Stores not just for storage. intracellular calcium release and synaptic plasticity.

Activation of most excitatory synapses of central neurons produces calcium release signals from intracellular stores. Synaptically evoked calcium release from stores is frequently triggered by the binding of glutamate to metabotropic receptors and the subsequent activation of IP(3) receptors in spines and dendrites. There is increasing evidence for the presence of local calcium signals caused by calcium-induced calcium release (CICR) through activation of ryanodine or IP(3) receptors. Recent work on mutant mice indicates that store signaling determines activity-dependent synaptic plasticity.

Intradendritic release of calcium induced by glutamate in cerebellar Purkinje cells.

The ability of excitatory amino acids to induce increases in the intracellular Ca2+ concentration ([Ca2+]i) of cerebellar Purkinje cells was examined by digital fluorescence ratio imaging of voltage-clamped Purkinje cells dialyzed with the Ca2+ indicator fura-2. Purkinje cells responded with large inward currents accompanied by increases in dendritic [Ca2+]i when challenged with the excitatory amino acid agonists glutamate and quisqualate. The rise in [Ca2+]i was transient and reached peak values of several hundred nanomolar. The response subsisted in the absence of extracellular Ca2+, a condition that eliminates Ca2+ entry through voltage-gated Ca2+ channels, indicating that Ca2+ arose in large part from an intracellular compartment. In support of this hypothesis, only the first agonist application elicited a [Ca2+]i increase in slices maintained in Ca(2+)-free medium, as expected if the intracellular stores become depleted. These results indicate that metabotropic glutamate receptors are functional in Purkinje cells and point to glutamate as a possible modulator of [Ca2+]i in these neurons.

Fractional contribution of calcium to the cation current through glutamate receptor channels.

The Ca2+ fraction of the ion current flowing through glutamatergic NMDA and AMPA/kainate receptor channels was determined in forebrain neurons of the medial septum. The neurons were overloaded with the Ca2+ indicator dye fura-2 (1 mM) via the recording patch pipettes. This approach allowed the direct determination of the Ca2+ influx from changes in the Ca(2+)-sensitive fura-2 fluorescence. We found that, at negative membrane potentials and at an extracellular free Ca2+ concentration of 1.6 mM, the Ca2+ fraction of the current through the NMDA receptor channels is only 6.8%, about 2-fold lower than previously estimated from reversal potential measurements. Interestingly, a quite high fractional Ca2+ current of 1.4% was determined for the linearly conducting AMPA/kainate receptor channels found in these neurons.

Large-scale oscillatory calcium waves in the immature cortex.

Two-photon imaging of large neuronal networks in cortical slices of newborn rats revealed synchronized oscillations in intracellular Ca2+ concentration. These spontaneous Ca2+ waves usually started in the posterior cortex and propagated slowly (2.1 mm per second) toward its anterior end. Ca2+ waves were associated with field-potential changes and required activation of AMPA and NMDA receptors. Although GABAA receptors were not involved in wave initiation, the developmental transition of GABAergic transmission from depolarizing to hyperpolarizing (around postnatal day 7) stopped the oscillatory activity. Thus we identified a type of large-scale Ca2+ wave that may regulate long-distance wiring in the immature cortex.

Patch-clamping in slices of mammalian CNS.

A new method has been developed that permits high-resolution patch-clamp recordings in neurones from slice preparations of virtually every region of the brain or spinal cord. Since in these preparations most of the synaptic contacts are intact, the method is particularly interesting for the investigation of the molecular mechanisms of synaptic transmission at identified central neurones.

Dendritic signal integration.

Recent studies have identified various forms of active dendritic signals that may contribute to neuronal integration. One of the most remarkable findings is the demonstration of highly localized Ca2+ transients that are limited to small dendritic segments and even to single dendritic spines. In addition, through use of the powerful two-photon excitation imaging technique, it has been possible to reveal the existence of dendritic Ca2+ signals under in vivo conditions. Finally, active backpropagation of action potentials into dendrites has been shown to boost dendritic Ca2+ signals supralinearly and, thus, to contribute to the induction of long-term potentiation.

