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Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK
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(Received 20 March 2004;
accepted after revision 16 April 2004;
first published online 23 April 2004)
Corresponding author T. S. Sihra: Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK. Email: t.sihra{at}ucl.ac.uk
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Introduction |
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Studies on the effect of the activation of kainate receptors on evoked excitatory postsynaptic currents (eEPSCs) in the hippocampus have produced diverse results. While some authors describe kainate receptor activation as producing a decrease in the amplitude of eEPSCs in CA1 (Chittajallu et al. 1996; Vignes et al. 1998; Kamiya & Ozawa, 1998; Frerking et al. 2001) and in the mossy fibre–CA3 region of the hippocampus (Kamiya & Ozawa, 2000; Schmitz et al. 2000; Contractor et al. 2000), others report that the activation of kainate receptors produces an increase of the amplitude of eEPSCs (Contractor et al. 2000; Schmitz et al. 2001; Lauri et al. 2001a,b). Interestingly, with regard to the bidirectionality of the reported effects of kainate (KA), some studies indicate that kainate receptor activation has a biphasic effect, such that low (20–50 nM) KA concentrations produce an increase in glutamatergic transmission, whereas higher concentrations produce a decrease in eEPSCs (see Kullmann, 2001; Lerma, 2003; Huettner, 2003 for reviews). With respect to the potentiating effects, kainate receptors have been suggested to contribute to the strong facilitation observed at mossy fibre synapses when the presynaptic axons are stimulated at intermediate frequencies (Schmitz et al. 2001). Contractor et al. (2000) verified the involvement of kainate receptors by demonstrating that the deletion of glutamate GluR6 receptor subunits reduced this frequency-dependent facilitation. Further evidence for a presynaptic facilitatory role for kainate receptors has come from reports implicating these receptors in mossy fibre long-term potentiation (LTP) (Bortolotto et al. 1999; Contractor et al. 2001; Lauri et al. 2001b; Schmitz et al. 2001), a form of LTP defined as being independent of postsynaptic NMDA receptors (Nicoll & Malenka, 1995).
The exact mechanism by which kainate receptors produce an increase in glutamate release remains unclear. Indeed, the precise localization of receptors that are responsible for this facilitation remains to be demonstrated. In the present work, we have examined the effect of KA on glutamate release from isolated hippocampal nerve terminals (synaptosomes), a preparation with which any confounding postsynaptic effects of KA on glutamate release are obviated by the minimal presence of functional postsynaptic elements. Additionally, we have used a more intact preparation, in the form of hippocampal slices, to study the effects of low concentrations of KA on glutamate release at the mossy fibre–CA3 synapse. Finally, we determined the mechanism involved in the effects of KA observed with the two complementary preparations. We found that the facilitation of glutamate release shows a major sensitivity to the stimulation of adenylyl cyclase (AC) activity leading to cAMP-mediated activation of protein kinase A (PKA). This indicates that kainate receptors are coupled to a cAMP cascade in some parts of the hippocampus, including, at least, mossy fibre terminals.
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Methods |
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Synaptosomes were prepared from the cerebral hippocampi of 2-month-old male rats (N= 35) essentially as previously described (Sihra, 1997). Animals were killed by stunning followed by decapitation under procedures covered by a project licence issued by the Home Office, under the Animals (Scientific Procedures) Act 1986. The final synaptosomal fraction was resuspended in Hepes-buffered incubation medium (HBM) containing (mM): 140 NaCl, 5 KCl, 5 NaHCO3, 1 MgCl2·6H2O, 1.2 Na2HPO4, 10 glucose, 20 Hepes (pH 7.4). Protein concentration was then determined using the Bradford assay. Synaptosomes were centrifuged in the final wash to obtain synaptosomal pellets with 0.5 mg protein. Synaptosomal pellets were stored on ice and used within 1–2 h.
