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Transient brain insults, including status epilepticus (SE), can trigger a period of epileptogenesis during which functional and structural reorganization of neuronal networks occurs resulting in the onset of focal epileptic seizures. In recent years, mechanisms that regulate the dynamic transcription of individual genes during epileptogenesis and thereby contribute to the development of a hyperexcitable neuronal network have been elucidated. Our own results have shown early growth response 1 (Egr1) to transiently increase expression of the T-type voltage-dependent Ca2+channel (VDCC) subunit CaV3.2, a key proepileptogenic protein. However, epileptogenesis involves complex and dynamic transcriptomic alterations; and so far, our understanding of the transcriptional control mechanism of gene regulatory networks that act in the same processes is limited. Here, we have analyzed whether Egr1 acts as a key transcriptional regulator for genes contributing to the development of hyperexcitability during epileptogenesis. We found Egr1 to drive the expression of the VDCC subunit α2δ4, which was augmented early and persistently after pilocarpine-induced SE. Furthermore, we show that increasing levels of α2δ4 in the CA1 region of the hippocampus elevate seizure susceptibility of mice by slightly decreasing local network activity. Interestingly, we also detected increased expression levels of Egr1 and α2δ4 in human hippocampal biopsies obtained from epilepsy surgery. In conclusion, Egr1 controls the abundance of the VDCC subunits CaV3.2 and α2δ4, which act synergistically in epileptogenesis, and thereby contributes to a seizure-induced “transcriptional Ca2+channelopathy.”SIGNIFICANCE STATEMENTThe onset of focal recurrent seizures often occurs after an epileptogenic process induced by transient insults to the brain. Recently, transcriptional control mechanisms for individual genes involved in converting neurons hyperexcitable have been identified, including early growth response 1 (Egr1), which activates transcription of the T-type Ca2+channel subunit CaV3.2. Here, we find Egr1 to regulate also the expression of the voltage-dependent Ca2+channel subunit α2δ4, which was augmented after pilocarpine- and kainic acid-induced status epilepticus. In addition, we observed that α2δ4 affected spontaneous network activity and the susceptibility for seizure induction. Furthermore, we detected corresponding dynamics in human biopsies from epilepsy patients. In conclusion, Egr1 orchestrates a seizure-induced “transcriptional Ca2+channelopathy” consisting of CaV3.2 and α2δ4, which act synergistically in epileptogenesis.

The size and structure of the dendritic arbor play important roles in determining how synaptic inputs of neurons are converted to action potential output. The regulatory mechanisms governing the development of dendrites, however, are insufficiently understood. The evolutionary conserved Ste20/Hippo kinase pathway has been proposed to play an important role in regulating the formation and maintenance of dendritic architecture. A key element of this pathway, Ste20-like kinase (SLK), regulates cytoskeletal dynamics in non-neuronal cells and is strongly expressed throughout neuronal development. However, its function in neurons is unknown. We show that, during development of mouse cortical neurons, SLK has a surprisingly specific role for proper elaboration of higher, ≥ third-order dendrites both in male and in female mice. Moreover, we demonstrate that SLK is required to maintain excitation-inhibition balance. Specifically, SLK knockdown caused a selective loss of inhibitory synapses and functional inhibition after postnatal day 15, whereas excitatory neurotransmission was unaffected. Finally, we show that this mechanism may be relevant for human disease, as dysmorphic neurons within human cortical malformations revealed significant loss of SLK expression. Overall, the present data identify SLK as a key regulator of both dendritic complexity during development and inhibitory synapse maintenance. SIGNIFICANCE STATEMENT We show that dysmorphic neurons of human epileptogenic brain lesions have decreased levels of the Ste20-like kinase (SLK). Decreasing SLK expression in mouse neurons revealed that SLK has essential functions in forming the neuronal dendritic tree and in maintaining inhibitory connections with neighboring neurons.

