Glutamate receptors of the kainate-type are well established as mediators of ionotropic post-synaptic synaptic transmission. Pre-synaptically, kainate receptors (KARs) have a modulatory role in regulating neurotransmitter release. In the latter regard, KARs have been shown to have a metabotropic capacity whereby they effect the regulation of both glutamate and GABA release (see Rodríguez-Moreno and Sihra 2007a,b; Jane et al. 2009 for review). At certain excitatory glutamatergic synapses, KARs activation can actually effect biphasic modulation, whereby low agonist concentrations facilitate glutamate release, while high concentrations decrease the release of the neurotransmitter (see Rodríguez-Moreno and Sihra 2007a,b for review). Mechanistic details of how this is achieved are subject of investigation and, indeed, the subcellular location of KARs responsible for pre-synaptic modulation remains contentious.
KARs are known to be highly expressed at somatosensory synapses (Bettler and Mulle 1995; Kerchner et al. 2001; Daw et al. 2007), but the precise actions of KARs at thalamocortical projection synapses require elucidation. Thalamocortical inputs have been described to participate in short-term depression (Kidd et al. 2002), but more recently, activation of KARs at synapses between axon terminals of the ventrobasal thalamus and stellate cells in Layer 4 (L4) of the somatosensory barrel cortex, has been shown to mediate a bi-directional modulation of transmission. Thus, at ‘low’ concentrations, KA (3 μM) produced an increase in glutamate release, while higher concentrations of agonist (5–30 μM) depressed neurotransmitter release (Jouhanneau et al. 2011); the extent of KAR activation evidently determining the mode of regulation. The mechanisms that mediate these diametrically opposite actions of KA at thalamocortical synapses are unclear.
Here, we have examined the mechanism underlying the facilitatory effects of KA using two preparations. Firstly, we examined the effect of KA on glutamate release from isolated cerebral nerve terminals (synaptosomes). Utilization of isolated and purified cortical nerve terminal obviates any confounding somatodendtric (post-synaptic) effects of KA on glutamate release and allows specific establishment of the functioning of definitively nerve terminal resident KARs. Secondly, we sought to elucidate the mechanistic details in a defined thalamocortical projection more specifically, and using a more intact synaptic preparation. We utilized thalamocortical slices incorporating synapses established between axon terminals from ventrobasal thalamus and stellate cells in L4 of the somatosensory barrel cortex in adult mice. These synapses are known to be important for plasticity during development, and for somatosensory integration and network activity (see Feldman et al. 1999 for review).
Using the two complementary preparations, we first established that there are common mechanistic features involved in the facilitation of glutamate release/synaptic transmission. Thus, we found that the KARs effecting facilitation of glutamate release and synaptic transmission in synaptosomes and slices have a congruent pharmacological profile, with both showing a mandatory dependence on adenylyl cyclase (AC) and cAMP-mediated protein kinase A (PKA) activity. Extending the mechanistic dissection with thalamocortical synapses in slices, we demonstrated that the KAR-mediated facilitation of transmission is contingent on both external Ca2+ permeation into the cytosol through KARs, and downstream intracellular Ca2+-store mobilization. Finally, a major sensitivity of thalamocortical facilitation to calmodulin inhibition suggests that KARs are coupled through a Ca2+-calmodulin/AC/cAMP/PKA pathway in thalamic terminals synapsing onto stellate L4 cortical neurons.
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Our results, employing electrophysiological studies in thalamocortical slices, informed by biochemical studies in isolated cortical nerve terminals (synaptosomes), show that the activation of pre-synaptic KARs at some cortical synapses produces a facilitation of synaptic transmission/glutamate release, and suggest a mechanistic coupling involving Ca2+-calmodulin/AC/cAMP/PKA activity, but independent of any G-protein activation.