Glutamate release assay
Glutamate release was assayed by on-line fluorometry (Nicholls & Sihra, 1986). Pelleted synaptosomes were resuspended at a protein concentration of 0.5 mg ml–1 in HBM containing 16 µM bovine serum albumin (BSA) and incubated in a stirred and thermostatted cuvette at 37°C in a Perkin-Elmer LS-3B spectrofluorimeter. NADP+ (1 mM), glutamate dehydrogenase (50 units ml–1) and CaCl2 (1 mM) were added after 3 min. After a further 10 min of incubation, 1 mM 4-aminopyridine (4-AP) was added to stimulate glutamate release. The oxidative deamination of released glutamate, leading to the reduction of NADP+, was monitored by measuring NADPH fluorescence at excitation and emission wavelengths of 340 and 460 nm, respectively. Data were accumulated at 2 s intervals. A standard of exogenous glutamate (5 nmol) was added at the end of each experiment and the fluorescence change produced by the standard addition was used to calculate the released glutamate as nanomoles glutamate per milligram synaptosomal protein. Release traces are shifted vertically to align the point of depolarization as zero release. Release values quoted in the text are levels attained at ‘steady-state’ after 4 min of depolarization (nmol (mg protein)–1 (4 min)–1). Cumulative data were analysed using Lotus 1-2-3 spreadsheets and MicroCal Origin. Statistical analysis was performed by Student's t test (* denotes P < 0.05; different from control).
Hippocampal slices
Hippocampal slices were prepared from 21- to 24-day-old rats (N= 20), as described in detail previously (Rodríguez-Moreno et al. 1997; Rodríguez-Moreno & Lerma, 1998). The whole brain containing the two hippocampi was positioned on the stage of a vibratome slicer and cut to obtain 350 µm thick transverse brain slices, which were maintained continously oxygenated for at least 1 h before use.
Electrophysiological recordings
Electrophysiological recordings were performed from neurones visually identified by IR-DIC microscopy using a 40 x water immersion objective. All experiments were carried out at room temperature (23–26°C). Slices were continously perfused with a solution consisting of (mM): 124 NaCl, 2.69 KCl, 1.25 KH2PO4, 2 MgSO4,1.8 CaCl2, 26 NaHCO3 and 10 glucose (pH 7.3, 300 mosmol l–1), supplemented with antagonists as required. Drugs were applied by gravity, switching between four perfusion lines. To evoke mossy fibre EPSCs, electrical pulses were applied by a bipolar electrode made from a glass pipette, placed in stratum lucidum just adjacent to the limit of the dentate gyrus and always within 100–200 µm from the recording site. Tight-seal (> 1 G
) whole-cell recordings were obtained from the cell body of neurones situated in the CA3 pyramidal layer. Patch electrodes were fabricated from borosilicate glass and had a resistance of 5–10 M when filled with (mM): 120 CsCl, 8 NaCl, 1 MgCl2, 0.2 CaCl2, 10 Hepes, 2 EGTA (pH 7.3, 287 mosmol l–1)). In all experiments, 20 mM QX-314 was included in the pipette solution to avoid firing of unclamped cell compartments. Neurones were voltage clamped using an Axopatch 200B amplifier (Axon Instruments). Access resistance (8–30 M) was regularly monitored during recordings and cells were rejected if it changed more than 15% during the experiment. Data were filtered at 2 kHz, digitized and stored on a computer using pCLAMP (Axon Instruments).
Compounds
Bicuculline methobromide, kainate, naloxone, and salts were purchased from Sigma; 2-hydroxy-saclofen, SYM2206, GYKI52466, DPCPX, D-2-phosphonovaleric acid (D-APV), MPPG, MCPG and L-CCG-1 were obtained from Tocris. Forskolin, dideoxyforskolin, IBMX, H-89 and Rp-Br-cAMP were purchased from Calbiochem.
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Results |
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Using an on-line enzymatic assay for measuring glutamate, we observed KA-mediated facilitation of glutamate release from hippocampal synaptosomes (Fig. 1A). We applied KA at different concentrations to hippocampal synaptosomes in the presence of the non-competitive AMPA receptor antagonists GYKI52466 or SYM2206 (100 µM) to avoid the activation of AMPA receptors by KA. The application of 10 and 100 µM KA produced a slight (6 ± 0.7 and 21 ± 4.5%, respectively) and statistically insignificant facilitation of glutamate release evoked by 1 mM 4-AP (nmol (mg protein)–1 (4 min)–1: Control, 4.7 ± 0.2, n= 20; 10 µM KA, 5.0 ± 0.4; 100 µM KA, 5.7 ± 0.5). The application of 200, 300 and 1000 µM KA, all produced a clear (30 ± 2.3, 72 ± 4.2 and 74 ± 4.9%, respectively) and statistically significant facilitation of glutamate release (nmol (mg protein)–1 (4 min)–1: 200 µM KA, 6.1 ± 0.2, n= 3; 300 µM KA, 8.1 ± 0.4, n= 7; 1000 µM KA, 8.2 ± 0.6, n= 2) (Fig. 1B). We never observed a decrease in glutamate release at any of the KA concentrations used. Given that in our hands 300 and 1000 µM KA produced similar extents of potentiation of glutamate release (72 and 74%), to minimize any effects of KA on glutamate transporters at high concentrations, we use 300 µM KA forthwith in our study.
