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A Konnerth

Publications and source records attributed to A Konnerth.

At least 55 records · Page 3Linked to original sources
PubMed1995

Subthreshold synaptic Ca2+ signalling in fine dendrites and spines of cerebellar Purkinje neurons.

The conventional view of synaptic integration is that it results from the simple summation of electrical signals produced by each active synapse innervating a given neuron. However, because synaptic action can go beyond the production of postsynaptic electrical signals, to include intracellular biochemical events such as the generation of second messengers, it is possible that synaptic integration could occur at another level. We have considered this possibility by examining changes in the dendritic concentration of the second messenger, calcium, resulting from subthreshold excitatory synaptic activity in cerebellar Purkinje neurons. We report here clear evidence that such non-electrical synaptic integration occurs and that it takes place in restricted dendritic compartments consisting of spines and adjacent fine dendrites.

Action Potentials↗
PubMed1995

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.

Animals↗
PubMed1994

Calcium requirement of long-term depression and rebound potentiation in cerebellar Purkinje neurons.

Cerebellar Purkinje neurons (PNs) receive two main excitatory inputs, from climbing fibers and parallel fibers, and inhibitory inputs, from GABAergic interneurons. The synapses formed by parallel fibers and by inhibitory interneurons on PNs are able to undergo long-lasting changes in efficacy. Thus, the excitatory parallel fiber-PN synapse undergoes long-term depression when it is activated in conjunction with climbing fibers. Synaptic inhibition can be potentiated by climbing fiber activity by a mechanism named rebound potentiation, resulting in a more powerful inhibitory effect of GABAergic interneurons. The induction of both long-term depression and rebound potentiation requires a transient elevation of the cytoplasmic calcium concentration ([Ca2+]i). The [Ca2+]i-transient is caused by Ca2+ entry through voltage-gated Ca2+ channels and, possibly, by release of Ca2+ from IP3- and ryanodine-sensitive stores. Direct Ca2+ entry through synaptic AMPA receptor channels seems not to contribute significantly to the Ca2+ signal mediating the induction of both long-term depression and rebound potentiation.

Animals↗
PubMed1993

A single amino acid determines the subunit-specific spider toxin block of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor channels.

Joro spider toxin (JSTX) is one of the most potent antagonists of glutamatergic AMPA/KA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate) receptor channels in invertebrates and vertebrates. A differential blocking effect on certain types of glutamatergic synapses--e.g., parallel and climbing fiber synaptic inputs to rat cerebellar Purkinje neurons--has been shown by using a synthetic analog of the spider toxin. By investigating the molecular basis of the JSTX action on the recombinant AMPA/KA receptors GluR1-GluR4 and GluR6 expressed in Xenopus oocytes, we found that submicromolar concentrations of JSTX exert a subunit-specific block. Thus, receptor subunits forming a receptor channel with a linear current-voltage (I-V) relationship (GluR1/2, GluR2/3, and GluR6) were not affected, while receptor subunits with rectifying I-V relationships (GluR1, GluR3, GluR4, and GluR1/3) were reversibly blocked by JSTX. By using receptor-subunit mutants obtained by site-directed mutagenesis, we have identified a single amino acid position (glutamine in the proposed second transmembrane domain) that is critical for the JSTX block. Since this site has previously been shown to control the I-V relationship of the AMPA/KA receptor channel and to participate in the regulation of the channel's permeability for calcium ions, our findings suggest that JSTX binds close to the central pore region of the channel.

Amino Acid Sequence↗
PubMed1993

Identification of a subunit-specific antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor channels.

