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Objective Assess occurrence of the dendritic spine scaffolding protein Drebrin as a pathophysiologically relevant autoantibody target in patients with recurrent seizures and suspected encephalitis as leading symptoms. Methods Sera of 4 patients with adult onset epilepsy and suspected encephalitis of unresolved etiology and equivalent results in autoantibody screening were subjected to epitope identification. We combined a wide array of approaches, ranging from immunoblotting, immunoprecipitation, mass spectrometry, subcellular binding pattern analyses in primary neuronal cultures, and immunohistochemistry in brains of wild-type and Drebrin knockout mice to in vitro analyses of impaired synapse formation, morphology, and aberrant neuronal excitability by antibody exposure. Results In the serum of a patient with adult onset epilepsy and suspected encephalitis, a strong signal at ∼70kDa was detected by immunoblotting, for which mass spectrometry revealed Drebrin as the putative antigen. Three other patients whose sera also showed strong immunoreactivity around 70kDa on Western blotting were also anti-Drebrin-positive. Seizures, memory impairment, and increased protein content in cerebrospinal fluid occurred in anti-Drebrin-seropositive patients. Alterations in cerebral magnetic resonance imaging comprised amygdalohippocampal T2-signal increase and hippocampal sclerosis. Diagnostic biopsy revealed T-lymphocytic encephalitis in an anti-Drebrin-seropositive patient. Exposure of primary hippocampal neurons to anti-Drebrin autoantibodies resulted in aberrant synapse composition and Drebrin distribution as well as increased spike rates and the emergence of burst discharges reflecting network hyperexcitability. Interpretation Anti-Drebrin autoantibodies define a chronic syndrome of recurrent seizures and neuropsychiatric impairment as well as inflammation of limbic and occasionally cortical structures. Immunosuppressant therapies should be considered in this disorder. ANN NEUROL 2020;87:869-884.

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.

Summary: Synaptically released glutamate is largely cleared by glutamate transporters localized on perisynaptic astrocyte processes. Therefore, the substantial variability of astrocyte coverage of individual hippocampal synapses implies that the efficacy of local glutamate uptake and thus the spatial fidelity of synaptic transmission is synapse dependent. By visualization of sub-diffraction-limit perisynaptic astrocytic processes and adjacent postsynaptic spines, we show that, relative to their size, small spines display a stronger coverage by astroglial transporters than bigger neighboring spines. Similarly, glutamate transients evoked by synaptic stimulation are more sensitive to pharmacological inhibition of glutamate uptake at smaller spines, whose high-affinity N-methyl-D-aspartate receptors (NMDARs) are better shielded from remotely released glutamate. At small spines, glutamate-induced and NMDAR-dependent Ca2+ entry is also more strongly increased by uptake inhibition. These findings indicate that spine size inversely correlates with the efficacy of local glutamate uptake and thereby likely determines the probability of synaptic crosstalk.

Mossy fiber long-term depression (LTD) has been shown to be triggered by either pharmacological or synaptic activation of Group II metabotropic glutamate receptors (mGluRs) whereas other studies indicate that synaptic activation of mGluRs is very limited. Therefore, we reexamined the role of Group II mGluRs for the induction of mossy fiber LTD. The complete depression of field potentials (fEPSPs) by 1 microM (2S,2'R,3'R)-2-(2',3'-Dicarboxycyclopropyl)glycine (DCG-IV) only partially reversed upon removal of the drug but fEPSPs were completely restored by the Group II antagonist 2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid (LY341495) (3 microM). In contrast, fEPSPs returned back to baseline within 30 min after a brief application of 0.2 microM DCG-IV suggesting that the incomplete reversal of higher concentrations may be due to a residual receptor occupancy rather than to an induction of LTD. LY341495 itself did not increase fEPSPs and also blocked the inhibition of (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine (L-CCG-I) (20 microM) and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) (10 microM) and its effect was mimicked by CPPG (50 microM). Furthermore, stimulation at 1 Hz for 15 min induced an LTD of 81% +/- 3% and 80% +/- 4% in the absence and presence of LY341495, respectively (n = 7, 5). Finally, we found that synaptic activation of Group II mGluRs during 15 min of 1-Hz stimulation only produces an inhibition of release by 8% +/- 1% (30 degrees C, n = 3). Our data suggests that pharmacological activation of Group II mGluRs is fully reversible per se and does not produce a long lasting depression and that activation of Group II mGluRs is neither necessary nor sufficient for the induction of mossy fiber LTD.