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The kainate-induced increase in glutamate release involves the cAMP cascade in hippocampal synaptosomes
The mechanism underlying the facilitation of glutamate release by KA remains to be elucidated. Having confirmed the selectivity of the action of KA on glutamate release, we further explored the mechanism underlying the effect. We looked at the possibility that a second messenger system acts as a mediator of this effect. Lauri et al. (2001b), in studies with mossy fibre–CA3 synapses, have suggested that a signalling cascade leading to protein kinase C (PKC) activation is not involved because the facilitatory effects of KA are still present in the presence of the selective PKC inhibitor calphostin C. Given that the cAMP cascade is one of the major second messenger systems regulating glutamate release at several hippocampal synapses, we analysed the effects of upregulating and inhibiting this pathway on KA-mediated facilitation of glutamate release from hippocampal synaptosomes.
We first analysed the effect of the inhibition of the cAMP-dependent protein kinase (PKA) on glutamate release by using the cell-permeable and selective inhibitor H-89 (100 µM). H-89 itself produced an inhibition of glutamate release of 30% (data not shown), but subsequent application of 300 µM KA increased glutamate release by 30% (i.e. to a level of release comparable to control) versus the 72% facilitation of glutamate release obtained without H-89. These results indicate that inhibition of the activation of PKA somewhat prevents the action of KA (Fig. 2A and B).
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To confirm the occlusion of the effects of KA by cAMP production, we also performed the reverse experiment with KA and forskolin, in which KA was added prior to forskolin. The application of 300 µM KA produced an increase in glutamate release of 62 ± 6% (n= 4). The subsequent addition of forskolin + IBMX (100 and 50 µM, respectively) induced a facilitation of glutamate release of 15 ± 3%– indicating that KA occludes the action of the forskolin (Fig. 2B). The results indicate that the actions of forskolin and those of KA reciprocally occlude each other, suggesting their dependent use of a common intracellular mechanism to produce a facilitation of glutamate release.
The activation of kainate receptors by low concentrations of KA produces an increase in the amplitude of NMDA- and AMPA-evoked postsynaptic currents in transverse hippocampal slices
While the results from studies with hippocampal slices looking at the effect of the activation of kainate receptors on eEPSCs have been diverse (Chittajallu et al. 1996; Vignes et al. 1998; Kamiya & Ozawa, 1998, 2000; Schmitz et al. 2000, 2001; Contractor et al. 2000; Frerking et al. 2001; Lauri et al. 2001a,b), in all cases a presynaptic locus of the action for KA was proposed, postulating that both facilitatory and inhibitory effects are mediated by the activation of presynaptic kainate receptors. We reproduced these results and determined whether the mechanisms of action of KA that we observed in synaptosomes are similar to those responsible for the increases in eEPSC amplitude that we observed in slice recordings from CA3 pyramidal neurones receiving mossy fibre inputs (Fig. 3A).
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As noted for synaptosomal experiments, the effect of KA on mossy fibre terminals could be indirect and occurring in response to the activation of presynaptic receptors by neurotransmitter(s) released secondarily to KA application. To minimize the participation of putative presynaptic receptors, with the current experiments we treated the slices with a cocktail which included the mGluR antagonists MCPG and MPPG (1.5 mM each), as well as naloxone (100 µM), bicuculline (25 µM), 2-OH-saclofen (150 µM), atropine sulphate (50 µM), propanolol (100 µM) and DPCPX (0.1 µM), to block metabotropic glutamate receptors, and opioid, GABAA, GABAB, muscarinic, ß-adrenergic and adenosine receptors, respectively. Under these blocking conditions, KA was equally effective, with –30 nM KA producing a 46 ± 5%(n= 5) increase in the EPSCs. This effectively eliminated the possibility of an indirect action of KA on glutamate release through, at the least, the aforementioned receptors as presynaptic regulators (Fig. 3C).