Excitatory synaptic transmission in the mammalian central nervous system is mediated predominantly by glutamate receptor (GluR) channels of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate (AMPA/KA) receptor type. A major improvement in our understanding of glutamatergic synaptic transmission has been achieved after the identification of quinoxalinediones (e.g., 6-cyano-7-nitroquinoxaline-2,3-dione) as specific antagonists of AMPA/KA receptors. In addition to their effects on neurons, quinoxalinediones were also shown to block glutamate-induced responses mediated by recombinant AMPA/KA receptor channels expressed in heterologous systems, irrespective of their particular subunit composition. Here we report the identification of an AMPA/KA receptor antagonist that selectively blocks a subset of AMPA/KA receptors. We found that Evans blue, a biphenyl derivative of naphthalene disulfonic acid, blocks at low concentrations (IC50 = 355 nM for the subunit combination GluR1,2) KA-mediated responses of the subunits GluR1, GluR1,2, GluR1,3, and GluR2,3 expressed in Xenopus oocytes but not responses of GluR3 or GluR6. The blocking action of Evans blue was partially reversible and did not compete with KA for the agonist binding site. These findings suggest not only that Evans blue is a potent tool for elucidating the functional role of specific AMPA/KA receptor subtypes for excitatory synaptic transmission but also that it may also represent a powerful starting point for clinically useful drugs that are able to reduce the excitatory drive in specific neuronal populations of the central nervous system.

Animals↗
PubMed1993

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).

Animals↗
PubMed1993

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.

Animals↗
PubMed1992

Brief dendritic calcium signals initiate long-lasting synaptic depression in cerebellar Purkinje cells.

We have performed experiments designed to test the hypothesis that long-term depression (LTD) of excitatory synaptic transmission in the cerebellar cortex is caused by a rise in postsynaptic Ca concentration. These experiments combined measurements of synaptic efficacy, performed with the thin slice patch clamp technique, with fura-2 measurements of intracellular Ca concentration ([Ca]i) in single cerebellar Purkinje cells. Simultaneous activation of the climbing fiber and parallel fibers innervating single Purkinje cells caused a LTD of transmission of the parallel fiber-Purkinje cell excitatory synapse. This LTD was associated with large and transient rises in [Ca]i in the Purkinje cell and apparently was due to Ca entry through voltage-gated Ca channels in the Purkinje cell dendrites. The rise in [Ca]i produced by climbing fiber activity was necessary for LTD, because addition of the Ca chelator bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) to the interior of the Purkinje cell blocked LTD. Further, elevation of [Ca]i, produced by depolarizing pulses delivered in conjunction with parallel fiber activation, induced a depression of synaptic activity that closely resembled LTD in both time course and magnitude. Thus, a rise in [Ca]i appears to be sufficient to initiate LTD. From these results, we conclude that LTD of the parallel fiber-Purkinje cell synapse is initiated by a brief, climbing fiber-mediated rise in postsynaptic [Ca]i and that LTD is maintained by other, longer-lived processes that are triggered by the rise in postsynaptic [Ca]i.

Animals↗
PubMed1992

Sodium action potentials in the dendrites of cerebellar Purkinje cells.

We report here that in cerebellar Purkinje cells from which the axon has been removed, positive voltage steps applied to the voltage-clamped soma produce spikes of active current. The spikes are inward, are all-or-none, have a duration of approximately 1 ms, and are reversibly eliminated by tetrodotoxin, a Na channel poison. From cell to cell, the amplitude of the spikes ranges from 4 to 20 nA. Spike latency decreases as the depolarizing step is made larger. These spikes clearly arise at a site where the voltage is not controlled, remote from the soma. From these facts we conclude that Purkinje cell dendrites contain a sufficient density of Na channels to generate action potentials. Activation by either parallel fiber or climbing fiber synapses produces similar spikes, suggesting that normal input elicits Na action potentials in the dendrites. These findings greatly alter current views of how dendrites in these cells respond to synaptic input.

Action Potentials↗
PubMed1992

Synaptic excitation produces a long-lasting rebound potentiation of inhibitory synaptic signals in cerebellar Purkinje cells.

