Trafficking of presynaptic AMPA receptors mediating neurotransmitter release: neuronal selectivity and relationships with sensitivity to cyclothiazide

Postsynaptic glutamate AMPA receptors (AMPARs) can recycle between plasma membrane and intracellular pools. In contrast, trafficking of presynaptic AMPARs has not been investigated. AMPAR surface expression involves interactions between the GluR2 carboxy tail and various proteins including glutamate receptor-interacting protein (GRIP), AMPA receptor-binding protein (ABP), protein interacting with C kinase 1 (PICK1), N-ethyl-maleimide-sensitive fusion protein (NSF). Here, peptides known to selectively block the above interactions were entrapped into synaptosomes to study the effects on the AMPA-evoked release of [3H]noradrenaline ([3H]NA) and [3H]acetylcholine ([3H]ACh) from rat hippocampal and cortical synaptosomes, respectively. Internalization of pep2-SVKI to prevent GluR2-GRIP/ABP/PICK1 interactions potentiated the AMPA-evoked release of [3H]NA but left unmodified that of [3H]ACh. Similar potentiation was caused by pep2-AVKI, the blocker of GluR2-PICK1 interaction. Conversely, a decrease in the AMPA-evoked release of [3H]NA, but not of [3H]ACh, was caused by pep2m, a selective blocker of the GluR2-NSF interaction. In the presence of pep2-SVKI the presynaptic AMPARs on noradrenergic terminals lost sensitivity to cyclothiazide. AMPARs releasing [3H]ACh, but not those releasing [3H]NA, were sensitive to spermine, suggesting that they are GluR2-lacking AMPARs. To conclude: (i) release-regulating presynaptic AMPARs constitutively cycle in isolated nerve terminals; (ii) the process exhibits neuronal selectivity; (iii) AMPAR trafficking and desensitization may be interrelated.     

Voltage-dependent block of native AMPA receptor channels by dicationic compounds

1. The kinetics of open channel block of GluR2-containing and GluR2-lacking AMPA receptors (AMPAR) by dicationic compounds (IEM-1460, IEM-1754, and IEM-1925) have been studied in rat hippocampal neurones using whole-cell patch clamp recording and concentration-jump techniques. Neurones were isolated from hippocampal slices by vibrodissociation. 2. The dicationic compounds were approximately 100 - 200 times more potent as blockers of GluR2-lacking AMPAR than as blockers of GluR2-containing AMPAR. The subunit specificity of channel block is determined by the blocking rate constant of a dicationic compound, whereas differences in unblocking rate constants account for differences in potency. 3. Hyperpolarization may decrease the block produced by IEM-1460 and IEM-1754 block due to the voltage-dependence of the unblocking rate constants for these compounds. This suggests that dicationic compounds permeate the AMPAR channel at negative membrane potentials. The effect was particularly apparent for GluR2-lacking AMPAR. These findings indicate that the presence of GluR2-subunit(s) in AMPAR hinders the binding of the cationic compounds and their permeation through the channel. 4. The most potent compound tested was IEM-1925. The presence of a phenylcyclohexyl moiety instead of an adamantane moiety, as in IEM-1460 and IEM1754, is probably responsible for the higher potency of IEM-1925. Dicationic compounds are important not only as pharmacological tools, but also as templates for the synthesis of new selective AMPAR blockers which may be potential therapeutic agents.     

Chronic antidepressant treatment increases the membrane expression of AMPA receptors in rat hippocampus