Depolarization of presynaptic terminals stimulates calcium influx, which evokes neurotransmitter release and activates phosphorylation-based signalling. Here, we present the first global temporal profile of presynaptic activity-dependent phospho-signalling, which includes two KCl stimulation levels and analysis of the poststimulus period. We profiled 1,917 regulated phosphopeptides and bioinformatically identified six temporal patterns of co-regulated proteins. The presynaptic proteins with large changes in phospho-status were again prominently regulated in the analysis of 7,070 activity-dependent phosphopeptides from KCl-stimulated cultured hippocampal neurons. Active zone scaffold proteins showed a high level of activity-dependent phospho-regulation that far exceeded the response from postsynaptic density scaffold proteins. Accordingly, bassoon was identified as the major target of neuronal phospho-signalling. We developed a probabilistic computational method, KinSwing, which matched protein kinase substrate motifs to regulated phosphorylation sites to reveal underlying protein kinase activity. This approach allowed us to link protein kinases to profiles of co-regulated presynaptic protein networks. Ca2+- and calmodulin-dependent protein kinase IIα (CaMKIIα) responded rapidly, scaled with stimulus strength, and had long-lasting activity. Mitogen-activated protein kinase (MAPK)/extracellular signal–regulated kinase (ERK) was the main protein kinase predicted to control a distinct and significant pattern of poststimulus up-regulation of phosphorylation. This work provides a unique resource of activity-dependent phosphorylation sites of synaptosomes and neurons, the vast majority of which have not been investigated with regard to their functional impact. This resource will enable detailed characterization of the phospho-regulated mechanisms impacting the plasticity of neurotransmitter release., Analysis of activity-dependent phosphorylation-based signalling in synaptosomes revealed six patterns of long-lasting presynaptic regulation from 1,917 phosphopeptides. The authors identified patterns most likely to be regulated by CamKII and MAPK/ERK and showed the active zone scaffold protein bassoon to be a major signalling target., Author summary Neurobiological processes are altered by linking neuronal activity to regulated changes in protein phosphorylation levels that influence protein function. Although some of the major targets of activity-dependent phospho-signalling have been identified, a large number of substrates remain unknown. Here, we have screened systematically for these substrates and extended the list from hundreds to thousands of phosphorylation sites, thereby providing a new depth of understanding. We monitored phospho-signalling for 15 min after the stimulation, which to our knowledge had not been attempted at a large scale. We focused on presynaptic protein substrates of phospho-signalling by isolating the presynaptic terminal. We also stimulated hippocampal neurons but did not monitor the poststimulus. Although the phospho-signalling is immensely complex, the findings could be simplified through data exploration. We identified distinct patterns of presynaptic phospho-regulation across the time course that may constitute co-regulated protein networks. In addition, we found a subset of proteins that had many more phosphorylation sites than the average and high-magnitude responses, implying major signalling or functional roles for these proteins. We also determined the likely protein kinases with the strongest responses to the stimulus at different times using KinSwing, a computational tool that we developed. This resource reveals a new depth of activity-dependent phospho-signalling and identifies major signalling targets, major protein kinases, and co-regulated phosphoprotein networks.

The large isoforms of the Rab3 interacting molecule (RIM) family, RIM1α/β and RIM2α/β, have been shown to be centrally involved in mediating presynaptic active zone function. The RIM protein family contains two additional small isoforms, RIM3γ and RIM4γ, which are composed only of the RIM-specific C-terminal C2B domain and varying N-terminal sequences and whose function remains to be elucidated. Here, we report that both, RIM3γ and RIM4γ, play an essential role for the development of neuronal arborization and of dendritic spines independent of synaptic function. γ-RIM knock-down in rat primary neuronal cultures andin vivoresulted in a drastic reduction in the complexity of neuronal arborization, affecting both axonal and dendritic outgrowth, independent of the time point of γ-RIM downregulation during dendrite development. Rescue experiments revealed that the phenotype is caused by a function common to both γ-RIMs. These findings indicate that γ-RIMs are involved in cell biological functions distinct from the regulation of synaptic vesicle exocytosis and play a role in the molecular mechanisms controlling the establishment of dendritic complexity and axonal outgrowth.