Persistent changes in synaptic efficacy are thought to underlie the formation of learning and memory in the brain. High-frequency activation of an afferent excitatory fibre system can induce long-term potentiation, and conjunctive activation of two distinct excitatory synaptic inputs to the cerebellar Purkinje cells can lead to long-term depression of the synaptic activity of one of the inputs. Here we report a new form of neural plasticity in which activation of an excitatory synaptic input can induce a potentiation of inhibitory synaptic signals to the same cell. In cerebellar Purkinje cells stimulation of the excitatory climbing fibre synapses is followed by a long-lasting (up to 75 min) potentiation of gamma-aminobutyric acid A (GABAA) receptor-mediated inhibitory postsynaptic currents (i.p.s.cs), a phenomenon that we term rebound potentiation. Using whole-cell patch-clamp recordings in combination with fluorometric video imaging of intracellular calcium ion concentration, we find that a climbing fibre-induced transient increase in postsynaptic calcium concentration triggers the induction of rebound potentiation. Because the response of Purkinje cells to bath-applied exogenous GABA is also potentiated after climbing fibre-stimulation with a time course similar to that of the rebound potentiation of i.p.s.cs, we conclude that the potentiation is caused by a calcium-dependent upregulation of postsynaptic GABAA receptor function. We propose that rebound potentiation is a mechanism by which in vivo block of climbing fibre activity induces an increase in excitability in Purkinje cells. Moreover, rebound potentiation of i.p.s.cs is a cellular mechanism which, in addition to the long-term depression of parallel fibre synaptic activity, may have an important role for motor learning in the cerebellum.

Animals↗
PubMed1992

Calcium influx through subunits GluR1/GluR3 of kainate/AMPA receptor channels is regulated by cAMP dependent protein kinase.

Excitatory synaptic transmission in the central nervous system (CNS) is mediated by three major classes of glutamate receptors, namely the ionotropic NMDA (N-Methyl-D-Aspartate) and KA/AMPA (kainate/alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid) receptors and the metabotropic receptor type. Among the ionotropic receptors, NMDA receptors are thought to mediate their physiological response mainly through the influx of extracellular calcium, while KA/AMPA receptor channels are mainly thought to carry the influx of monovalent cations. Recently, we have challenged this view by showing that cloned KA/AMPA receptor subunits GluR1 and GluR3 form ion channels which are permeable to calcium. We now directly demonstrate large increases in intracellular calcium concentrations induced by calcium fluxes through KA/AMPA receptor channels in solutions with physiological calcium concentrations. Calcium fluxes were observed through glutamate receptor channels composed of the subunits GluR1 and GluR3, which are both abundantly present in various types of central neurones. The calcium influx was fluorometrically monitored in Xenopus oocytes injected with the calcium indicator dye fura-2. Bath application of the membrane permeable analogue of adenosine cyclic monophosphate (cAMP) potentiated the current and also the flux of calcium through open KA/AMPA receptor channels. Further pharmacological experiments suggested that this effect was mediated by the activation of protein kinase A. Our results provide a molecular interpretation for the function of calcium permeable KA/AMPA receptor channels in neurones and identify two of the subunits of the KA/AMPA receptor channel which are regulated by the cAMP dependent second messenger system.

Animals↗
PubMed1992

GABA-mediated synaptic transmission in neuroendocrine cells: a patch-clamp study in a pituitary slice preparation.