First one in, last one out: the role of gabaergic transmission in generation and degeneration.

This paper is the result of discussions between scientists working in widely separated areas, united by an interest in the hippocampus. The discussions focused on the possible role of GABA in the development and maturation of the hippocampus and in neurodegeneration in Alzheimer's disease (AD). GABA neurons are among the first to differentiate in the hippocampus and the properties of GABA neurotransmission in the developing hippocampus are distinct from those in the adult. GABAergic transmission may play a role in the clustering and maturation of GABA receptors, as well as of receptors for other neurotransmitters. The development and maturation of synaptic connections involves changes in the organization of the cytoskeleton, and mechanical force generation is probably required to establish appropriate points of contact. This generation of force may require coupling of specific receptors to the cytoskeleton through specialized proteins. In AD, much of the developmental process is progressively unraveled in the hippocampus, as afferent fibers, most notably from entorhinal excitatory neurons and from basal forebrain cholinergic cells, degenerate. This denervation undoubtedly has consequences for receptor systems, dendritic morphology and the underlying cytoskeleton. GABA neurons remain in the AD hippocampus, and may actually contribute to abnormal firing and degeneration of remaining pyramidal neurons. This attempt to bring together data from different areas of research has allowed the development of a scheme which identifies significant specific gaps in our knowledge, which could be readily filled by focused experimental work.

Gamma-frequency oscillations: a neuronal population phenomenon, regulated by synaptic and intrinsic cellular processes, and inducing synaptic plasticity.

Neurons are extraordinarily complicated devices, in which physical and chemical processes are intercoupled, in spatially non-uniform manner, over distances of millimeters or more, and over time scales of < 1 msec up to the lifetime of the animal. The fact that neuronal populations generating most brain activities of interest are very large-perhaps many millions of cells-makes the task of analysis seem hopeless. Yet, during at least some population activities, neuronal networks oscillate synchronously. The emergence of such oscillations generates precise temporal relationship between neuronal inputs and outputs, thus rendering tractable the analysis of network function at a cellular level. We illustrate this idea with a review of recent data and a network model of synchronized gamma frequency (> 20 Hz) oscillations in vitro, and discuss how these and other oscillations may relate to recent data on back-propagating, action potentials, dendritic Ca2+ transients, long-term potentiation and GABAA receptor-mediated synaptic potentials.

NMDA receptor-mediated Na+ signals in spines and dendrites.

Spines and dendrites of central neurons represent an important site of synaptic signaling and integration. Here we identify a new, synaptically mediated spine signal with unique properties. Using two-photon Na(+) imaging, we show that suprathreshold synaptic stimulation leads to transient increases in Na(+) concentration in postsynaptic spines and their adjacent dendrites. This local signal is restricted to a dendritic domain near the site of synaptic input. In presumed active spines within this domain, the Na(+) level increases by 30-40 mm even during short bursts of synaptic stimulation. During a long-term potentiation induction protocol (100 Hz, 1 sec), the Na(+) level in the active spines reaches peak amplitudes of approximately 100 mm. We find that the Na(+) transients are mainly mediated by Na(+) entry through NMDA receptor channels and are detected during the coincident occurrence of synaptic potentials and backpropagating action potentials. The large amplitudes of the Na(+) transients and their location on dendritic spines suggest that this signal is an important determinant of electrical and biochemical spine characteristics.

NMDA receptor-mediated subthreshold Ca(2+) signals in spines of hippocampal neurons.