Synaptic signaling to NG2-expressing oligodendrocyte precursor cells (NG2 cells) could be key to rendering myelination of axons dependent on neuronal activity, but it has remained unclear whether NG2 glial cells integrate and respond to synaptic input. Here we show that NG2 cells perform linear integration of glutamatergic synaptic inputs and respond with increasing dendritic calcium elevations. Synaptic activity induces rapid Ca2+ signals mediated by low-voltage activated Ca2+ channels under strict inhibitory control of voltage-gated A-type K+ channels. Ca2+ signals can be global and originate throughout the cell. However, voltage-gated channels are also found in thin dendrites which act as compartmentalized processing units and generate local calcium transients. Taken together, the activity-dependent control of Ca2+ signals by A-type channels and the global versus local signaling domains make intracellular Ca2+ in NG2 cells a prime signaling molecule to transform neurotransmitter release into activity-dependent myelination. DOI: http://dx.doi.org/10.7554/eLife.16262.001

Vesicular release of neurotransmitter is the universal output signal of neurons in the brain. It is generally believed that fast transmitter release is restricted to nerve terminals that contact postsynaptic cells in the gray matter. Here we show in the rat brain that the neurotransmitter glutamate is also released at discrete sites along axons in white matter in the absence of neurons and nerve terminals. The propagation of single action potentials along axons leads to rapid vesicular release of glutamate, which is detected by ionotropic glutamate receptors on local oligodendrocyte precursor cells. Axonal release of glutamate is reliable, involves highly localized calcium microdomain signaling and is strongly calcium cooperative, similar to vesicle fusion at synapses. This axonal transmitter release represents a widespread mechanism for high-fidelity, activity-dependent signaling at the axon-glia interface in white matter.

Hippocampal mossy fiber synapses show unique molecular features and dynamic range of plasticity. A recent paper proposed that the defining features of mossy fiber synaptic plasticity are caused by a local buildup of extracellular adenosine (Moore et al., 2003). In this study, we reassessed the role of ambient adenosine in regulating mossy fiber synaptic plasticity in mouse and rat hippocampal slices. Synaptic transmission was highly sensitive to activation of presynaptic adenosine A1receptors (A1Rs), which reduced transmitter release by >75%. However, most of A1Rs were not activated by ambient adenosine. Field potentials increased only by 20-30% when A1Rs were fully blocked with the A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (1 μm). Moreover, blocking A1Rs hardly altered paired-pulse facilitation, frequency facilitation, or posttetanic potentiation. Frequency facilitation was similar inA1R-/-mice and when measured with NMDA receptor-mediated EPSCs in CA3 pyramidal cells in the presence of DPCPX. Additional experiments suggested that the results obtained by Moore et al. (2003) can partially be explained by their usage of a submerged recording chamber and elevated divalent cation concentrations. In conclusion, a reduction of the basal release probability by ambient adenosine does not underlie presynaptic forms of plasticity at mossy fiber synapses.

Hybrid clamp protocols, in which a discontinuous single electrode voltage clamp (dSEVC) amplifier is switched from current to voltage clamp during the recording, are frequently used to investigate conductance changes after high frequency trains of action potentials. This technique is advantageous because it combines physiological stimulation of the cell with the possibility of analyzing the consecutive conductance changes quantitatively. In this study an improved hybrid clamp protocol, called dynamic hybrid clamp, is developed that enables the experimenter to study tail currents after single action potentials. The protocol employs real time action potential detection to assure precise timing of the mode switch and utilizes an external sample and hold amplifier to avoid voltage steps during the switch to voltage clamp. With the use of whole-cell patch clamp recordings and high switching frequencies (or =25 kHz), dSEVC can easily be started with a minimal delay (1.5 ms) after single action potentials and tail currents underlying afterhyperpolarisations (AHPs) and afterdepolarisations ensuing single spikes are clearly resolved. The dynamic hybrid clamp should also be useful for analysis of spontaneously occurring events such as intrinsic or population bursts.

