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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.

NG2 expressing oligodendroglial precursor cells are ubiquitous in the central nervous system and the only cell type cycling throughout life. Previous fate mapping studies have remained inconsistent regarding the question whether NG2 cells are capable of generating certain types of neurons. Here, we use CNP-Cre mice to map the fate of a sub-population of NG2 cells assumed to be close to differentiation. When crossing these mice with the ROSA26/YFP Cre-reporter line we discovered large numbers of reporter-expressing pyramidal neurons in the piriform and dorsal cortex. In contrast, when using Z/EG reporter mice to track the fate of Cnp-expressing NG2 cells only oligodendroglial cells were found reporter positive. Using BrdU-based birth dating protocols and inducible NG2CreER:ROSA26/YFP mice we show that YFP positive neurons are generated from radial glial cells and that these radial glial cells display temporary and low level activity of certain oligodendroglial genes sufficient to recombine the Cre-inducible reporter gene in ROSA26/YFP but not in Z/EG mice. Taken together, we did not obtain evidence for generation of neurons from NG2 cells. Our results suggest that with an appropriate reporter system Cnp activity can be used to define a proliferative subpopulation of NG2 cells committed to generate oligodendrocytes. However, the strikingly different results obtained from ROSA26/YFP versus Z/EG mice demonstrate that the choice of Cre-reporter line can be of crucial importance for fate mapping studies and other applications of the Cre-lox technology. GLIA 2017;65:342-359.

Zusammenfassung NG2- Gliazellen repräsentieren einen häufig vorkommenden glialen Zelltyp im Gehirn, der das Proteoglykan NG2 exprimiert, dessen funktionelle Bedeutung jedoch weitgehend unklar ist. Ein Großteil dieser Zellen ist auch im erwachsenen Gehirn noch teilungsaktiv. Diese Zellen besitzen eine Vielzahl von Ionenkanalen und Transmitter-Rezeptoren, die es ihnen ermöglichen, neuronale Aktivität zu detektieren. Bemerkenswerterweise werden NG2 - Gliazellen durch glutamaterge und GABAerge Neurone synaptisch innerviert. Da die postsynaptischen glialen Strome vergleichsweise klein sind, kommt der räumlichen und zeitlichen Integration dieser Signale möglicherweise eine wichtige Rolle zu. In der weißen Hirnsubstanz differenzieren viele NG2 - Gliazellen in Oligodendrozyten , und vermutlich wird dieser Prozess über die Aktivität der Neuron-Glia -Synapsen beeinflusst. Es zeichnet sich immer deutlicher ab, dass sich die Eigenschaften der NG2 - Glia in verschiedenen Hirnregionen unterscheiden, obgleich wir die Bedeutung dieser Heterogenität noch nicht verstehen.

Although NG2 glial cells represent a frequent glial cell type in the brain, characterized by expression of the NG2 proteoglycan, the functional impact of these cells is still enigmatic. A large proportion of NG2 glia are proliferatively active throughout life. These cells express a plethora of ion channels and transmitter receptors, which enable them to detect neuronal activity. Intriguingly, NG2 glial cells receive synaptic input from glutamatergic and GABAergic neurons. Since these postsynaptic glial currents are very small, their spatial and temporal integration might play an important role. In white matter, most NG2 glial cells differentiate into oligodendrocytes and this process might be influenced through the activity of the aforementioned neuron-glia synapses. Increasing evidence suggests that the properties of NG2 glia vary across brain regions; however, the impact of this variability is not understood yet.

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

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.

Progenitor cells expressing the proteoglycan NG2 represent approximately 5% of the total cells in the adult brain, and are found both in grey and white matter regions where they give rise to oligodendrocytes. The finding that these cells receive synaptic contacts from excitatory and inhibitory neurons has not only raised major interest in the possible roles of these synapses, but also stimulated further research on the developmental and cellular functions of NG2-expressing (NG2+) progenitors themselves in the context of neural circuit physiology. Here we review recent findings on the functional properties of the synapses on NG2+ cells in grey and white matter regions of the brain. In this review article we make an attempt to integrate current knowledge on the cellular and developmental properties of NG2+ progenitors with the functional attributes of their synapses, in order to understand the physiological relevance of neuron–NG2+ progenitor signal transmission. We propose that, although NG2+ progenitors receive synaptic contact in all brain regions where they are found, their synapses might have different developmental and functional roles, probably reflecting the distinct functions of NG2+ progenitors in the brain.

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.