The observed KA-mediated enhancement of eEPSCs at thalamocortical synapses effected was because of increased glutamate release as a similar change in NMDA receptor-mediated current (in the presence of the AMPA receptor antagonist GYKI53655), and AMPA receptor-mediated current (in the presence of the NMDA receptor antagonist AP5) was recorded. That the KA-mediated modulation displayed some common characteristics pharmacologically in synaptosomes and at a specific synapse in thalamocortical slices (i.e. AC/cAMP/PKA involvement), lends support to a mechanistic commonality, while not necessarily attributing this KA modulation to a particular nerve terminal type in the former preparation. In both, synaptosome and slice models, the facilitation by KA was blocked by CNQX, under conditions where AMPA receptors were already antagonized by GYKI53655. This supports the specific role of KARs in the regulation, particularly as a cocktail of receptor antagonists, designed to inhibit the effects of secondarily release neurotransmitters, had no effect.
In considering synaptic regulation, it remains paramount to identify the subcellular location of the receptor postulated. The strength of the synaptosome model is that, with functional post-synaptic elements eliminated in the preparation, observed pre-synaptic actions by KA must, by definition, be because of nerve terminal resident KARs. Using thalamocortical slices, although modulation could be examined at a defined synapse, to gain further insight into the locus of KAR-mediated facilitation of synaptic transmission, further analysis was warranted. Thus, to determine the pre- or post-synaptic presence and activity of KARs at thalamocortical synapses, we employed electrophysiological analysis using four different approaches. Firstly, we analysed the pair-pulse ratio of consecutive eEPSCs in thalamocortical slices. A clear increase in paired-pulse ratio of eEPSCs observed with KA application was suggestive of a change in release probability (definitively a pre-synaptic component in synaptic transmission/regulation). Secondly, we observed that the increase in the mean eEPSCs amplitude was paralleled by an increase in 1/CV2, again suggesting a pre-synaptic effect of KA.
Thus, thirdly, we determined the effect of KA on the proportion of synaptic failures. When KA was present, the proportion of failures was clearly decreased, consistent with an increase in pre-synaptic transmitter release probability, in line with the facilitation seen. Finally, similar effects of KA observed for NMDA- and AMPA receptor-mediated currents are indicative of a pre-synaptic mode of action for KARs, as no equivalence would otherwise be expected if the modulation was post-synaptic. Altogether, all four of these independent analyses confirmed and emphasized a pre-synaptic locus of action of KARs operating at thalamocortical synapses.
The question as to whether, the pre-synaptic regulation by KARs in thalamocortical neurons reflects the activity of nerve terminal/axonal or somatodendritically localized receptor, requires further deliberation. Considering the possibility that modulation mediated by putative somatodentric KARs might reflect a change in the spike threshold, we have conducted some preliminary field recording experiments to address this issue. In these experiments, consistent with Jouhanneau et al. (2011), we found no correlation between changes in fibre volley and the effect of KA on the fEPSP amplitude, and indeed, the former operated with distinct temporal characteristics in comparison to the KA-dependent synaptic modulation reported. Thus, the indications are that changes of excitability cannot explain the facilitation by KA reported herein, but rather we are moved to hypothesize the presence and operation of terminal or axonal KARs in the regulation. While, additional electrophysiological analyses could offer some elaboration of the characteristics of the thalamocortical synapse along similar lines to Jouhanneau et al. (2011), subcellular electrophysiological or pharmacological isolation of KARs proves difficult (space clamp and agonist spillover considerations respectively). Therefore, the unequivocal delineation of KAR compartmentalization prompts more direct approaches. Although technically challenging paradigms outwith the remit of the present study, to explicitly address pre-synaptic compartmentalization of KARs, two approaches warranting future consideration are: (i) The use of immunogold-based receptor localization studies; contingent on the availability of KAR antibodies with sufficient specificity and avidity. (ii) The use of targetable caged-blockers of KARs; pending development of such reagents (cf NMDA receptors studies, Rodríguez-Moreno et al. 2011). In lieu of the necessary innovations to examine KAR compartmentalization in thalamocortical neurons, the use of cortical synaptosomes does at least allow the demonstration that the KAR-mediated signalling posited does in fact operate in pre-synaptic terminals, that is, a model devoid of other functional compartments, though the shortcoming here is that a homogeneous preparation of a single terminal type is not feasible at present.
