regulating receptors and dependency on ionotropic glutamate receptor functioning, suggest that nitric oxide stimulates exocytotic-like, activity-dependent glutamate release. Activation of ionotropic glutamate receptors/nitric oxide synthase/guanylyl cyclase pathway by endogenously released glutamate was involved in the cGMP response to 1 microM SNAP, as blockade of NMDA/non-NMDA receptors, nitric oxide synthase or guanylyl cyclase, abolished the cGMP response. To conclude, in adult rat cerebellar slices low-nanomolar exogenous nitric oxide could facilitate glutamate exocytotic-like release possibly from parallel fibers that subsequently activated the glutamate ionotropic receptors/nitric oxide/cGMP pathway. Presynaptic 5-HT1D receptors could regulate the nitric oxide-evoked release of glutamate and subsequent cGMP production.