It has been proposed that potentiation of AMPA receptor (AMPAR) function may be useful in the treatment of depression. Here we studied the acute and chronic effect of the antidepressants desipramine and paroxetine, which differentially affect monoamine reuptake, on the expression of the AMPAR subunits GluR1 and GluR2/3, analyzed by Western blot, both in total and in membrane-enriched extracts from rat hippocampus. Acute antidepressant treatment did not produce any change in the expression of AMPAR subunits. In chronic treatments, rats were daily treated with the antidepressants (10 mg/kg/day) for 7, 14, or 21 days. In rats receiving either of the two antidepressant treatments for 21 consecutive days and killed 24 h after the last injection, an increase in GluR1 and GluR2/3 levels was found in the membrane fraction, with no significant change in the total extract, suggesting a trafficking of the AMPAR subunits from intracellular pools to synaptic sites in the hippocampus. This appeared to be a region-specific effect since no change in AMPAR subunit expression was found in the frontal cortex. Previously reported modifications in phosphorylating enzymes by chronic antidepressants could perhaps play a role in hippocampal membrane insertion of AMPAR subunits. When the survival time after the 21-day-treatment was longer — 72 instead of 24 h — the hippocampal membrane expression of GluR1, but not of GluR2/3 subunits, was still increased, as could be expected from the distinct mechanisms operating in synaptic delivery of GluR1 and GluR2/3 subunits. The antidepressant-induced increase in the number of GluR1- and GluR2/3-containing AMPARs at the synapses may indicate an enhanced AMPAR-mediated synaptic transmission which could help to counteract the alterations in neuronal connectivity which appear to underlie the pathophysiology of mood disorders.     

Sequential changes in BDNF mRNA expression and synaptic levels of AMPA receptor subunits in rat hippocampus after chronic antidepressant treatment

Increased expression of brain-derived neurotrophic factor (BDNF) appears to be involved in the mechanism of action of antidepressant drugs. It has also been proposed that potentiation of the AMPA receptor (AMPAR) function may be useful in the treatment of depression. Here we looked for the time course of the effect of different doses of two antidepressants, desipramine (DMI) and paroxetine (PAR), which differentially affect monoamine reuptake, on BDNF mRNA expression in hippocampal subfields (CA1, CA3 and dentate gyrus) and levels of AMPAR subunits in total and membrane-enriched extracts from rat hippocampus. Acute antidepressant treatment changed neither BDNF mRNA expression nor AMPAR subunit levels. In chronic treatments, rats were treated daily with the antidepressants for 7–21 days. PAR produced a time- and dose-dependent increase of BDNF expression in the three hippocampal subfields examined. On the contrary, the effect of DMI on BDNF mRNA was neither dose- nor time-dependent. In rats receiving the same chronic antidepressant treatments, PAR produced a dose-dependent increase of GluR1 and GluR2/3 levels in the membrane fraction after a 3-week treatment, and not at earlier times. DMI increased the membrane levels of AMPAR subunits after a 3-week treatment with the lower dose tested. However, a higher dose, 15 mg/kg, did not produce any change in AMPAR subunits and reduced membrane levels of -tubulin and PSD-95, possibly indicating a disorganization of membrane scaffolding proteins. The results suggest that paroxetine, but not desipramine, enhances synaptic plasticity in the hippocampus by increasing BDNF mRNA expression, which determines a later AMPAR subunit trafficking to synaptic membranes.     

Selective enhancement of AMPA receptor-mediated function in hippocampal CA1 neurons from chronic benzodiazepine-treated rats

Two days following one-week administration of the benzodiazepine, flurazepam (FZP), rats exhibit anticonvulsant tolerance in vivo, while reduced GABA(A) receptor-mediated inhibition and enhanced EPSP amplitude are present in CA1 pyramidal neurons in vitro. AMPA receptor (AMPAR)-mediated synaptic transmission in FZP-treated rats was examined using electrophysiological techniques in in vitro hippocampal slices. In CA1 pyramidal neurons from FZP-treated rats, the miniature excitatory postsynaptic current (mEPSC) amplitude was significantly increased (33%) without change in frequency, rise time or decay time. Moreover, mEPSC amplitude was not elevated in dentate granule neurons following 1-week FZP treatment or in CA1 pyramidal neurons following acute desalkyl-FZP treatment. Regulation of AMPAR number was assessed by quantitative autoradiography with the AMPAR antagonist, [(3)H]Ro48-8587. Specific binding was significantly increased in stratum pyramidale of hippocampal areas CA1 and CA2 and in proximal dendritic fields of CA1 pyramidal neurons. Regulation of AMPAR subunit proteins was examined using immunological techniques. Neither abundance nor distribution of GluR1-3 subunit proteins was different in the CA1 region following FZP treatment. These findings suggest that enhanced AMPAR currents, mediated at least in part by increased AMPAR number, may contribute to BZ anticonvulsant tolerance. Furthermore, these studies suggest an interaction between GABAergic and glutamatergic systems in the CA1 region which may provide novel therapeutic strategies for restoring BZ effectiveness.     