We investigated the electrophysiology of morphologically identified human granule cells with conventional current-clamp recordings. Slices were prepared from 14 human epileptic sclerotic hippocampi. Granule cells appeared to have a diverse electrophysiology. Each cell was distinguished by the shape of the afterhyperpolarization following single action potentials. Two types could be discerned: type I afterhyperpolarizations were monophasic and brief (typically 10-40 ms), whilst type II afterhyperpolarizations were biphasic and long (typically 50-100 ms). The two types also differed in their repetitive firing behaviour and action potential morphology: type I cells had significantly weaker spike frequency adaptation, lower action potential amplitude and smaller action potential upstroke/downstroke ratio. Thus, the firing pattern of type I cells resembled that of rodent dentate interneurons. In contrast, the corresponding parameters of type II cells were comparable to rodent dentate granule cells. Despite the distinct firing patterns, membrane properties were not different. The two types of cells also differed in their synaptic responses to stimulation of the perforant path. At strong suprathreshold stimulation intensity, type I cells always generated multiple action potentials, whereas type II cells usually spiked once only. Slow inhibitory postsynaptic potentials were not detected in type I neurons, but were easily identified in type II neurons. Extracellular recordings of perforant path-evoked field potentials in the cell layer confirmed that the majority of granule cells showed multiple discharges even when we recorded simultaneously from a type II cell that generated one action potential only. The morphology of both types of cells was characteristic of what has been described for primate dentate granule cells. Based on comparisons with previous studies on rodent and human granule cells, we tentatively hypothesize that: (i) the majority of granule cells from sclerotic hippocampus display an hyperexcitable epileptogenic electrophysiology; (ii) there is a subset of granule cells whose electrophysiology is preserved and is more comparable to granule cells from non-epileptic hippocampus.

It has remained difficult to ascribe a specific functional role to immobile or fixed intracellular calcium buffers in central neurons because the amount of these buffers is unknown. Here, we explicitly isolated the fixed buffer fraction by prolonged whole-cell patch-clamp dialysis and quantified its buffering capacity in murine hippocampal slices using confocal calcium imaging and the “added-buffer” approach. In dentate granule cells, the calcium binding ratio (κ) after complete washout of calbindin D28k(Cb), κfixed, displayed a substantial value of ∼100. In contrast, in CA1 oriens lacunosum moleculare (OLM) interneurons, which do not contain any known calcium-binding protein(s), κfixedamounted to only ∼30. Based on these values, a theoretical analysis of dendritic spread of calcium after local entry showed that fixed buffers, in the absence of mobile species, decrease intracellular calcium mobility 100- and 30-fold in granule cells and OLM cells, respectively, and thereby strongly slow calcium signals. Although the large κfixedalone strongly delays the spread of calcium in granule cells, this value optimizes the benefits of additionally expressing the mobile calcium binding protein Cb. With such high κfixed, Cb effectively increases the propagation velocity to levels seen in OLM cells and, contrary to expectation, does not affect the peak calcium concentration close to the source but sharpens the spatial and temporal calcium gradients. The data suggest that the amount of fixed buffers determines the temporal availability of calcium for calcium-binding partners and plays a pivotal role in setting the repertoire of cellular calcium signaling regimens.