To examine whether the observed effects of KA on EPSCs were presynaptic, we plotted the change in the coefficient of variation (CV) of synaptic responses versus the change in their averaged amplitude. Consistent with a presynaptic effect of KA, the increase in the mean EPSC (AMPA receptor-mediated) amplitude was paralleled by an increase in 1/CV2, a parameter known to vary as a function of quantal content rather than of quantal size (Forsythe & Clements, 1990; reviewed by Thomson & Deuchars, 1995). Indeed, the change in 1/CV2 was proportional to the change in mean EPSC amplitude, implying that a change in release probability accounts for most of the observed change in amplitude (Fig. 3D). The same result was obtained from NMDA-mediated currents (data not shown). These results indicate that the selective activation of a presynaptic rather than postsynaptic kainate receptor at mossy fibre synapses is instrumental in the facilitation of glutamate release, seen here as an increase in EPSC amplitude.
In hippocampal slices, as in synaptosomes, kainate receptor-mediated facilitation of glutamate release involves a cAMP-dependent cascade
Having confirmed the selectivity of the action of KA, we further explored the possibility that the same second messenger system that acts to mediate effects in synaptosomes also operates in the facilitation of EPSCs in slices.
Firstly, we studied the effect of the PKA inhibitor H-89 on glutamate release. H-89 (100 µM) itself produces an inhibition of a glutamate release of 16 ± 3% in slices; the subsequent application of KA increased glutamate release by 7% (versus the 45 ± 5% effect of KA (30 nM) without H-89) (Fig. 4A). The effect of H89 was apparent across the full range of KA concentrations producing facilitation (Fig. 4A, inset). Thus, the inhibition of PKA prevents the action of KA. We also used the PKA inhibitor Rp-Br-cAMP (100 µM). In the presence of Rp-Br-cAMP, 30 nM KA produced a small and statistically insignificant increase of a 3.8 ± 4% increase, indicating a complete abolition of the effect of KA on eEPSC amplitudes (Fig. 4A).
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As confirmed for synaptosomes previously (Wang & Sihra, 2003), to ensure in slices that the observed forskolin effects were due to cAMP production rather than any non-specific effect of the diterpene, we performed control experiments using 1,9-dideoxyforskolin, the inactive analogue of forskolin. In the presence of dideoxyforskolin (100 µM), KA (30 nM) produced an increase of 48 ± 6%(n= 4) in the amplitude of the eEPSCs, thus displaying no significant effect on eEPSCs compared to KA alone (Fig. 4A). This points to a large part of the forskolin effect on KA-mediated modulation being attributable to increases in cAMP levels produced by adenylyl cyclase activation.
Once again, we confirmed that the downstream effects of forskolin were mediated through PKA by measuring the effect of Rp-Br-cAMP on forskolin-mediated enhancement of EPSCs. Slices pre-incubated with 100 µM Rp-Br-cAMP for 30 min (the cyclic nucleotide was retained in the experimental buffer during forskolin treatment) showed a virtually complete abrogation of the forskolin-mediated enhancement of EPSCs. Together, these results indicate that forskolin, through an adenylyl cyclase (AC)–cAMP–PKA pathway, occludes the action of KA and vice versa, and that the modulation seen with these two regulators may involve a common intracellular mechanism to produce a facilitation of glutamate release.
Adenylyl cyclase I (AC I) is specifically expressed in large amounts in mossy fibres (Xia et al. 1991). The activation of the AC I to produce an increase in glutamate release can be mediated by the activation of the calcium–calmodulin-dependent protein kinase II (CaMKII), as proposed by Weisskopf et al. (1994) to explain increases in glutamate release and LTP at mossy fibre-CA3 synapses. We tested for this possibility in slices treated with the membrane-permeable inhibitor of CaMKII, KN-62. In the presence of 5 µM KN-62, KA was, however, found to be equally effective at increasing the eEPSC amplitude (41 ± 6%versus 45 ± 5% in control) (Fig. 4C). This therefore indicates that the proposed signalling pathway activated by CaMKII does not converge with the pathway triggered by kainate receptors at mossy fibre terminals.