We have used rapid confocal microscopy to investigate the mechanism of Ca(2+) signals in individual dendritic spines of hippocampal CA1 pyramidal cells. The experiments focused on the signals that occur during single weak synaptic responses that were subthreshold for triggering postsynaptic action potentials. These Ca(2+) signals were not strongly affected by blocking the EPSPs with the AMPA receptor antagonist CNQX. The signals were also not strongly reduced by blocking T-type voltage-gated Ca(2+) channels (VGCCs) with Ni(2+) or by blocking a broad range of VGCCs with intracellular D890. The spine Ca(2+) signals were blocked by NMDA receptor channel (NMDAR) antagonist and had the voltage dependence characteristic of these channels. Neither ryanodine nor cyclopiazonic acid (CPA), substances known to deplete intracellular Ca(2+) stores, substantially reduced the amplitude of synaptically evoked Ca(2+) signals. CPA slowed the recovery phase of Ca(2+) signals in spines produced by synaptic stimulation or by backpropagating action potentials, suggesting a role of intracellular stores in Ca(2+) reuptake. Thus, we find that Ca(2+) release from intracellular stores is not required to produce spine Ca(2+) signals. We conclude that synaptic Ca(2+) signals in spines are primarily caused by Ca(2+) entry through NMDARs. Although these channels are largely blocked by Mg(2+) at voltages near the resting potential, they can nevertheless produce significant Ca(2+) elevation. The resulting Ca(2+) signals are an integral component of individual evoked or spontaneous synaptic events and may be important in the maintenance of synaptic function.

Roles of glutamate receptor delta 2 subunit (GluRdelta 2) and metabotropic glutamate receptor subtype 1 (mGluR1) in climbing fiber synapse elimination during postnatal cerebellar development.

Climbing fiber (CF) synapse formation onto cerebellar Purkinje cells (PCs) is critically dependent on the synaptogenesis from parallel fibers (PFs), the other input to PCs. Previous studies revealed that deletion of the glutamate receptor delta2 subunit (GluRdelta2) gene results in persistent multiple CF innervation of PCs with impaired PF synaptogenesis, whereas mutation of the metabotropic glutamate receptor subtype 1 (mGluR1) gene causes multiple CF innervation with normal PF synaptogenesis. We demonstrate that atypical CF-mediated EPSCs (CF-EPSCs) with slow rise times and small amplitudes coexisted with typical CF-EPSCs with fast rise times and large amplitudes in PCs from GluRdelta2 mutant cerebellar slices. CF-EPSCs in mGluR1 mutant and wild-type PCs had fast rise times. Atypical slow CF responses of GluRdelta2 mutant PCs were associated with voltage-dependent Ca(2+) signals that were confined to PC distal dendrites. In the wild-type and mGluR1 mutant PCs, CF-induced Ca(2+) signals involved both proximal and distal dendrites. Morphologically, CFs of GluRdelta2 mutant mice extended to the superficial regions of the molecular layer, whereas those of wild-type and mGluR1 mutant mice did not innervate the superficial one-fifth of the molecular layer. It is therefore likely that surplus CFs of GluRdelta2 mutant mice form ectopic synapses onto distal dendrites, whereas those of wild-type and mGluR1 mutant mice innervate proximal dendrites. These findings suggest that GluRdelta2 is required for consolidating PF synapses and restricting CF synapses to the proximal dendrites, whereas the mGluR1-signaling pathway does not affect PF synaptogenesis but is involved in eliminating surplus CF synapses at the proximal dendrites.

Distinct roles of Galpha(q) and Galpha11 for Purkinje cell signaling and motor behavior.

G-protein-coupled metabotropic glutamate group I receptors (mGluR1s) mediate synaptic transmission and plasticity in Purkinje cells and, therefore, critically determine cerebellar motor control and learning. Purkinje cells express two members of the G-protein G(q) family, namely G(q) and G11. Although in vitro coexpression of mGluR1 with either Galpha11 or Galpha(q) produces equally well functioning signaling cascades, Galpha(q)- and Galpha11-deficient mice exhibit distinct alterations in motor coordination. By using whole-cell recordings and Ca2+ imaging in Purkinje cells, we show that Galpha(q) is required for mGluR-dependent synaptic transmission and for long-term depression (LTD). Galpha11 has no detectable contribution for synaptic transmission but also contributes to LTD. Quantitative single-cell RT-PCR analyses in Purkinje cells demonstrate a more than 10-fold stronger expression of Galpha(q) versus Galpha11. Our findings suggest an expression level-dependent action of Galpha(q) and Galpha11 for Purkinje cell signaling and assign specific roles of these two G(q) isoforms for motor coordination.