NG2-expressing oligodendrocyte precursor cells (OPCs) are ubiquitous and generate oligodendrocytes throughout the young and adult brain. Previous work has shown that virtually every NG2 cell receives synaptic input from many axons, but the meaning of this signaling is not understood. In particular, it is unclear whether neurons specifically synapse onto OPCs or whether OPCs merely trace adjacent neurotransmitter release sites and are not recognized by the presynaptic neuron. Here, we show with whole-cell recordings from distinct developmental stages of oligodendroglial cells in brain slices that synaptic input essentially disappears as soon as OPCs differentiate into premyelinating oligodendrocytes (NG2 − , DM20/PLP + , O1 + ). Uncaging experiments and tracer loading revealed that premyelinating oligodendrocytes still express a substantial number of AMPA/kainate receptors and many processes, but spontaneous and stimulated synaptic currents are mostly absent. Nevertheless, in a minority of premyelinating cells, electrical stimulation evoked small synaptic currents with an unusual behavior: their amplitude compared well with the quantal amplitude in OPCs but they occurred asynchronously and with the remarkable latency of 40–100 ms, indicating that the presynaptic release machinery has become ineffective. Mature myelinating oligodendrocytes completely lack AMPA/kainate receptors and respond to uncaging and synaptic stimulation with glutamate transporter currents. Our data show that neurons selectively synapse onto only one of several coexisting developmental stages of glial cells and thereby indicate that neurons indeed specifically signal to OPCs and are able to modulate transmitter output by regulating the local release machinery in a manner specific to the developmental stage of the postsynaptic glial cell.

Removal of neurotransmitter from the extracellular space is crucial for normal functioning of the central nervous system. In this study, we have used high-affinity metabotropic glutamate receptors (mGluRs) expressed by hippocampal CA1 pyramidal cells to test how far bath-applied glutamate penetrates into slice tissue before being removed by uptake mechanisms. Activation of group I mGluRs by 100 microM DHPG produced an inward current of -48+/-10pA (I(mGluR)), which was blocked by application of group I mGluR antagonists. In contrast, bath application of 100 microM glutamate in the presence of a ionotropic glutamate receptor antagonist and TTX did not activate I(mGluR) in CA1 cells patch-clamped at a depth of approximately 30 microm. Similarly, sole inhibition of glutamate transporters by the broad-spectrum glutamate transporter antagonist TBOA did not induce I(mGluR) under the same conditions. Only if glutamate was co-applied with TBOA an I(mGluR) of -39+/-8pA was recorded which was also blocked by group I antagonists. The data suggest that TBOA-sensitive uptake mechanisms are able to maintain a steep concentration gradient of glutamate to such a degree that a CA1 neuron at a depth of 30 microm is exposed to low extracellular glutamate levels that are not sufficient to induce a detectable activation of group I mGluRs (2 microM).

The precise shape of action potentials in cortical neurons is a key determinant of action potential-dependent Ca2+influx, as well as of neuronal signaling, on a millisecond scale. In cortical neurons, Ca2+-sensitive K+channels, or BK channels (BKChs), are crucial for action potential termination, but the precise functional interplay between Ca2+channels and BKChs has remained unclear. In this study, we investigate the mechanisms allowing for rapid and reliable activation of BKChs by single action potentials in hippocampal granule cells and the impact of endogenous Ca2+buffers. We find that BKChs are operated by nanodomains of single Ca2+channels. Using a novel approach based on a linear approximation of buffered Ca2+diffusion in microdomains, we quantitatively analyze the prolongation of action potentials by the Ca2+chelator BAPTA. This analysis allowed us to estimate that the mean diffusional distance for Ca2+ions from a Ca2+channel to a BKCh is ∼13 nm. This surprisingly short diffusional distance cannot be explained by a random distribution of Ca2+channels and renders the activation of BKChs insensitive to the relatively high concentrations of endogenous Ca2+buffers in hippocampal neurons. These data suggest that tight colocalization of the two types of channels permits hippocampal neurons to regulate global Ca2+signals by a high cytoplasmic Ca2+buffer capacity without affecting the fast and brief activation of BKChs required for proper repolarization of action potentials.