Combined β-adrenergic and corticosteroid receptor activation regulates AMPA receptor function in hippocampal neurons

Shortly after stress, limbic neurons are exposed to high levels of noradrenaline and corticosterone. These hormones are necessary for optimal behavioural adaptation. Behavioural effects critically depend on noradrenaline acting via β-adrenergic receptors, but these effects are strongly modulated by corticosterone, indicating putative interactions between the two hormones. Since both noradrenaline and corticosterone are known to quickly affect properties of AMPA-type glutamate receptors (AMPAR), we here examined - in hippocampal neurons - three parameters which give insight in the functionality of AMPARs: phosphorylation, surface expression and spontaneous synaptic transmission. In homogenates of adult hippocampal slices, application of corticosterone (30 nM for 15 min) by itself did not affect phosphorylation of the AMPAR GluA1 subunit at S845 or S831. Co-application of the β-adrenergic receptor agonist isoproterenol (10 μM) largely increased S845 (but not S831) phosphorylation. Corticosterone also did not change GluA1 and GluA2 surface expression in hippocampal primary cultures. However, combined administration of corticosterone and 1 μM isoproterenol - which by itself was ineffective - enhanced surface expression. Interestingly, 10 μM isoproterenol alone enhanced GluA1 surface expression, but this was decreased by corticosterone. Finally, in hippocampal primary cultures, the inter-event interval of miniature excitatory postsynaptic currents (mEPSCs) was decreased by the combination of 1 μM isoproterenol and corticosterone (which were ineffective by themselves) while the same combination did not affect the amplitude. We conclude that AMPAR phosphorylation, surface expression and mEPSC inter-event interval respond most strongly to a combination of corticosterone and β-adrenergic receptors. These combined hormonal effects on glutamate transmission might contribute to their memory-enhancing effects.     

An ER retention signal explains differences in surface expression of NMDA and AMPA receptor subunits.

The molecular mechanisms that control the surface expression of NMDA receptors (NMDARs) and AMPA receptors (AMPARs) are unknown. To determine the role of the intracellular C-terminal tails of glutamate receptor subunits in the synaptic targeting of AMPARs and NMDARs, we fused the tails of the AMPAR subunits, GluR1 and GluR2, and the NMDAR subunit, NR1, to the human T lymphocyte membrane protein CD8 and expressed these constructs in HEK293 cells and cultured hippocampal neurons. The GluR1 and GluR2 fusion proteins exhibited robust surface expression in the plasma membrane of neurons at synapses as did CD8 alone. In contrast, the NR1 fusion protein was retained intracellularly in both HEK293 cells and neurons because of the presence of an ER retention signal in the C1 cassette. This ER retention signal was overridden either by the addition of a PDZ domain-binding motif or by mimicking phosphorylation at a site adjacent to the retention signal. These results provide further evidence that the intracellular trafficking of AMPAR and NMDAR subunits are regulated independently at least in part because of differences in the protein-protein interactions of their intracellular C-terminal tails.     

Transient synaptic activation of NMDA receptors leads to the insertion of native AMPA receptors at hippocampal neuronal plasma membranes.

The molecular mechanisms underlying long-term potentiation (LTP) of excitatory synaptic transmission in the hippocampus are not well understood. Transient depolarisation of cultured postnatal hippocampal neurones (3x1 s exposure to 90 mM K+) induces a form of LTP that is manifest primarily as an increase in mEPSC frequency. Site-directed antibodies that recognise an extracellular region of all AMPA receptor (AMPAR) subunits (GluR1-4) were used for the immunolabelling of living neurones. These antibodies were raised in two species to enable sequential immunofluorescent labelling of individual living neurones before and after the induction of LTP. High K+ treatment resulted in the appearance of new AMPAR clusters at sites on the neuronal surface that previously lacked detectable AMPARs. The appearance of new AMPAR clusters was NMDA receptor (NMDAR)-dependent since it was antagonised by the application of NMDAR antagonists. Our data indicate that the transient synaptic activation of NMDARs can lead to the insertion of native AMPARs at sites on the neuronal membrane that initially lacks AMPARs.     