Impairment of mossy fiber long-term potentiation and associative learning in pituitary adenylate cyclase activating polypeptide type I receptor-deficient mice.

The pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor (PAC1) is a G-protein-coupled receptor binding the strongly conserved neuropeptide PACAP with 1000-fold higher affinity than the related peptide vasoactive intestinal peptide. PAC1-mediated signaling has been implicated in neuronal differentiation and synaptic plasticity. To gain further insight into the biological significance of PAC1-mediated signaling in vivo, we generated two different mutant mouse strains, harboring either a complete or a forebrain-specific inactivation of PAC1. Mutants from both strains show a deficit in contextual fear conditioning, a hippocampus-dependent associative learning paradigm. In sharp contrast, amygdala-dependent cued fear conditioning remains intact. Interestingly, no deficits in other hippocampus-dependent tasks modeling declarative learning such as the Morris water maze or the social transmission of food preference are observed. At the cellular level, the deficit in hippocampus-dependent associative learning is accompanied by an impairment of mossy fiber long-term potentiation (LTP). Because the hippocampal expression of PAC1 is restricted to mossy fiber terminals, we conclude that presynaptic PAC1-mediated signaling at the mossy fiber synapse is involved in both LTP and hippocampus-dependent associative learning.

Localized calcium signalling and neuronal integration in cerebellar Purkinje neurones.

The use of high resolution imaging techniques has revealed new forms of dendritic signal integration in neurones. In contrast to electrical signals that have a more widespread influence on the cell, brief Ca2+ transients resulting from synaptic activation are often restricted to a small part of the dendritic tree. In cerebellar Purkinje neurones, different levels of Ca2+ signalling have been observed that may involve the entire neurone or be spatially limited to fine dendritic structures. The Ca2+ signals accompanying subthreshold excitatory postsynaptic potentials resulting from stimulation of the excitatory parallel fibre input can be restricted to regions as small as a spiny dendrite or a single dendritic spine. With the recruitment of increasing numbers of inputs there is a summation of Ca2+ signals in highly restricted regions of the spiny dendrites that is independent of electrical summation at the soma. Of a number of potential sources that could provide the Ca2+ responsible for the postsynaptic changes, Ca2+ entry through voltage-gated Ca2+ channels has received the most support, although other sources like Ca2+ entry through ligand-gated channels and especially Ca2+ release from intracellular stores need to be considered.

Glutamate- and AMPA-mediated calcium influx through glutamate receptor channels in medial septal neurons.

The Ca(2+)-fraction of the ion current flowing through glutamate receptor channels activated either by glutamate or by AMPA was determined in forebrain neurons of the rat medial septum. By combining whole-cell patch-clamp and fura-2 fluorometric measurements we found that, at negative membrane potentials and at an extracellular free Ca(2+)-concentration of 1.6 mM, the Ca(2+)-fraction of the current activated by glutamate is 5.7%. A pharmacological analysis of responses produced by ionophoretically-released glutamate demonstrated a large contribution of NMDA-receptors but a small contribution of AMPA/kainate receptors to these responses. Interestingly, also AMPA-mediated currents were associated with significant changes in Ca(2+)-sensitive fluorescence. The fractional Ca2+ current of AMPA-induced responses was 1.2 +/- 0.4% (n = 5).

Potentiation of GABA-mediated currents by cAMP-dependent protein kinase.

Recent reports described a down-regulation of gamma-aminobutyric acid (GABA)-receptor function in several types of central neurones by cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA). Surprisingly, we found that in cerebellar Purkinje cells (PCs) the membrane permeable-compound 8-bromo-cAMP (500 microM) induced a long-lasting potentiation of both, whole-cell current responses to bath-applied GABA and amplitudes of miniature inhibitory synaptic currents (mIPSCs). When dialyzing the PCs with the specific protein kinase inhibitor peptide (PKIP, 400 micrograms ml-1), the same manipulation failed to induce a potentiation. These results strongly suggest that, in contrast to its action in other types of neurones, activation of PKA up-regulates the GABAA receptor function in cerebellar PCs.