In the hippocampus of chronic temporal lobe epilepsy, many abnormalities in structure and function have been described but their pathophysiological relevance often is poorly understood. In this study, we asked whether there may be a link between changes in the firing pattern and the loss of the calcium binding protein calbindin-D28k in epileptic hippocampal granule cells. Using the perforated patch-clamp technique, we investigated granule cells in slices prepared from human hippocampi removed for the treatment of pharmacoresistant temporal lobe epilepsy. Granule cells in hippocampi without significant signs of structural damage (lesion group) displayed a firing pattern indistinguishable from that of rodent granule cells and were strongly labeled with anti-calbindin-D28k antibodies. In contrast, half of granule cells in sclerotic hippocampi (HS group) showed an altered firing pattern and a severe loss of calbindin-D28k. While these cells show passive membrane properties comparable to cells of the rodent and lesion group, they lack the medium afterhyperpolarization and display only a weak spike frequency adaptation. On the other hand, granule cells in the HS group have an increased action potential threshold and an enlarged fast afterhyperpolarization. Applying post-recording immunohistochemistry to individual electrophysiologically characterized granule cells, we show that the loss of calbindin-D28k is not causally related to any of the changes in firing pattern. Both alterations seem to occur during the course of temporal lobe epilepsy, with the firing pattern being affected earlier than the calbindin-D28k content. In conclusion, we propose that it is the combination of the altered intrinsic excitability of granule cells with the amplified and prolonged synaptic input from perforant path fibers previously described in the epileptic dentate area which promotes tonic, non-adapting, high frequency firing of granule cells and thereby strongly augments the excitability of the hippocampus.

To test whether the slow afterhyperpolarization (sAHP) underlies the filter function of hippocampal granule cells (GCs), we compared the sAHP and spike frequency adaptation between granule cells and CA3 pyramidal cells (PCs) in hippocampal slices employing gramicidin perforated patch clamp recordings to best preserve the physiological cytoplasmic constitution. sAHPs were evoked in GCs and PCs with trains of action potentials in current clamp mode and showed comparable kinetics in both types of cells. The threshold frequency (500 ms firing) triggering a detectable sAHP was approximately 10 Hz and approximately 3 Hz in GCs and PCs, respectively. Half maximal sAHPs were reached at 30 Hz and 8 Hz in GCs and PCs, respectively. Maximal amplitude of sAHPs in GCs amounted to approximately 3.5 mV, was approximately 2-fold smaller than in PCs and could not be further increased with higher firing frequencies. The time course of sAHP activation was investigated with 50 Hz trains of action potentials applied for increasing durations. In both types of cells, the sAHP amplitude increased with a time constant of approximately 400 ms. Nevertheless, sAHP never exceeded 4 mV in GCs but rose to approximately 12 mV in PCs when cells fired for 3 s. The repetitive firing pattern of GCs and PCs was compared by injecting current amplitudes adjusted to provoke an initial firing frequency of 50 Hz. In GCs firing frequency declined slower (tau = 229 ms) and leveled off at a higher tonic firing frequency (28 Hz) when compared to PCs (tau = 126 ms, 18 Hz). We conclude that the intrinsic excitability of GCs cannot be primarily regulated by the sAHP. The sAHP in GCs is minimal most likely due to a small sAHP-channel density as well as to a more rigid control of intracellular Ca(2+) levels.

Ca2+-binding proteins are ubiquitously expressed throughout the CNS and serve as valuable immunohistochemical markers for certain types of neurons. However, the functional role of most Ca2+-binding proteins has to date remained obscure because their concentration in central neurons is not known. In this study, we investigate the intracellular concentration of the widely expressed Ca2+-binding protein calbindin-D28kin adult hippocampal slices using patch-clamp recordings and immunohistochemistry. First, we show that calbindin-D28kfreely exchanges between patch pipette and cytoplasm during whole cell patch-clamp recordings with a time constant of ∼10 min. Substituting known concentrations of recombinant calbindin-D28kin patch pipettes enabled us to determine the endogenous calbindin-D28kconcentration by postrecording immunohistochemistry. Using this calibration procedure, we find that mature granule cells (doublecortin-) contain ∼40 μm, and newborn granule cells (doublecortin+) contain 0-20 μmcalbindin-D28k. CA3 stratum radiatum interneurons and CA1 pyramidal cells enclose ∼47 and ∼45 μmcalbindin-D28k, respectively. Numerical simulations showed that 40 μmcalbindin-D28kis capable of tuning Ca2+microdomains associated with action potentials at the mouth of single or clustered Ca2+channels: calbindin-D28kreduces the increment in free Ca2+at a distance of 100 and 200 nm by 20 and 35%, respectively, and strongly accelerates the collapse of the Ca2+gradient after cessation of Ca2+influx. These data suggest that calbindin-D28kequips hippocampal neurons with ∼160 μmmobile, high-affinity Ca2+-binding sites (κS∼200) that slow and reduce global Ca2+signals while they enhance the spatiotemporal fidelity of submicroscopic Ca2+signals.