Notwithstanding the still unresolved issue about the subcellular localization of pre-synaptic thalamocortical KARs, aligning the mechanistic details of the KA-mediated regulation in synaptosomes and thalamocortical slices showed remarkable congruence. As with our previous synaptosomal studies in the hippocampus (Rodríguez-Moreno and Sihra 2004), inhibition of PKA activation using the cell-permeant cyclic nucleotide analogue Rp-Br-cAMP, led to an elimination of KA-mediated enhancement of synaptic transmission/glutamate release at thalamocortical synapses. The correspondence of mechanism was also emphasized in the current studies in that inhibition of the catalytic activity of PKA by H-89 suppressed the KA-mediated facilitation in both synaptosome and slice models of glutamate release. Likewise, direct activation of AC by forskolin (+IBMX) produced occlusion of the effects of KA in both preparations. Together, these data consistently point to an AC/cAMP/PKA signalling cascade underpinning the facilitatory regulation of glutamate release, which nonetheless remained recalcitrant to inhibition of G-proteins.
The facilitatory effects of KA on glutamate release from synaptosomes and the eEPSC in slices qualitatively display conspicuous similarity with respect to the involvement of AC/cAMP/PKA. However, the key detractor from an unequivocal suggestion of commonality of mechanism underlying the facilitation in the two models is clearly the different dose-dependencies observed. Although the undeniable difference in complexity of the two preparations likely contributes to the discrepancy, several specific reasons can be posited for the exquisite sensitivity to KA in slices compared to synaptosomes. Firstly, axonal localization of KARs has been indicated by GluK2/3 immunolabelling studies, albeit described in the hippocampus (Petralia et al. 1994). While axonal or axo-dendritic KARs would be activated by KA in a slice preparation, in isolated nerve terminals, depleted of any axonal compartment for purpose, the contribution of these receptors would be severely attenuated, if not completely eliminated. Also likely contributing to the relative sensitivity of the slices to KA compared with synaptosomes, is the enhancement of response in the former through heterosynaptic interaction of synapses through pre-synaptic, juxtasynaptic KARs (Schmitz et al. 2000). This latter phenomena, thought to contribute to frequency facilitation of glutamate responses mediated through pre-synaptic KARs in the hippocampus (Schmitz et al. 2001), would clearly not occur in the dissociated nerve terminal situation, where indeed the nature of release assay obviates any effects due to endogenously released glutamate. Secondly, the frugal possibility remains that higher concentrations of KA required to obtain facilitation in synaptosomal model reflects an uncoupling/inactivation of functional receptors because of preparative procedures. The requirement for the relatively higher concentrations of agonist in the synaptosomal preparation compared with slices is indeed a feature of studies examining KA-mediated pre-synaptic modulation using the biochemical methodology. Even in slice preparations, KA sensitivity can be seen to vary broadly depending on the synapse (autoreceptor activation in CA1 synapses in the hippocampus for example, requires 10-times higher kainate than elsewhere [Kamiya and Ozawa 1998; ]) and subunit constitution (GluK3-subunit containing receptors show 10-fold lower affinity than other non-NMDA receptors [Schiffer et al. 1997]). These foregoing observations 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. This is not to imply, however, that the synaptosome data obtained from a heterogenous population of terminals specifically reflect, in quantitative terms, the behaviour of thalamocortical terminals being examined in slices. In the absence of a specific marker, it is indeed not possible to quantitate how much of the synaptosome population represents thalamocortical terminals. Aside the aforementioned differences, the synaptosome model has been useful in several ways. First, it has allowed us to directly ascertain whether pre-synaptic KARs, as terminal-resident receptors, exist and modulate the release of glutamate, and determine the specific intracellular signalling pathway(s) involved in the regulation. Secondly, the consistent absence of KA-mediated depression espouses the hypothesis that pre-synaptic KARs involved in depression of glutamate release are not terminal-resident and, indeed, infer that the distinct populations of KAR mediating respective facilitation and depression of release are separate entities in different compartments.