Long-term potentiation as a mechanism of functional synapse induction in the developing hippocampus.

During the first 2 days of postnatal development, CA1 hippocampal glutamatergic synaptic transmission is based almost exclusively on NMDA receptors and is non-functional at resting potential. Within the following days an increasing number of functionally mature synapses, containing both NMDA and AMPA receptors, were observed. We found that the maturation of the NMDA receptor-mediated synapses could be induced experimentally with a pairing protocol, a process termed functional synapse induction. Our data provide evidence that a LTP-like mechanism involved in the activity-dependent formation of functional glutamergic synapses in the developing hippocampus.

Depolarization-induced calcium signals in the somata of cerebellar Purkinje neurons.

Cerebellar Purkinje neurons express voltage-gated Ca2+ channels that are located on their somata and dendrites. Previous reports, based on microelectrode recordings and fura-2 Ca2+ imaging, suggested that depolarization-mediated intracellular Ca2+ signaling is confined almost completely to the dendrites. We investigated the spatial distribution of depolarization-induced Ca2+ signals in Purkinje neurons by applying whole-cell patch-clamp recordings combined with fluorometric Ca2+ imaging to cerebellar slices. Under our recording conditions, depolarizing pulses produced the dendritic but also large somatic Ca2+ signals. By selective perfusion of the slice with a Ca(2+)-free EGTA-containing solution, we could isolate experimentally Ca2+ signals in somata and dendrites, respectively. Moreover, experiments performed on cerebellar slices from young rats (up to postnatal day 6), in which Purkinje neurons are almost completely devoid of dendrites, showed that Ca2+ currents produced by the activation of somatic Ca2+ channels are associated with Ca2+ transients similar to those seen in the somata of adult Purkinje neurons. Our results strongly indicate that the depolarization-induced somatic Ca2+ signals are caused by Ca2+ entry through voltage-gated channels located on the somatic membrane of Purkinje neurons.

The flaginserter: a simple device for automatically marking events in video recordings.

Dynamic imaging of cellular responses often involves the use of standard video components. While these components (cameras, monitors, etc.) are relatively inexpensive and easily available, there is the difficulty of integrating them into experimental set-ups. Especially for video recordings of fast signals that occur in neurobiological preparations (e.g. synaptic responses), there is a critical need for accurate synchronisation of video images with external events, such as extracellular stimulation or intracellular depolarising voltage steps. We developed a circuit that allows such events within the video sequence to be marked by means of a white flag that is inserted into the video image. Depending on the mode of insertion, the time resolution is better than 17 ms or better than 65 micros (i.e. a single video field or video line, respectively). The circuit has been shown to work reliable in combination with video-rate confocal imaging and patch-clamp recordings.

Effects of GABA on presumed presynaptic Ca2+ entry in hippocampal slices.

Evoked field potentials and changes in [Ca2+]o were measured in the 'in vitro' hippocampal slice of the rat. When [Ca] in the perfusion medium was lowered to 0.2 mM synaptic transmission from Schaffer collateral/commissural fibers was blocked. Nevertheless, repetitive stimulation of afferent fibers still resulted in detectable decreases of [Ca2+]o. In contrast to findings in normal medium these decreases in [Ca2+]o could be larger in stratum radiatum than in stratum pyramidale, so mimicking the spatial distribution of activated afferent fibers. These findings suggest, that the loss of extracellular Ca2+ in low Ca2+ media is predominantly due to entry into presynaptic terminals. This permits to study effects of drugs on presynaptic endings. We found that iontophoretic application of GABA is capable to block this presumed presynaptic Ca2+ entry without affecting the electrical activity of the afferent fibers. This suggests, that presynaptic GABA receptors occur also in the Schaffer collateral/commissural fiber system.