Patch-clamp recording techniques were applied to thin slices of the rat pituitary gland in order to study synaptic transmission between hypothalamic nerve terminals and neuroendocrine cells of the intermediate lobe. Inhibitory postsynaptic currents (IPSCs) could be evoked by electrical stimulation of afferent neuronal fibres in the surrounding tissue of the slice. The IPSCs could be evoked in an all-or-nothing mode depending on the stimulus intensity, suggesting that single afferent fibres were stimulated. They had a chloride-dependent reversal potential and were blocked by bicuculline (Kd = 0.1 microM), indicating that they were mediated by gamma-aminobutyric acid A (GABAA) receptors. In symmetrical chloride solutions the current/voltage relation of the IPSC peak amplitudes was linear. The IPSCs were characterized by a fast (1-2 ms) rise time and a biexponential decay, with time constants of 21 +/- 4 ms and 58 +/- 14 ms at a holding potential of -60 mV (n = 6 cells). Both decay time constants increased with depolarization in an exponential manner. Spontaneously occurring IPSCs had a time course that was similar to that of evoked IPSCs. These miniature IPSCs, recorded in 1 microM tetrodotoxin, displayed an amplitude distribution that was well fitted by single Gaussian functions, with a mean value of its maxima of 18.1 +/- 2.3 pA (n = 4 cells). Amplitude histograms of evoked IPSCs were characterized by multiple peaks with a modal amplitude of about 18 pA (n = 6 cells). These findings indicate the quantal nature of GABAergic synaptic transmission in this system, with a quantal conductance step of about 280 pS. Single-channel currents underlying the IPSCs were studied by bath application of GABA to outside-out patches excised from intermediate lobe cells. Such GABA-induced currents revealed two conductance levels of 14 pS and 26 pS. In conclusion, GABAergic synaptic transmission in neuroendocrine cells of the pituitary has properties that are quite similar to those observed in neurones of the central nervous system.

Animals↗
PubMed1992

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.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Excitatory and inhibitory synaptic currents and receptors in rat medial septal neurones.

1. A thin-slice preparation was used to study the postsynaptic potentials and the underlying currents of visually identified rat medial septal (MS) neurones under tight-seal voltage- and current-clamp conditions. 2. Upon stimulation of the afferent fibres, all MS neurones exhibited a sequence of excitatory-inhibitory postsynaptic potentials (EPSP-IPSP). Under voltage clamp, with potassium glutamate as internal solution and at negative holding potentials (Vh), this synaptic pattern appeared as an initial inward current followed by a longer lasting outward current. 3. The inward postsynaptic current was completely abolished by 5 microM-6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) whereas the outward current disappeared in the presence of 10 microM-bicuculline. Thus the major excitatory and inhibitory synaptic inputs were identified as being due to activation of quisqualate/kainate glutamatergic and gamma-aminobutyric acid (GABAA) receptors, respectively. 4. At positive Vh a CNQX-resistant component of the excitatory postsynaptic current (EPSC) was revealed. This component was slower than the one mediated by the quisqualate receptor and was abolished by 3-3(2-carboxypiperazine-4-yl)propyl-1-phosphonate (CPP), indicating that N-methyl-D-aspartate (NMDA) receptors are involved in excitatory synaptic transmission in MS cells. The existence of the two main subtypes (NMDA and non-NMDA) of glutamatergic receptors in MS neurones was also confirmed by the responses of the neurones to bath application of the different agonists (glutamate, quisqualate, kainate and NMDA). 5. The CNQX-sensitive EPSC had a reversal potential near 0 mV. The fast rise time (approximately 0.7 ms) indicates a somatic location of the excitatory synapses. The relaxation kinetics of the fast EPSC were fitted by a single exponential function with a time constant of 1.13 +/- 0.1 ms. This parameter was independent of Vh. Fast EPSCs were blocked by CNQX in a dose-dependent manner (dissociation constant, KD = 0.2 microM). 6. Inhibitory postsynaptic currents (IPSCs) were studied in symmetrical chloride solutions after blockade of the excitatory receptors. The current-voltage relation was linear and reversed at 0 mV. The IPSCs had a fast rise time and their decay was best fitted by the sum of two exponentials with time constant of approximately 20 and 50 ms (Vh = -60 mV). The IPSCs were abolished by bicuculline (KD = 1 microM), a selective antagonist of GABAA receptors. As expected, bath application of GABA produced large whole-cell currents. 7. In many cells, in addition to the usual EPSP-IPSP sequence, failures of either the EPSP or the IPSP were frequently observed during the experimental protocol.(ABSTRACT TRUNCATED AT 400 WORDS)

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1991

Action potential broadening and frequency-dependent facilitation of calcium signals in pituitary nerve terminals.

