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B Sakmann

Publications and source records attributed to B Sakmann.

At least 91 records · Page 5Linked to original sources
PubMed1994

Nerve-dependent induction of AChR epsilon-subunit gene expression in muscle is independent of state of differentiation.

To determine if the expression of the epsilon-subunit of the acetylcholine receptor by the subsynaptic nuclei in skeletal muscle is dependent on the state of differentiation of the muscle, we have compared the spatiotemporal distribution of epsilon-subunit transcripts during synapse formation in fetal and adult muscle. Both during "ontogenic" synaptogenesis in the fetus and during "ectopic" synaptogenesis in the adult animal the motor nerve induced focally the expression of the epsilon-subunit mRNA in subsynaptic nuclei. The temporal expression patterns at both types of developing synapses were similar. The results support the view that in muscle developing in vivo epsilon-subunit gene transcription and its stabilization in subsynaptic nuclei is exclusively controlled by the motor neuron, independently of the developmental state of the muscle nuclei. Thus, both nerve and muscle remain plastic in their respective abilities to induce and express the synapse-specific combination of AChR subunit genes.

Age Factors↗
PubMed1994

Developmental and regional expression in the rat brain and functional properties of four NMDA receptors.

An in situ study of mRNAs encoding NMDA receptor subunits in the developing rat CNS revealed that, at all stages, the NR1 gene is expressed in virtually all neurons, whereas the four NR2 transcripts display distinct expression patterns. NR2B and NR2D mRNAs occur prenatally, whereas NR2A and NR2C mRNAs are first detected near birth. All transcripts except NR2D peak around P20. NR2D mRNA, present mainly in midbrain structures, peaks around P7 and thereafter decreases to adult levels. Postnatally, NR2B and NR2C transcript levels change in opposite directions in the cerebellar internal granule cell layer. In the adult hippocampus, NR2A and NR2B mRNAs are prominent in CA1 and CA3 pyramidal cells, but NR2C and NR2D mRNAs occur in different subsets of interneurons. Recombinant binary NR1-NR2 channels show comparable Ca2+ permeabilities, but marked differences in voltage-dependent Mg2+ block and in offset decay time constants. Thus, the distinct expression profiles and functional properties of NR2 subunits provide a basis for NMDA channel heterogeneity in the brain.

Animals↗
PubMed1994

Differences in Ca2+ permeability of AMPA-type glutamate receptor channels in neocortical neurons caused by differential GluR-B subunit expression.

Fast excitatory synaptic transmission in the CNS is mediated by AMPA-type glutamate receptor (GluR) channels. Heterologous expression suggested that the Ca2+ permeability of these receptors critically depends on the subunit composition. Using patch-clamp techniques in brain slices, we found that the Ca2+ permeability of native AMPA-type GluRs was markedly higher in nonpyramidal (PCa/PK approximately 0.63) than in pyramidal (PCa/PK approximately 0.05) neurons of rat neocortex. Analysis of mRNA in single cells indicated that the relative abundance of GluR-B-specific mRNA was significantly lower in nonpyramidal (GluR-B/GluR-non-B approximately 0.3) than in pyramidal (GluR-B/GluR-non-B approximately 3) cells. This suggests that differences in relative abundance of GluR-B-specific mRNA generate functional diversity of AMPA-type GluRs in neurons with respect to Ca2+ permeability.

Animals↗
PubMed1994

Quantal analysis of excitatory postsynaptic currents at the hippocampal mossy fiber-CA3 pyramidal cell synapse.

Unitary excitatory postsynaptic current (EPSC) amplitude histograms obtained from synapses between mossy fibers (MF) and CA3 pyramidal cells in hippocampus were analyzed with the aim of deriving the mean peak current of quantal events, its coefficient of variation CVq, the number of release sites NS, and the release probability PR. Unitary EPSC histograms with multiple peaks were fitted satisfactorily under the assumption that quantal events have a slightly skewed amplitude distribution and that the release mechanism is described by models with either binomial (standard), nonuniform, or nonstationary statistics. The average peak current of the quantal event derived from these fits reflected the opening of between 15 and 65 glutamate receptor (GluR) channels of the AMPAR subtype. The variability in the amplitude of quantal events is characterized by a CVq of 25% to 30%. The best fits to multiple peak EPSC histograms are obtained if it is assumed that the number of release sites contained within a single MF-bouton is between 8 and 21, a value comparable to the published number of morphologically measured presynpatic active zones and postsynaptic densities (PSDs) of individual MF-CA3 synapses. This suggests that at the MF-CA3 synapse each presynaptic zone and its associated PSD act as independent units, contributing one quantal event to unitary EPSCs. Simulations of unitary EPSC distributions, based on measurements of PSD size could, however, indicate that the interpretation of multipeak EPSC amplitude distributions at MF-CA3 synapses in terms of fluctuating release probabilities might be an over-simplification, and that postsynaptic factors may contribute significantly to fluctuations of unitary EPSC amplitudes.

Animals↗
PubMed1994

Detailed passive cable models of whole-cell recorded CA3 pyramidal neurons in rat hippocampal slices.