Increased expression, but not postsynaptic localisation, of ionotropic glutamate receptors during the late-phase of long-term potentiation in the dentate gyrus in vivo

Long-term potentiation (LTP) is extensively studied as a cellular mechanism of information storage in the brain. The induction and early expression mechanisms of LTP depend on activation and rapid modulation of ionotropic glutamate receptors. However, the mechanisms that underlie maintenance of LTP over the course of days or longer are poorly understood. Here, we have investigated the overall expression of AMPA- and NMDA-type glutamate receptors (AMPARs and NMDARs, respectively), as well as their levels at the synaptic surface membrane and in the postsynaptic density (PSD), in the dentate gyrus at 48 h following the induction of LTP at perforant path synapses in awake rats. We found a high-frequency stimulation-dependent increase in the overall levels of AMPAR subunits GluA1 and GluA2, but not GluA3 in the dentate gyrus. The increases in GluA1 and GluA2 levels were partially NMDAR-dependent, but were not found in biochemically isolated synaptic surface membrane or PSD fractions. In contrast, we found that the core NMDAR subunit, GluN1, increased in the synaptic surface-membrane fraction but it also was not targeted to the PSD. The GluA1 and GluA2 expression and the surface localisation of GluN1 returned to baseline levels by 2 weeks post-LTP induction. These data suggest that the late-phase LTP is not mediated by an overt increase in the AMPAR content of perforant path synapses. The increase in surface expression NMDARs may influence thresholds for future plasticity events.     

Subunit dependencies of N-methyl-D-aspartate (NMDA) receptor-induced alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor internalization

N-Methyl-D-aspartate (NMDA) receptor (NMDAR) activity regulates the net number of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPAR) at the cell surface by modulating the balance between AMPAR membrane insertion and endocytosis. In this study, we addressed the role of NMDAR subtypes and of NMDAR-mediated Ca2+ influx in the NMDAR-induced endocytosis of GluR2-containing AMPARs in primary murine hippocampal neurons. We found that NMDAR activation enhanced the endocytosis of AMPARs containing the GluR2 splice variants with short, but not long, cytoplasmic tails. NMDA-induced GluR2 endocytosis was completely inhibited by pharmacological block of NR2B-containing NMDARs. In turn, preferential block of NR2A-containing NMDARs did not affect NMDA-induced AMPAR endocytosis, indicating that AMPAR internalization is controlled by a restricted set of NMDARs. The NMDA-induced GluR2 internalization was also observed in the absence of extracellular Na+ ions, suggesting that membrane depolarization is not a prerequisite for this effect. Furthermore, the activation of Ca2+-impermeable NMDARs containing the mutant NR1(N598R) subunit failed to enhance AMPAR endocytosis, indicating a requirement of Ca2+ influx directly through the NMDAR channels. In summary, our findings suggest that the NMDAR-induced selective internalization of short C-terminal GluR2-containing AMPARs requires a Ca2+ signal that originates from NMDAR channels and is processed in an NMDAR subtype-restricted manner.     

The Role of Glutamate Receptor Redistribution in Locomotor Sensitization to Cocaine

α-Amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR) surface expression in the nucleus accumbens (NAc) is enhanced after withdrawal from repeated cocaine exposure. However, it is unclear whether this contributes to the expression of locomotor sensitization and whether similar changes can be observed in other striatal regions. In this study we examined the relationship between AMPAR surface expression in the NAc and locomotor sensitization. We also examined AMPAR distribution in the dorsolateral striatum (DS) and NMDA receptor (NMDAR) distribution in the NAc and DS. Trends but no significant changes in NMDAR distribution were found in the NAc after withdrawal. No NMDAR changes were observed in the DS. AMPAR surface expression was increased in the NAc 15 days after the last exposure to cocaine, but decreased in the DS. Re-exposure to cocaine on withdrawal day 14 decreased AMPAR surface expression in the NAc 24 h, but not 30 min, after challenge, but increased it in the DS 24 h and 30 min after challenge. Locomotor sensitization was evaluated at times associated with increased or decreased AMPAR surface expression in the NAc. The magnitude of sensitization did not vary with changes in the level of AMPAR surface expression, nor was it significantly reduced by decreasing AMPAR transmission through intra-NAc infusion of CNQX before cocaine challenge. On the basis of our results, and other findings, we suggest that the expression of sensitization has no clear relationship to altered AMPAR surface expression in the NAc, although the latter may have a role in the enhanced pursuit and self-administration of drugs observed in sensitized rats.     