It is well established that NG2 cells throughout the young and adult brain consistently detect the release of single vesicles filled with glutamate from nearby axons. The released neurotransmitter glutamate electrically excites NG2 cells via non-NMDA (N-methyl-D-aspartic acid) glutamate receptors but the individual contribution of AMPA and kainate receptors to neuron-NG2 cell signalling, is not well understood. Here we pharmacologically block AMPA-type glutamate receptors and investigate whether hippocampal NG2 cells also express the kainate subtype of glutamate receptors and what may be their contribution to synaptic connectivity. It has been shown previously that vesicular glutamate release does not lead to a detectable activation of kainate receptors on NG2 cells. Here we report that while bath application of 250 nM–1 μM kainate does not have a major effect on NG2 cells it consistently induces a small and persistent depolarising current. This current was not mimicked by ATPA, suggesting that this current is carried by non-GluR5 containing kainate receptors. In addition to this inward current, nanomolar concentrations of kainate also produced a dramatic increase in the frequency of spontaneous GABA-A receptor-mediated synaptic currents (IPSCs) in NG2 cells. This increase in spontaneous IPSC frequency was even more pronounced on application of the GluR5-specific agonist ATPA (approximately 15-fold increase in frequency). In contrast, mono-synaptic stimulated IPSCs recorded in NG2 cells were unaffected by kainate receptor activation. Those and further experiments show that the occurrence of the high frequency of IPSCs is due to action potential firing of hippocampal interneurons caused by activation of GluR5 receptors on the somatodendritic membrane of the interneurons. Our data suggest that hippocampal kainate receptors are not only important for communication between neurons but may also play a dual and subtype-specific role for neuron–glia signalling: Firstly, extra-synaptic non-GluR5 kainate receptors in the membrane of NG2 cells are ideally suited to instruct NG2 cells on the population activity of local excitatory neurons via ambient glutamate. Secondly, based on the known importance of GluR5 receptors on hippocampal interneurons for the generation of network rhythms and based on our finding that these interneurons heavily project onto NG2 cells, it appears that synaptic activation of interneuronal GluR5 receptors triggers signalling to NG2 cells which transmits the phase and frequency of ongoing network oscillations in the developing hippocampus.

Specific agonists of metabotropic glutamate receptors (mGluRs) provide powerful tools to discriminate afferent fibers originating from different presynaptic neurons. The group II mGluR agonists L-CCG-I ((2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine) and DCG-IV ((2S,2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl)glycine) are commonly used to distinguish between mossy fiber and associational-commissural (A/C) fiber input to the hippocampal CA3 region because only on the former group II mGluRs are expressed. Since previous reports indicated that L-CCG-I can activate group III mGluRs as well, we investigated whether L-CCG-I depresses A/C field potentials. L-CCG-I (10-300 microM) exhibited a significant dose-dependent and reversible reduction of A/C field potentials by 8 +/- 4% (10 microM), by 32 +/- 4% (100 microM, p0.001) and by 38 +/- 7% (300 microM, p0.05) that was accompanied by a concomitant increase in paired-pulse facilitation. Moreover, the selective group III antagonist (R,S)-alpha-methylserine-O-phosphate (MSOP; 100 microM) significantly reduced the field potential inhibition by L-CCG-I (100 microM) to 9 +/- 4% (p0.05). In contrast, DCG-IV did not affect A/C field potentials. In conclusion, the purported group II mGluR agonist L-CCG-I depresses A/C synaptic transmission by activation of group III mGluRs. For this reason, DCG-IV should be the drug of choice when aiming to discriminate between mossy fiber and A/C input to CA3 pyramidal cells.