Finally, given that slices also displayed an inhibitory effect of KA, albeit at higher concentrations of the agonist, we carried out one final control to ensure that this effect in no way impinged on the facilitation we observed. The inhibitory effects of KA have been attributed to a metabotropic mechanism involving the inhibitory G-proteins Gi/o. However, at the concentrations of KA producing facilitating effects, we observed no effect of pertussis toxin (PTX) on the response (Fig. 4C).
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Discussion |
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Presynaptic kainate receptors
From our results, it is apparent that the activation of kainate receptors produces an increase in glutamate release (at all concentrations of KA that we used in synaptosomes and at nanomolar concentrations in slices). Considering that the other glutamate receptors that can be activated by KA are blocked by SYM2206 (or GYKI52466 in some cases), and that the block of other metabotropic and ionotropic receptors potentially present did not prevent the action of KA, we can conclude that the effect that we observe is mediated by the activation of glutamate receptors of the kainate type. Additionally, CNQX, which in our experimental conditions (in synaptosomes and in slices when recording NMDA-mediated currents) was used as a kainate receptor antagonist (with AMPA receptors being blocked by SYM2206), prevented the action of KA, thus again indicating that the modulation was due to kainate receptors. Given that, in the synaptosome preparation, the presence of postsynaptic membranes is minimal and that, in slices, the change in mean current amplitude was proportional to the change in the coefficient of variation, a presynaptic mode of action for KA was indicated.
The facilitatory effects of KA on glutamate release from synaptosomes and the eEPSCs in slices qualitatively display remarkable similarity with respect to the involvement of cAMP–PKA. However, the key detractor from the unequivocal proposal of a commonality of mechanism underlying the facilitation in the two models, is the different dose dependencies observed. This discrepancy is perhaps not surprising given the undeniable difference in nature of the two preparations; however, several specific reasons can be posited for the exquisite sensitivity to KA of slices compared to synaptosomes. Firstly, axonal localization of kainate receptors has been indicated by GluR6/7 immunolabelling studies of the mossy fibre–CA3 synapse, for instance (Petralia et al. 1994). While these receptors would obviously be activated by KA in slice preparations, in isolated nerve terminals devoid of an axonal compartment, the contributions of these receptors would be severely attenuated, if not completely removed. Also likely contributing to the relative sensitivity to KA of the slices compared to synaptosomes is the potential enhancement of response in the former by heterosynaptic interactions of synapses through presynaptic, juxtasynaptic kainate receptors (Schmitz et al. 2000). This latter phenomena, thought to contribute to frequency facilitation of glutamate responses mediated through presynaptic kainate receptors (Schmitz et al. 2001), would clearly not occur in the dissociated nerve terminal situation, where indeed the very nature of the release assay used here obviates any potential effects due to endogenously released glutamate. Secondly, the parsimonious possibility remains that the higher concentrations of KA required to obtain facilitation in the synaptosomal model reflect an uncoupling/inactivation of functional receptors due to the preparative procedures. Indeed, the requirement for the relatively higher concentrations of agonist in the synaptosomal preparation compared to slices is a feature of most studies that have examined KA-mediated presynaptic modulation using biochemical methodology. Notwithstanding this, even in slice preparations, KA sensitivity can be seen to vary broadly depending on the synapse (autoreceptor activation in CA1 synapses requires 10 times higher KA than elsewhere: Kamiya & Ozawa, 1998) and subunit constitution (GluR7 subunit-containing receptors show 10-fold lower affinity that other non-NMDA receptors: Schiffer et al. 1997). These observations together reason against the rejection of a commonality of mechanism between the two models used here, purely on the basis of differing concentration dependencies of modulation.