The dentate gyrus is believed to play an important pathophysiological role during experimentally induced kindling. In this study, we investigated whether an altered content of the calcium binding protein calbindin-D(28k) or an increased intrinsic excitability of hippocampal granule cells contribute to the induction of the kindling phenomenon. We determined the firing pattern of granule cells in hippocampal slices using perforated patch-clamp recordings in current clamp mode. The expression of calbindin-D(28k) and glutamic acid decarboxylase (GAD(67)) by granule cells was analyzed immunohistochemically. Rats developed secondarily generalized limbic seizures within approximately 11 days of twice-daily stimulation of the amygdala. As reported for other kindling paradigms, this protocol induced a clear up-regulation of GAD(67) in granule cells, indicating their involvement in the induced neuronal activity. However, when comparing kindled and control rats, we could not detect any differences in intrinsic excitability: Firing frequency, after-hyperpolarisations, action potentials, input resistance and membrane potentials were nearly identical between both groups. Furthermore, we did not observe any differences in the calbindin-D(28k) immunoreactivity between groups. In every slice, virtually all granule cells were found to be strongly calbindin-D(28k) positive, and there was no apparent reduction in the general level of calbindin-D(28k) expression. We conclude that changes in intrinsic membrane properties or in the calbindin-D(28k) content of granule cells are not necessary for the development of amygdala kindling.

The ionotropic and cytolytic P2X7receptor is typically found on immune cells, where it is involved in the release of cytokines. Recently, P2X7receptors were reported to be localized to presynaptic nerve terminals and to modulate transmitter release. In the present study, we reassessed this unexpected role of P2X7receptors at hippocampal mossy fiber-CA3 synapses. In agreement with previous findings, the widely used P2X7agonist 2′-3′-O-(4-benzoylbenzoyl)-adenosine-5′-triphosphate (BzATP) clearly depressed field potentials (fEPSPs); however, no evidence for an involvement of P2X7receptors could be obtained. First, depression of fEPSPs by BzATP was unchanged in P2X7-/-mice. Second, experiments using P2X7-/-mice, immunohistochemistry, and electron microscopy showed that the antigen detected by frequently used P2X7antibodies is not compatible with a plasmalemmal P2X7receptor. Third, BzATP did not alter Ca2+levels in synaptic terminals. In contrast, the depression of fEPSPs by BzATP was fully blocked by adenosine (A1) receptor antagonists. Furthermore, the application of BzATP also activated postsynaptic A1receptor-coupled K+channels. This effect of BzATP was mimicked by ATP and adenosine and was completely prevented by enzymes specifically degrading adenosine. Activation of A1-coupled K+channels by BzATP was dependent on ecto-nucleotidases, extracellular enzymes that convert ATP to adenosine. Moreover, the opening of A1-coupled K+channels by BzATP was dependent on nucleoside transporters. Taken together, our results indicate that BzATP is extracellularly catabolized to Bz-adenosine and subsequently hetero-exchanged for intracellular adenosine and then depresses mossy fiber fEPSPs through presynaptic A1receptors rather than through P2X7receptors. Thus, the present study casts doubts on the neuronal localization of P2X7receptors in rodent hippocampus.