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.

The ubiquitous presynaptic metabotropic glutamate receptors (mGluRs) are generally believed to primarily inhibit synaptic transmission through blockade of Ca(2+) entry. Here, we analyzed how mGluR8 achieves a nearly complete inhibition of glutamate release at hippocampal synapses. Surprisingly, presynaptic Ca(2+) imaging and miniature excitatory postsynaptic current recordings showed that mGluR8 acts without affecting Ca(2+) entry, diffusion, and buffering. We quantitatively compared the Ca(2+) dependence of the inhibition of release by mGluR8 with the inhibition by ω-conotoxin GVIA. These calculations suggest that the inhibition produced by mGluR8 may be explained by a decrease in the apparent Ca(2+) affinity of the release sensor and, to a smaller extent, by a reduction of the maximal release rate. Upon activation of mGluR8, phasic transmitter release toward the end of a train of action potentials is greater as compared with presynaptic inhibition induced by blocking Ca(2+) entry, which is consistent with the important role of Ca(2+) in accelerating the replenishment of released vesicles. The action of mGluR8 was resistant to blockers of classical G-protein transduction pathways including inhibition of adenylate cyclase and may represent a direct effect on the release machinery. In conclusion, our data identify a mode of presynaptic inhibition which allows mGluR8 to profoundly inhibit vesicle fusion while not diminishing vesicle replenishment and which thereby differentially changes the temporal transmission properties of the inhibited synapse.

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.

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 development of resistance to pharmacological treatment is common to many human diseases. In chronic epilepsy, many patients develop resistance to anticonvulsant drug treatment during the course of their disease, with the underlying mechanisms remaining unclear. We have studied cellular mechanisms underlying drug resistance in resected hippocampal tissue from patients with temporal lobe epilepsy by comparing two groups of patients, the first displaying a clinical response to the anticonvulsant carbamazepine and a second group with therapy-resistant seizures. Using patch-clamp recordings, we show that the mechanism of action of carbamazepine, use-dependent block of voltage-dependent Na(+) channels, is completely lost in carbamazepine-resistant patients. Likewise, seizure activity elicited in human hippocampal slices is insensitive to carbamazepine. In marked contrast, carbamazepine-induced use-dependent block of Na(+) channels and blocked seizure activity in vitro in patients clinically responsive to this drug. Consistent with these results in human patients, we also show that use-dependent block of Na(+) channels by carbamazepine is absent in chronic experimental epilepsy. Taken together, these data suggest that a loss of Na(+) channel drug sensitivity may constitute a novel mechanism underlying the development of drug-resistant epilepsy.

AMPA-receptor (AMPA-R) currents were recorded from CA1 pyramidal neurons in situ and after acute isolation from the hippocampus of 3- to 45-day-old rats. Membrane currents were analyzed by combining the patch clamp method with fast application techniques. The complete block of receptor currents by GYKI 53655 and the absence of modulation by Concanavalin A indicated that the cells exclusively expressed non-NMDA glutamate receptors of the AMPA subtype while functional kainate receptors could not be detected. The lowest sensitivity to kainate and NBQX was observed at postnatal day (p) 18. These changes might reflect a lower abundance of GluR1 at that developmental stage. A decrease of potentiation of receptor currents by cyclothiazide (CTZ), an acceleration of the recovery from CTZ potentiation and a faster and more complete desensitization of glutamate-evoked currents suggest an up-regulation of flop splice variants with increasing age. These functional data indicate that AMPA-R expression in CA1 pyramidal neurons varies during postnatal development which can be expected to influence the kinetics of synaptic transmission and the excitotoxic vulnerability as well.

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.