Several previous reports have indicated that kainate receptor activation has a biphasic effect on synaptic transmission, whereby low KA concentrations (20–50 nM) produced an increase in glutamate release, but higher concentrations effected a decrease in eEPSC. While the latter inhibition was also apparent in our hands (data not shown), the hippocampal synaptosomes studied herein never displayed a decrease in glutamate release, regardless of the KA concentration used. This observation, our previous observations using cortical synaptosomes (Perkinton & Sihra, 1999), and other synaptosomal studies, including those with hippocampal synaptosomes (Malva et al. 1996; Poli et al. 1985; Zhou et al. 1995), have all supported the ‘classical studies’ with slices from a variety of brain areas (Coyle, 1983) showing facilitation of glutamate release in response to KA. In contrast to this body of literature is a prominently cited report of KA-mediated inhibition of [3H]-glutamate release from hippocampal synaptosomes (Chittajallu et al. 1996). Notwithstanding the notable fact that, while the former studies mentioned measured endogenous neurotransmitter, the latter study utilized radiolabelled tracer to assay release (with the inherent ambiguities of the methodology discussed in Perkinton & Sihra, 1999 and Nicholls & Sihra, 1986), numerous electrophysiological observations have indicated a clear inhibitory effect of KA on excitatory synaptic transmission (see Lerma, 2003; Huettner, 2003 for reviews). The question of how these studies can be reconciled with the large majority of aforementioned biochemical studies showing KA-mediated increases in neurotransmitter release therefore remains unanswered. The notion that the inhibitory kainate receptors/effects are in fact localized exclusively postsynaptically is certainly not supportable from the available evidence. However, another rather facile explanation for the lack of inhibition of glutamate release, at least in synaptosomes, could be that the ‘inhibitory kainate receptor’ is somehow lost or inactivated during the synaptosomal preparation. Yet another possibility is that presynaptic terminals need a postsynaptically released messenger to produce inhibition, and such a retrograde messenger is lost in the preparation and purification of synaptosomes. Perhaps the most tenable explanation of all arises from considering the bimodal effects of KA in slice preparations. Thus, while facilitation might occur at low concentrations of agonist through potential mechanisms discussed below, at higher concentrations, the increased ionotropic effects of KA may reasonably be suggested to cause the inactivation of voltage-dependent ion channels supporting nerve terminal excitability and thereby inhibit glutamate release (MacDermott et al. 1999; Miller, 1998; Chittajallu et al. 1996; Kamiya & Osawa, 1998). Isolated nerve terminals would not be subject to such inactivation effects to same degree, given their lack of axonal input and consequent dependence on the use of chemical activators/secretagogues for stimulation.
Mechanism of facilitatory actions of kainate on excitatory synaptic transmission
If the observed facilitation in the synaptosome and slice models studied herein does have commonality as we contend, what then are the potential mechanism(s) that might underlie facilitatory modulation? In elucidating this question, we demonstrate that the activation of the adenylyl cyclase occludes the effect of KA facilitating glutamate release. Moreover our observations that: (i) the reverse experiment (i.e. forskolin applied after KA) did not produced any additional increase in glutamate release (current study) (ii) the inactive analog of forskolin (dideoxyforskolin) did not affect glutamate release (Wang & Sihra, 2003) and (iii) the effect of KA in the presence of dideoxyforskolin in slices is unchanged (current study) taken together, suggest that the activation of the adenylyl cyclase is linked to the action of KA. Our demonstration that, following the inhibition of PKA in synaptosomes or slices, using H-89 or Rp-Br-cAMP, forskolin and KA fail to affect glutamate release, invokes an occlusion and implies that the effects of these agents are mediated by a common intracellular cascade involving the activation of PKA.
From the foregoing results discussed, the clear possibility arising is that the activation of kainate receptors somehow leads to the stimulation of the adenylyl cyclase or vice versa. What then might be the basis of such an interaction or dependence? Invoking the established ionotropic properties of kainate receptors, one immediate possibility is that a rise in intracellular Ca2+, either through a depolarization-dependent activation of Ca2+ channels or by direct kainate receptor-mediated conduction, effects a Ca2+-dependent activation of adenylate cyclase (Weisskopf et al. 1994; Cooper, 2003). The evidence regarding the entry of Ca2+ following kainate receptor activation is varied in both synaptosomes and slices, with some studies showing measurable increases in intracellular Ca2+ (Malva et al. 1995; Lauri et al. 2003) and others no effect (Perkinton & Sihra, 1999; Kamiya et al. 2002), or even decreases (Kamiya & Ozawa, 1998, 2000). One reason for the disparity may be that determination of changes in cytosolic Ca2+ in some cases is confounded by the relative inability of Ca2+-probes to detect plasma membrane localized changes in the concentration of the cation. Nevertheless, localized Ca2+ entry may certainly be sufficient or even necessary to activate a plasma membrane resident enzymes such as, for instance, Ca2+/calmodulin-sensitive isotypes of adenylate cyclase including AC1 and AC8 (Cooper, 2003).