The anticonvulsants lamotrigine, riluzole, and valproate differentially regulate AMPA receptor membrane localization: relationship to clinical effects in mood disorders.

A growing body of data suggests that the glutamatergic system may be involved in the pathophysiology and treatment of severe mood disorders. Chronic treatment with the antimanic agents, lithium and valproate, resulted in reduced synaptic expression of the AMPA(-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor subunit GluR1 in the hippocampus, while treatment with an antidepressant (imipramine) enhanced the synaptic expression of GluR1. The anticonvulsants, lamotrigine (6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5-diamine) and riluzole (2-amino-6-trifluoromethoxybenzothiazole), have been demonstrated to have efficacy in the depressive phase of bipolar disorder. We therefore sought to determine the role of these anticonvulsants, compared to that of the predominantly antimanic anticonvulsant valproate, on AMPA receptor localization. We found that the agents with a predominantly antidepressant profile, namely lamotrigine and riluzole, significantly enhanced the surface expression of GluR1 and GluR2 in a time- and dose-dependent manner in cultured hippocampal neurons. By contrast, the predominantly antimanic agent, valproate, significantly reduced surface expression of GluR1 and GluR2. Concomitant with the GluR1 and GluR2 changes, the peak value of depolarized membrane potential evoked by AMPA was significantly higher in lamotrigine- and riluzole-treated neurons, supporting the surface receptor changes. Phosphorylation of GluR1 at the PKA (cAMP-dependent protein kinase) site (S845) was enhanced in both lamotrigine- and riluzole-treated hippocampal neurons, but reduced in valproate-treated neurons. In addition, lamotrigine and riluzole, as well as the traditional antidepressant imipramine, also increased GluR1 phosphorylation at GluR1 (S845) in the hippocampus after chronic in vivo treatment. Our findings suggest that regulation of GluR1/2 surface levels and function may be responsible for the different clinical profile of anticonvulsants (antimanic or antidepressant), and may suggest avenues for the development of novel therapeutics for these illnesses.     

Consolidation of Remote Fear Memories Involves Corticotropin-Releasing Hormone (CRH) Receptor Type 1-Mediated Enhancement of AMPA Receptor GluR1 Signaling in the Dentate Gyrus

Persistent dreadful memories and hyperarousal constitute prominent psychopathological features of posttraumatic stress disorder (PTSD). Here, we used a contextual fear conditioning paradigm to demonstrate that conditional genetic deletion of corticotropin-releasing hormone (CRH) receptor 1 within the limbic forebrain in mice significantly reduced remote, but not recent, associative and non-associative fear memories. Per os treatment with the selective CRHR1 antagonist DMP696 (3 mg/kg) attenuated consolidation of remote fear memories, without affecting their expression and retention. This could be achieved, if DMP696 was administered for 1 week starting as late as 24 h after foot shock. Furthermore, by combining electrophysiological recordings and western blot analyses, we demonstrate a delayed-onset and long-lasting increase in AMPA receptor (AMPAR) GluR1-mediated signaling in the dentate gyrus (DG) of the dorsal hippocampus 1 month after foot shock. These changes were absent from CRHR1-deficient mice and after DMP696 treatment. Inactivation of hippocampal GluR1-containing AMPARs by antisense oligonucleotides or philantotoxin 433 confirmed the behavioral relevance of AMPA-type glutamatergic neurotransmission in maintaining the high levels of remote fear in shocked mice with intact CRHR1 signaling. We conclude that limbic CRHR1 receptors enhance the consolidation of remote fear memories in the first week after foot shock by increasing the expression of Ca²⁺-permeable GluR1-containing AMPARs in the DG. These findings suggest both receptors as rational targets for the prevention and therapy, respectively, of psychopathology associated with exaggerated fear memories, such as PTSD.     