The diffusion of calcium inside neurons is determined in part by the intracellular calcium binding species that rapidly bind to free calcium ions upon entry. It has long been known that some portion of a neuron’s intracellular calcium binding capacity must be fixed or poorly mobile, as calcium diffusion is strongly slowed in the intracellular environment relative to diffusion in cytosolic extract. The working assumption was that these immobile calcium binding sites are provided by structural proteins bound to the cytoskeleton or intracellular membranes and may thereby be relatively similar in composition and capacity across different cell types. However, recent evidence suggests that the immobile buffering capacity can vary greatly between cell types and that some mobile calcium binding proteins may alter their mobility upon binding calcium, thus blurring the line between mobile and immobile. The ways in which immobile buffering capacity might be relevant to different calcium domains within neurons has been explored primarily through modeling. In certain regimes, the presence of immobile buffers and the interaction between mobile and immobile buffers have been shown to result in complex spatiotemporal patterns of free calcium. In total, these experimental and modeling findings call for a more nuanced consideration of the local intracellular calcium microenvironment. In this review we focus on the different amounts, affinities, and mobilities of immobile calcium binding species; propose a new conceptual category of physically diffusible but functionally immobile buffers; and discuss how these buffers might interact with mobile calcium binding partners to generate characteristic calcium domains.

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.

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.

Metabotropic glutamate receptors (mGluRs) control excitatory neurotransmission as inhibitory autoreceptors at many synapses throughout the CNS. Since pharmacological activation of mGluRs potently depresses excitatory transmission, anticonvulsive effects were found in a number of experimental epilepsies. However, although native rodent mGluRs and heterologously expressed human mGluRs have so far been investigated in great detail, our knowledge about native human mGluRs in situ is limited. Here we used acute human hippocampal slices prepared from hippocampi surgically removed for the treatment of temporal lobe epilepsy in order to investigate the modulation of glutamatergic transmission by human mGluRs at the perforant path-granule cell synapse. The broad spectrum mGluR agonist (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) profoundly and reversibly reduced field EPSPs (fEPSPs) with an EC50 of 30±7.4 μM. Paired-pulse depression of fEPSPs was converted into strong facilitation. The inhibition of fEPSPs by ACPD was mimicked by the specific group II mGluR agonist (2S, 2′R, 3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV), while the specific group I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) was ineffective. The effect of ACPD was blocked by group II antagonist (2S,3S,4S)-2methyl-2-(carboxycyclopropyl)glycine (MCCG) but was not changed by coapplication of the specific group III antagonist (S)2 amino2methyl4phosphonobutanoic acid (MAP4). ACPD reduced pharmacologically isolated intracellular EPSPs in granule cells to the same extent as fEPSPs, whereas a specific group III agonist had no effect on EPSPs. Whole-cell recordings from morphologically identified granule cells revealed that DCG-IV significantly reduced the frequency of miniature EPSCs (mEPSCs) in granule cells while the mean amplitude of mEPSCs was not affected. We conclude that in human dentate gyrus mGluR2/3 can almost completely depress glutamate release by a presynaptic mechanism which acts downstream of presynaptic voltage gated calcium-entry and most likely involves a direct modulation of the release machinery.

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.

NG2 expressing oligodendrocyte precursor cells stand out from other types of glial cells by receiving classical synaptic contacts from many neurons. This unconventional form of signalling between neurons and glial cells enables NG2 cells to receive information about the activity of presynaptic neurons with high temporal and spatial precision and has been postulated to be involved in activity-dependent myelination. While this still unproven concept is generally compelling, how NG2 cells may integrate synaptic input has hardly been addressed to date. Here we review the biophysical characteristics of synaptic currents and membrane properties of NG2 cells and discuss their capabilities to perform complex temporal and spatial signal integration and how this may be important for activity-dependent myelination.

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.

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.

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.

Astrocytes have long been regarded as essentially unexcitable cells that do not contribute to active signaling and information processing in the brain. Contrary to this classical view, it is now firmly established that astrocytes can specifically respond to glutamate released from neurons. Astrocyte glutamate signaling is initiated upon binding of glutamate to ionotropic and/or metabotropic receptors, which can result in calcium signaling, a major form of glial excitability. Release of so-called gliotransmitters like glutamate, ATP and D-serine from astrocytes in response to activation of glutamate receptors has been demonstrated to modulate various aspects of neuronal function in the hippocampus. In addition to receptors, glutamate binds to high-affinity, sodium-dependent transporters, which results in rapid buffering of synaptically-released glutamate, followed by its removal from the synaptic cleft through uptake into astrocytes. The degree to which astrocytes modulate and control extracellular glutamate levels through glutamate transporters depends on their expression levels and on the ionic driving forces that decrease with ongoing activity. Another major determinant of astrocytic control of glutamate levels could be the precise morphological arrangement of fine perisynaptic processes close to synapses, defining the diffusional distance for glutamate, and the spatial proximity of transporters in relation to the synaptic cleft. In this review, we will present an overview of the mechanisms and physiological role of glutamate-induced ion signaling in astrocytes in the hippocampus as mediated by receptors and transporters. Moreover, we will discuss the relevance of astroglial glutamate uptake for extracellular glutamate homeostasis, focusing on how activity-induced dynamic changes of perisynaptic processes could shape synaptic transmission at glutamatergic synapses.