In a recent paper, Lauri et al. (2003) have proposed that kainate receptors that are permeable to Ca2+ may be responsible for the facilitation of synaptic transmission and LTP seen at the mossy fibre-CA3 synapses, putatively through the release of intracellular Ca2+ stores, although, notably, other authors do not observe any role of Ca2+ during LTP induction at this type of synapse (Kamiya et al. 2002). While it remains to be established whether the facilitatory mechanism that we describe here is in any way involved in the induction of LTP by kainate receptors described in the aforementioned preparation, one clear indication from our data is the that facilitation is at least not sensitive to the Ca2+/calmodulin-dependent kinase II (Ca2+/CAM KII) inhibitor, KN62. Indeed the latter inhibitor has previously been shown to be ineffective in mossy fibre LTP (Huang et al. 1994). The lack of involvement of downstream activation of Ca2+/CAM KII in the KA-mediated presynaptic facilitation seen here would therefore delineate this type of modulation from the synaptic enhancement underlying hippocampal LTP in the proposed models strongly invoking a role for the kinase (Weisskopf et al. 1994). Moreover, this observation obviates the involvement of Ca2+/CAM KII-mediated phosphorylation of kainate receptor subunits (Ghetti & Heinemann, 2000; Yakel et al. 1995) in the regulation reported here.
Other than a mechanism based on the ionotropic properties of KA, in principle, the facilitatory effect described herein may be mediated by the coupling of kainate receptors through a heterotrimeric G-protein, as has been described for the modulation of GABA release by KA application (Rodríguez-Moreno & Lerma, 1998; Rodríguez-Moreno et al. 2000). A metabotropic and G-protein dependent mechanism for the action of KA might in some ways more easily rationalize the observed interactions with the AC/PKA cascade. However, while the inhibitory modulation of GABA release by KA could be confirmed on the basis of sensitivity to the Gi/o inhibitor pertussis toxin (PTX), the same type of approach is not possible with a stimulatory G-protein as might be posited for a facilitatory response. Certainly in our hands, the stimulatory effect of KA on eEPSCs persisted with the Gs stimulator, cholera toxin (data not shown), and perhaps unsurprisingly, it was also insensitive to PTX. Although this might lead one to speculate on the involvement of an ‘atypical G-protein’, at this stage as discussed above, a G-protein independent mechanism, operating under the auspices of the classical ionotropic remit of kainate receptors, remains manifestly tenable.
While it was not the purpose of this study to assign facilitatory or inhibitory kainate receptor function on the basis of subunit composition of the receptors, it is of interest to note that, of the most prevalent transcripts of kainate receptor subunits in the hippocampus (GluR5 and GluR6), neurones projecting facilitatory KA inputs (principal and dentate granule cells to CA3 neurones), express GluR6 mRNA most abundantly (Paternain et al. 2000). The notion arising from this, that GluR6 subunits may subserve a key role in the facilitatory effects of kainate receptors, is supported by the sensitivity of synaptosomal glutamate release facilitation to NS-102, a selective GluR6 subunit antagonist (Perkinton & Sihra, 1999). Moreover, studies showing an impairment in the KA-mediated frequency facilitation of synaptic transmission and LTP at mossy fibre-CA3 synapses in GluR6 knockout mice (Contractor et al. 2001) also indicate as much. Taken together with observations that the facilitatory aspect of the bimodal modulation by KA is lost in KA2 subunit knockout mice (Contractor et al. 2003), a tentative assignment can me made of a facilitatory kainate receptor composed of oligomers of GluR6 and KA2 subunits. Intriguingly, with respect to the link of facilitatory kainate receptors to AC/PKA activation reported herein, GluR6 subunits are known to be PKA substrates (Raymond et al. 1993) and phosphorylation of this subunit-type leads to up-regulation of channels activity (Traynelis & Wahl, 1997; Wang et al. 1993). Be it at this locus or another, clearly future experiments are warranted to better understand the precise link between kainate receptors and the PKA pathway alluded to here, and thereby allow a more complete elucidation of the intracellular signalling cascade leading to the facilitation of glutamate release.
In summary, we provide support for the functional interaction of facilitatory kainate receptors with a second messenger cAMP-dependent signalling cascade leading to PKA activation.
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Acknowledgements |
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Author's present address
A. Rodríguez-Moreno: División de Neurociencias, Universidad Pablo de Olavide, Ctra. de Utrera Km. 1, 41013 Seville, Spain.
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