Metabotropic glutamate receptors (mGluRs) provide a powerful control of synaptic transmission in the hippocampus and may serve as a target for drug development in human temporal lobe epilepsies. Agonists and antagonists at these receptors influence the development and propagation of seizures in some animal models of epilepsy. Experimental seizures can change the level of expression of mGluRs in the rat hippocampus. In the human dentate gyrus of patients suffering from temporal lobe epilepsy (TLE), group III mGluR mediated inhibition of synaptic transmission is almost lost in the sub-group with Ammon's horn sclerosis. We tested the modulation of synaptic transmission by the group III mGluR specific agonist l (+)-2-amino-4-phosphonobutyric acid ( l -AP4) in the dentate gyrus outer molecular layer in control and amygdala-kindled rats, a common model for TLE. Extracellular field potential recordings upon subthreshold stimulation of lateral perforant path fibers were measured simultaneously in the outer molecular layer and granule cell layer. Analysis of `paired-pulse' characteristics in the absence and presence of l -AP4 and group III mGluR mediated inhibition of synaptic transmission in the lateral perforant path revealed no significant alterations in fully kindled rats. Since there is no evidence of altered l -AP4 responses, a loss of group III mGluR function, particularly that of subtype mGluR8, seems not necessary for the kindling epilepsy.

Metabotropic glutamate receptors (mGluRs) have emerged as an interesting family of eight different receptor subtypes that can be divided into three groups according to their pharmacology and sequence similarity. In the present study, the specific mGluR agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD) and L(+)-2-amino-4-phosphonobutyric acid (L-AP4) depressed field excitatory postsynaptic potentials (fEPSPs) in the rat dentate gyrus evoked by perforant path stimulation in a concentration-dependent, rapid and reversible manner (EC50: L-AP4 5.9 +/- 1.6 microM, (1S,3R)-ACPD 80 +/- 34 microM). In a 'paired-pulse' stimulation protocol, the first fEPSP showed a stronger reduction, resulting in 'paired-pulse' facilitation. The effects of L-AP4 but not of (1S,3R)-ACPD could be antagonized by the group III mGluR antagonists (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4) and (RS)-alpha-methyl-4-phosphonophenylglycine (MPPG). Moreover, (1S,3R)-ACPD was still potently depressing fEPSPs after preperfusion of near saturating concentrations of L-AP4. Together, the results suggest that both substances act on different mGluRs. The effects of (1S,3R)-ACPD could not be further differentiated by selective group I or group II mGluR agonists. Although (2S,1'S,2'S)-2-carboxycyclopropylglycine (L-CCG-I) blocked fEPSPs at concentrations >> 1 microM, these effects, as well as L-AP4 effects, were potently antagonized by MAP4. This suggests that mGluR8 might be responsible for the actions of L-AP4 and L-CCG-I. The two different mGluRs showed a distinct distribution when fEPSPs were recorded simultaneously in the outer and middle molecular layer (OML/MML): The L-AP4 sensitive receptor, possibly mGluR8, seems to be located in the OML while (1S,3R)-ACPD showed its main effect in the MML.

Ca2+ influx into presynaptic terminals via voltage-dependent Ca2+ channels triggers fast neurotransmitter release as well as different forms of synaptic plasticity. Using electrophysiological and genetic techniques we demonstrate that presynaptic Ca2+ entry through Cav2.3 subunits contributes to the induction of mossy fiber LTP and posttetanic potentiation by brief trains of presynaptic action potentials while they do not play a role in fast synaptic transmission, paired-pulse facilitation, or frequency facilitation. This functional specialization is most likely achieved by a localization remote from the release machinery and by a Cav2.3 channel-dependent facilitation of presynaptic Ca2+ influx. Thus, the presence of Cav2.3 channels boosts the accumulation of presynaptic Ca2+ triggering presynaptic LTP and posttetanic potentiation without affecting the low release probability that is a prerequisite for the enormous plasticity displayed by mossy fiber synapses.