Transient plasticity of hippocampal CA1 neuron glutamate receptors contributes to benzodiazepine withdrawal-anxiety.

Withdrawal from 1-week oral administration of the benzodiazepine (BZ), flurazepam (FZP) is associated with enhanced AMPA receptor (AMPAR)-mediated and reduced NMDA receptor (NMDAR)-mediated excitation in CA1 pyramidal neurons 2-days after cessation of FZP administration. The present study examined temporal regulation of glutamate receptor-mediated whole-cell currents in CA1 neurons from hippocampal slices prepared from 0-, 1-, 2-, and 4-day FZP-withdrawn rats in relation to expression of anxiety-like behavior during BZ withdrawal. AMPAR-mediated miniature excitatory postsynaptic current (mEPSC) amplitude was significantly increased in CA1 neurons from 1- and 2-day FZP-withdrawn rats, while evoked NMDAR EPSC amplitude was reduced only in neurons from 2-day FZP-withdrawn rats. Withdrawal-anxiety, measured in the elevated plus-maze, was observed 1 day, but not 0, 2, or 4 days, after FZP treatment with 1-day withdrawn rats spending significantly reduced time in open arms compared to controls. CA1 neuron hyperexcitability was evident from the significant increase in the frequency of extracellular, 4-AP-induced spike discharges in slices from 1-day FZP-withdrawn rats. Systemic injection of the NMDAR antagonist MK-801 (0.25 mg/kg) on day 1 of withdrawal prevented reduced NMDAR-mediated currents in CA1 neurons from 2-day FZP-withdrawn rats, whereas AMPAR-mediated currents remained upregulated. Furthermore, MK-801 'unmasked' withdrawal-anxiety in the same 2-day FZP-withdrawn rats. Systemic injection of the AMPAR antagonist GYKI-52466 (0.5 mg/kg) at the onset of withdrawal blocked increased AMPAR-mediated currents and withdrawal-anxiety in 1-day FZP-withdrawn rats. These findings suggest that increased CA1 neuron AMPAR-mediated excitation may contribute to hippocampal hyperexcitability and expression of withdrawal-anxiety after prolonged BZ exposure via NMDAR-mediated neural circuits.     

Calcium/Calmodulin-Dependent Protein Kinase II Mediates Hippocampal Glutamatergic Plasticity During Benzodiazepine Withdrawal

Benzodiazepine withdrawal anxiety is associated with potentiation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) currents in hippocampal CA1 pyramidal neurons attributable to increased synaptic incorporation of GluA1-containing AMPARs. The contribution of calcium/calmodulin-dependent protein kinase II (CaMKII) to enhanced glutamatergic synaptic strength during withdrawal from 1-week oral flurazepam (FZP) administration was further examined in hippocampal slices. As earlier reported, AMPAR-mediated miniature excitatory postsynaptic current (mEPSC) amplitude increased in CA1 neurons from 1- and 2-day FZP-withdrawn rats, along with increased single-channel conductance in neurons from 2-day rats, estimated by non-stationary noise analysis. Input–output curve slope was increased without a change in paired-pulse facilitation, suggesting increased AMPAR postsynaptic efficacy rather than altered glutamate release. The increased mEPSC amplitude and AMPAR conductance were related to CaMKII activity, as intracellular inclusion of CaMKIINtide or autocamtide-2-related inhibitory peptide, but not scrambled peptide, prevented both AMPAR amplitude and conductance changes. mEPSC inhibition by 1-naphthyl acetyl spermine and the negative shift in rectification index at both withdrawal time points were consistent with functional incorporation of GluA2-lacking AMPARs. GluA1 but not GluA2 or GluA3 levels were increased in immunoblots of postsynaptic density (PSD)-enriched subcellular fractions of CA1 minislices from 1-day FZP-withdrawn rats, when mEPSC amplitude, but not conductance, was increased. Both GluA1 expression levels and CaMKIIα-mediated GluA1 Ser⁸³¹ phosphorylation were increased in PSD-subfractions from 2-day FZP-withdrawn rats. As phospho-Thr²⁸⁶CaMKIIα was unchanged, CaMKIIα may be activated through an alternative signaling pathway. Synaptic insertion and subsequent CaMKIIα-mediated Ser⁸³¹ phosphorylation of GluA1 homomers contribute to benzodiazepine withdrawal-induced AMPAR potentiation and may represent an important hippocampal pathway mediating both drug-induced and activity-dependent plasticity.     