Although the synaptosome model provided an essential basis for our studies at the outset, to obviate issues with heterogeneous readout from a mixed population of terminals obtained with this preparation, we conducted the further dissection of the details of the observed KAR-mediated facilitation in the thalamocortical slice preparation, assaying a specific/defined glutamatergic synapse. Given the lack of evidence for an upstream G-protein mediated initiation/transduction of the proposed AC/cAMP/PKA cascade involved in pre-synaptic KAR-mediated enhancement of glutamate release, we considered the potential role of Ca2+ as the instigator of the signalling at defined thalamocortical synaptic model. Potentially, KARs can effect external Ca2+ entry through, either an ionotropic activity to depolarize nerve terminals (Perkinton and Sihra 1999) and thereby activate voltage-gated Ca2+ channels or, directly, through Ca2+ permeable KARs per se (Fletcher and Lodge 1996; Scott et al. 2008). Intriguingly, in thalamocortical synapses, a blockade of the latter by philanthotoxin eliminated the KA-mediated synaptic facilitation, suggesting a strict dependence of the modulation on external Ca2+ entry via KARs. Thus, although unedited Ca2+ permeable KARs usually prevail early in neuronal development, the receptors evidently persist at thalamocortical synapses in adult brains.
Continuing the analysis of requirements for KAR-mediated regulation, we evaluated the possibility that core, but likely limited entry of Ca2+ via KARs, may be amplified by intraterminal Ca2+ store mobilization as has been described at hippocampal synapses (Lauri et al. 2003; Scott et al. 2008). Indeed, a crucial role for intraterminal Ca2+ stores was emphasized by our observations that, in the presence of thapsigargin, which effectively depletes intracellular Ca2+ stores (Irving et al. 1992), the facilitation of glutamate release mediated by KARs was abolished. Furthermore, by treating the slices with ryanodine, and thereby selectively inhibiting Ca2+-induced Ca2+-release (Berridge 1998), we demonstrated that Ca2+ entering via KARs induces Ca2+ mobilization from intraterminal Ca2+ stores.
Using two different pharmacological approaches in thalamocortical slices, we have demonstrated that Ca2+ entering via KARs and release of Ca2+ from intraterminal stores is indeed obligatory for the facilitation of glutamate release produced by KA. The question remained, how might the increase in cytosolic [Ca2+] couple to the postulated AC/cAMP/PKA signalling underlying the facilitation by KAR. A tenable possibility is that the increased cytosolic [Ca2+] activates Ca2+-dependent ACs present in thalamocortical terminals. Numerous ACs have been described, but two members of the family, viz. AC1 and AC8, are known to be activated by Ca2+-calmodulin, are prevalent in the central nervous system (see Wang and Storm 2003; Cooper 2003 for reviews), and have been shown to be essential for Ca2+-stimulated elevation of cAMP in studies with double knockouts of AC1 and AC8 (Wong et al. 1999). Our data, showing that the calmodulin antagonists W-7 and CMZ abolished the pre-synaptic KAR-mediated modulation in thalamocortial slices, support the hypothesis that formation of a Ca2+-calmodulin complex following KAR activation might stimulate AC1 and/or AC8, and thereby instigate the AC/cAMP/PKA cascade in the promotion of facilitation of glutamate release at thalamocortical synapses.