Hormone release from nerve terminals in the neurohypophysis is a sensitive function of action potential frequency. We have investigated the cellular mechanisms responsible for this frequency-dependent facilitation by combining patch clamp and fluorimetric Ca2+ measurements in single neurosecretory terminals in thin slices of the rat posterior pituitary. In these terminals both action potential-induced changes in the intracellular Ca2+ concentration ([Ca2+]i) and action potential duration were enhanced by high-frequency stimuli, all with a frequency dependence similar to that of hormone release. Furthermore, brief voltage clamp pulses inactivated a K+ current with a very similar frequency dependence. These results support a model for frequency-dependent facilitation in which the inactivation of a K+ current broadens action potentials, leading to an enhancement of [Ca2+]i signals. Further experiments tested for a causal relationship between action potential broadening and facilitation of [Ca2+]i changes. First, increasing the duration of depolarization, either by broadening action potentials with the K(+)-channel blocker tetraethylammonium or by applying longer depolarizing voltage clamp steps, increased [Ca2+]i changes. Second, eliminating frequency-dependent changes in duration, by voltage clamping the terminal with constant duration pulses, substantially reduced the frequency-dependent enhancement of [Ca2+]i changes. These results indicate that action potential broadening contributes to frequency-dependent facilitation of [Ca2+]i changes. However, the small residual frequency dependence of [Ca2+]i changes seen with constant duration stimulation suggests that a second process, distinct from action potential broadening, also contributes to facilitation. These two frequency-dependent mechanisms may also contribute to activity-dependent plasticity in synaptic terminals.

Action Potentials↗
PubMed1991

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.

Animals↗
PubMed1991

Synaptic- and agonist-induced excitatory currents of Purkinje cells in rat cerebellar slices.

1. Postsynaptic currents originating from activation of the two major excitatory inputs to Purkinje cells were studied in thin slices of rat cerebellum, using the tight-seal whole-cell recording technique. Two types of excitatory postsynaptic currents were analysed: those evoked by stimulation of the granule cell-parallel fibre system (PF-EPSC) and those elicited by stimulation of the climbing fibres (CF-EPSC). 2. Both types of postsynaptic currents had a linear current-voltage relation, reversing at membrane potentials close to 0 mV. Their time course of activation was independent of the membrane potential. 3. For both types of postsynaptic currents, the time course of decay was well described by a single exponential function, with a time constant which increased as the membrane potential was made more positive. 4. Postsynaptic currents arising from stimulation of the climbing fibre generally had a slightly faster time course of onset and decay than those associated with stimulation of the granule cell-parallel fibre system. The average values of the 10-90% rise time were 1.8 +/- 0.4 ms (means +/- S.D., n = 7) for PF-EPSCs and 0.8 +/- 0.3 ms (n = 9) for CF-EPSCs. Time constants of decay, at a holding potential of -60 mV, had values of 8.3 +/- 1.6 ms (n = 7) and 6.4 +/- 1.1 ms (n = 9) for PF-EPSCs and CF-EPSCs respectively. 5. CF-EPSCs and PF-EPSCs had the characteristics described above in slices derived from animals aged 9-22 days old and 9-15 days old, respectively. The PF-EPSCs in animals older than 15 days had very slow time courses and positive apparent reversal potentials, suggesting that they originated from distal locations, not under accurate voltage control. 6. In order to assess the quality of the voltage clamp, responses to hyperpolarizing pulses from -70 mV were analysed. The capacitive currents could be fitted by the sum of two exponentials, and were interpreted with an equivalent electrical circuit comprising two main compartments (soma and proximal dendrites on one hand, distal dendrites on the other). Analysis of synaptic currents in terms of this model suggested that the recorded time course of decay was approximately correct. 7. CF-EPSCs as well as PF-EPSCs were insensitive to the NMDA receptor antagonist 3-3(2-carboxypiperazine-4-yl)propyl-1-phosphonate (CPP), but were blocked in a dose-dependent reversible manner by the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX).(ABSTRACT TRUNCATED AT 400 WORDS)

6-Cyano-7-nitroquinoxaline-2,3-dione↗