Cannabinoid receptor-mediated inhibition of acetylcholine release from hippocampal and cortical synaptosomes

In previous studies cannabinoid agonists have been found to inhibit and cannabinoid antagonists to enhance electrically-evoked [(3)H]-acetycholine (ACh) release in hippocampal slices. The present study was undertaken to determine if similar cannabinoid effects could be observed in synaptosomes. [(3)H]-ACh release was evoked by two methods, both sensitive to presynaptic receptor effects. The first involved the addition of 1.3 mM calcium following perfusion with calcium-free Krebs and the second the addition of 11 mM potassium following perfusion with normal Krebs. In hippocampal synaptosomes the 1.3 mM calcium-evoked release and the high potassium-evoked [(3)H]-ACh release were inhibited by the cannabinoid agonist, WIN 55212-2, by 59 and 39%, respectively, and with an EC(50) of approximately 1 nM. WIN 55212-2 produced a similar, although less potent, inhibition of [(3)H]-ACh release in cortical synaptosomes. No inhibitory effect of WIN 55212-2 on [(3)H]-ACh release in striatal synaptosomes was observed, supporting previous data collected in this area with brain slices. The cannabinoid antagonist, SR 141716A, produced a robust enhancement of 1.3 mM calcium-evoked [(3)H]-ACh release in hippocampal synaptosomes (EC(50)<0.3 nM) but had no effect on potassium-evoked release or on [(3)H]-ACh release in the cortex or striatum. In conclusion our data demonstrates the inhibitory effects of WIN 55212-2 observed on ACh release in brain slices can be observed in hippocampal and cortex synaptosomes, suggesting a direct action of these compounds on the synaptic terminals. The SR 141716A-induced enhancement of ACh release can similarly be observed in hippocampal synaptosomes and is probably due to an inverse agonist action at constitutively active receptors.     

Chronic benzodiazepine administration alters hippocampal CA1 neuron excitability: NMDA receptor function and expression(1)

Rats are tolerant to benzodiazepine (BZ) anticonvulsant actions two days after ending one-week administration of the BZ, flurazepam (FZP). Concurrently, GABA(A) receptor-mediated inhibition is reduced and AMPA receptor-mediated excitation is selectively enhanced in CA1 pyramidal neurons in hippocampal slices. In the present study, the effects of chronic FZP exposure on NMDA receptor (NMDAR) currents were examined in CA1 pyramidal neurons in hippocampal slices and following acute dissociation. In CA1 neurons from chronic FZP-treated rats, evoked NMDAR EPSC amplitude was significantly decreased (52%) in slices, and the maximal current amplitude of NMDA-induced currents in dissociated neurons was also significantly reduced (58%). Evoked NMDAR EPSCs were not altered following acute desalkyl-FZP treatment. Using in situ hybridization and immunohistochemical techniques, a selective reduction in NR2B subunit mRNA and protein expression was detected in the CA1 and CA2 regions following FZP treatment. However, total hippocampal NMDAR number, as assessed by autoradiography with the NMDAR antagonist, [(3)H]MK-801, was unchanged by FZP treatment. These findings suggest that reduced NMDAR-mediated currents associated with chronic BZ treatment may be related to reduced NR2B subunit-containing NMDARs in the CA1 and CA2 regions. Altered NMDAR function and expression after chronic BZ exposure may contribute to BZ anticonvulsant tolerance or dependence.     