The experiments with thalamocortical slices described herein show the ventrobasal thalamus-L4 stellate synapse to be a robust model for the study of KAR-mediated modulation. Interestingly, however, we observed a facilitation of synaptic transmission/glutamate release at 1 μM KA, contrary to previous study with the same synapse, where 3 μM KA was required (Jouhanneau et al. 2011). The likely reason for this discrepancy is the different age of animals used for the experiments (we used adult animals whereas Jouhanneau et al. 2011 utilized 1 week post-natal animals). The higher concentrations of KA necessary for the activation of KARs at juvenile synapses may well reflect the low efficacy of KA at KARs lacking GluK4 and GluK5 subunits as suggested (Jouhanneau et al. 2011). Conversely, the lower effective concentrations of KA at adult thalamocortical synapses may indicate compositional plasticity of KARs, with the addition of at least some GluK4 or GluK5 subunits increasing KAR affinity for KA at mature synapses.
Although it is clear from our results that pre-synaptic KAR function certainly persists in adult animals at thalamocortical synapses, and is not restricted to the first few post-natal days as reported previously (Kidd et al. 2002), the question remains as to why, functionally, juvenile KARs may subtend relatively low affinity for KA (and glutamate) (Jouhanneau et al. 2011) compared with KARs at the same synapse in adult animals. One explanation might be that, in developing animals, KARs have an autoreceptor role, with the levels of activation determining pre-synaptic facilitation (at low [KA]) and depression (at high [KA]), and thereby presumably affecting synapse consolidation. While the exact role(s) of these KARs in adult animals remains to be elucidated, the modulation of glutamate release reported herein might well be involved in some forms of plasticity (Negrete-Díaz et al. 2007).
In our experiments, we observe that KAR activation has a biphasic effect at ventrobasal thalamus-L4 synapses as previously reported (Jouhanneau et al. 2011), inducing a depression of glutamate release at relatively high concentrations (> 1–3 μM). While the scope of the present study was to determine the mechanisms involved in the observed facilitation, and not to determine the intracellular mechanisms involved in the depression, our results indicated that the transient depression observed with higher [KA] is abolished in the presence of Rp-Br-cAMP, and is not affected by philantothoxin, Tsg, CMZ or W-7. These results suggest that the observed depression likely involves the AC/cAMP/PKA pathway as described for facilitation, indicating that these KARs may have alternative modes of facilitatory and depressive action, respectively coupling to an increase in cAMP levels (and subsequent activation of PKA), or to a decrease in cAMP levels (and subsequent decrease in activation of PKA), as has been described in the mossy fiber-CA3 synapses of the hippocampus (Negrete-Díaz et al. 2006). Our present results suggest that, while the activation of AC, to mediate a facilitation of glutamate release, involves the Ca2+-calmodulin complex, these KARs can also be negatively coupled to the AC/cAMP/PKA pathway by decreasing the activation of AC. As we reported previously for the depression of glutamate release mediated by activation of KARs at mossy-fiber-CA3 synapses, the negative coupling of the receptor to the AC/cAMP/PKA pathway mediating the depression, might involve the action of a pertussis toxin sensitive G-protein (Negrete-Díaz et al. 2006), as we indeed confirm here. Finally, the possibility remains that these diametric mechanisms are mediated by two different types of KARs. Future study will determine the exact instruments involved in the observed modulation and whether one or two different populations of KARs exist pre-synaptically at thalamocortical synapses.
In conclusion, our data show that the activation of pre-synaptic KARs by KA in thalamocortical synapses results in the facilitation of glutamate release, pharmacologically and mechanistically congruent to that observed in isolated cortical nerve terminals. We propose that the mechanism of KA-mediated pre-synaptic facilitation involves an entry of Ca2+ through Ca2+ permeable KARs, which triggers the release of Ca2+ from internal stores in thalamocortical terminals. The raised Ca2+ binds to calmodulin to form a Ca2+-calmodulin complex, which then putatively activates AC1 or AC8 to produce an increase in cAMP levels and a resultant stimulation of PKA; now established to result in an enhancement of glutamate release and hence synaptic transmission.