Nobiletin, a citrus flavonoid with neurotrophic action, augments protein kinase A-mediated phosphorylation of the AMPA receptor subunit, GluR1, and the postsynaptic receptor response to glutamate in murine hippocampus

Nobiletin isolated from citrus peels prevents bulbectomy- and amyloid-beta protein-induced memory impairment in rodents. In the present study, using combined methods of biochemistry and electrophysiology, we examined the effects of nobiletin on phosphorylation of GluR1 receptor, the subunit of alpha-amino-3-hydroxy-5-methyl-D-aspartate (AMPA) receptors, and the receptor-mediated synaptic transmission in the hippocampus, a region implicated in memory formation, in culture and/or in slices. Western blot analysis showed that nobiletin-stimulated phosphorylation of multiple protein kinase A (PKA) substrates at 10 min following the treatment in cultured hippocampal neurons. In the cultured neurons, this natural compound also increased not only PKA activity, but also phosphorylation of GluR1 receptor at a PKA phosphorylation site, Ser 845, which has been demonstrated to be critical for synaptic plasticity, including enhancement of postsynaptic glutamate response, and important for spatial memory in vivo. The increased phosphorylation of GluR1 receptor at Ser 845 was abolished by H89 (N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride), the PKA inhibitor, but not U0126 (1,4-diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene), the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, in the cultured neurons. An increment of the phosphorylation of GluR1 receptor at Ser 845 was induced by nobiletin in the hippocampal slices as well. Furthermore, our electrophysiological analysis showed that nobiletin potentiated the AMPA receptor-mediated synaptic transmission at Schaffer collateral-CA1 pyramidal cell synapses in the hippocampal slices. This potentiation induced by the natural compound was not accompanied by the changes in paired-pulse ratio, and partially occluded the long-term potentiation, indicating the possible involvement of the postsynaptic mechanism. These findings suggest that nobiletin probably up-regulates synaptic transmission via the postsynaptic AMPA receptors at least partially by stimulation of PKA-mediated phosphorylation of GluR1 receptor in the hippocampus.     

Synaptic expression of glutamate receptor after encoding of fear memory in the rat amygdala

Fear conditioning has been ascribed to presynaptic mechanisms, particularly presynaptic facilitation of transmission at thalamo- and cortico-amygdala synapses. Here, by labeling surface receptors with biotin or using membrane fractionation approaches, we report that fear conditioning resulted in an increase in surface expression of GluR1 subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the amygdala, whereas total GluR1 mRNA and protein levels were unchanged. The control group that received conditioned stimulus (CS) and unconditioned stimulus in an unpaired fashion did not present any increase, indicating that GluR1 increase was specific to the learning component of the task. Conditioning-induced increase in surface expression of GluR1 depended on the activation of N-methyl-d-aspartate receptors and protein kinases and required the synthesis of new proteins. CS-alone trials applied 24 h before training attenuated fear-potentiated startle and prevented conditioning-induced increase in surface expression of GluR1. Increase in GluR1 was also observed in the amygdala slices after delivery of tetanic stimulation that elicited long-term potentiation of synaptic transmission. Proteasome inhibitor increased surface expression of GluR1 in a time- and dose-dependent manner. Furthermore, pretraining administration of proteasome inhibitor into the amygdala facilitated the fear-potentiated startle. These results suggest that long-term memory formation is correlated with the change in synaptic expression of GluR1, and trafficking of GluR1 to the synaptic sites contributes at least in part to the expression of fear memory.     

Protection with metabotropic glutamate 1 receptor antagonists in models of ischemic neuronal death: time-course and mechanisms.

In order to study the role of metabotropic glutamate 1 (mGlu1) receptors in ischemic neuronal death, we examined the effects of the recently characterized and relatively selective mGlu1 receptor antagonists 1-aminoindan-1,5-dicarboxylic acid (AIDA) and (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG) in murine cortical cell cultures and rat organotypic hippocampal slices exposed to oxygen glucose deprivation (OGD) and in vivo, following transient global ischemia in gerbils. AIDA and CBPG significantly reduced neuronal death when added to the incubation medium during the OGD insult and the subsequent recovery period. Neuroprotection was observed even when these compounds were added up to 60 min (in cortical neurons) or 30 min (in hippocampal slices) after OGD. In vivo, i.c.v. administration of AIDA and CBPG reduced hippocampal CA1 pyramidal cell injury following transient global ischemia. Neuroprotection was also observed when AIDA was added to the hippocampal perfusion fluid in microdialysis experiments, and this effect was associated with an increase in the basal output of GABA. These findings demonstrate that AIDA and CBPG are neuroprotective when administered during the maturation of ischemic damage and that different mechanisms are likely to be involved in mediating their effects following blockade of mGlu1 receptors in cortical and hippocampal neurons.