Tight-seal whole-cell recordings were made from cleaned somata of CA3 pyramidal cells deep in hippocampal slices from 19-21-d-old rats. The cells were filled with biocytin, and their voltage responses to short current pulses were recorded. After washout of initial sag, responses scaled linearly with injected current and were stable over time. The dendritic and axonal arbors of four cells were reconstructed and measured using light microscopy. Dendritic spines and axonal boutons were counted and the additional membrane area was incorporated into the relevant segments. The morphology of each neuron was converted into a detailed branching cable model by assuming values for specific membrane capacitance Cm and resistance Rm, and cytoplasmic resistivity Ri. These parameters were optimized for each cell by directly matching the model's response to that of the real cell by means of a modified weighted least-squares fitting procedure. By comparing the deviations between model and experimental responses to control noise recordings, approximate 95% confidence intervals were established for each parameter. If a somatic shunt was allowed, a wide range of possible Rm values produced acceptable fits. With zero shunt, Cm was 0.7-0.8 microFcm-2, Ri was 170-340 omega cm, and Rm ranged between 120 and 200 k omega cm2. The electrotonic lengths of the basal and oblique dendrites were 0.2-0.3 space constants, and those of the apical tufts were 0.4-0.7 space constants. The steady-state electrical geometry of these cells was therefore compact; average dendritic tip/soma relative synaptic efficacies were > 93% for the basal and oblique dendrites, and > 81% for the tufts. With fast transient synaptic inputs, however, the models produced a wide range of postsynaptic potential shapes and marked filtering of voltage-clamp currents.

Animals↗
PubMed1993

Patch-clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy.

A description is given of the implementation of infrared differential interference contrast (IR-DIC) video microscopy to an upright compound microscope. Using the improved resolution offered by IR-DIC a procedure is described for making patch-pipette recordings from visually identified neuronal somata and dendrites in brain slices. As an example of the application of this technique to electrophysiological recordings from small neuronal processes in brain slices we describe whole-cell current-clamp and cell-attached and excised patch-clamp recordings from the apical dendrites of layer V pyramidal neurons in slices of rat neocortex.

Animals↗
PubMed1993

Determinants of Ca2+ permeability in both TM1 and TM2 of high affinity kainate receptor channels: diversity by RNA editing.

GluR6, a subunit of high affinity kainate receptor channels in the mammalian CNS, carries a glutamine (Q) or arginine (R) residue in a critical position (Q/R site) of the putative channel-forming segment TM2. One form, GluR6(Q), is encoded by the GluR6 gene; the other, GluR6(R), is generated by RNA editing. Further analysis of cloned GluR6 cDNA revealed that two additional positions, located in transmembrane segment TM1, are diversified by RNA editing to generate either isoleucine (I) or valine (V) in one and tyrosine (Y) or cysteine (C) in the other TM1 position. In GluR6 channels, in contrast with AMPA receptor channels, the presence of Q in the TM2 Q/R site determines channels with low Ca2+ permeability, whereas an R determines a higher Ca2+ permeability if TM1 is fully edited. In the TM1 unedited form of GluR6, Ca2+ permeability is less dependent on the presence of either Q or R in TM2. Thus Ca2+ permeability of kainate receptor channels can vary, depending on editing of both TM1 and TM2.

Amino Acid Sequence↗
PubMed1993

Quantal components of unitary EPSCs at the mossy fibre synapse on CA3 pyramidal cells of rat hippocampus.

1. Excitatory postsynaptic currents (EPSCs) were recorded in CA3 pyramidal cells of hippocampal slices of 15- to 24-day-old rats (22 degrees C) using the whole-cell configuration of the patch clamp technique. 2. Composite EPSCs were evoked by extracellular stimulation of the mossy fibre tract. Using the selective blockers 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D-2-amino-5-phosphonopentanoic acid (APV), a major alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor-mediated component and a minor NMDA receptor-mediated component with slower time course were distinguished. For the AMPA/kainate receptor-mediated component, the peak current-voltage (I-V) relation was linear, with a reversal potential close to 0 mV. The half-maximal blocking concentration of CNQX was 353 nM. 3. Unitary EPSCs of the mossy fibre terminal (MF)-CA3 pyramidal cell synapse were evoked at membrane potentials of -70 to -90 mV by low-intensity extracellular stimulation of granule cell somata using fine-tipped pipettes. The EPSC peak amplitude as a function of stimulus intensity showed all-or-none behaviour. The region of low threshold was restricted to a few micrometres. This suggests that extracellular stimulation was focal, and that the stimulus-evoked EPSCs were unitary. 4. Latency and rise time histograms of EPSCs evoked by granule cell stimulation showed narrow unimodal distributions within each experiment. The mean latency was 4.2 +/- 1.0 ms, and the mean 20-80% rise time was 0.6 +/- 0.1 ms (23 cells). When fitted within the range 0.7 ms to 20 ms after the peak, the decay of the EPSCs with the fastest rise (rise time 0.5 ms or less) could be described by a single exponential function; the mean time constant was in the range 3.0-6.6 ms with a mean of 4.8 ms (8 cells). 5. Peak amplitudes of the EPSCs evoked by suprathreshold granule cell stimulation fluctuated between trials. The apparent EPSC peak conductance in normal extracellular solution (2 mM Ca2+, 1 mM Mg2+), excluding failures, was 1 nS. Reducing the Ca2+ concentration and increasing the Mg2+ concentration reduced the mean peak amplitude in a concentration-dependent manner. 6. Peaks in EPSC peak amplitude distributions were apparent in low Ca2+ and high Mg2+. Using the criteria of equidistance and the presence of peaks and dips in the autocorrelation function, five of nine EPSC peak amplitude distributions were judged to be quantal.(ABSTRACT TRUNCATED AT 400 WORDS)

2-Amino-5-phosphonovalerate↗
PubMed1992

Asymmetry of the rat acetylcholine receptor subunits in the narrow region of the pore.

The acetylcholine receptor (AChR) channel is a pentameric protein in which every subunit contributes to the conducting parts of the pore. Recent studies of rat nicotinic AChR channels mutated in the alpha-subunit revealed that a threonine residue (alpha T264) in the transmembrane segment M2 forms part of the narrow region of the channel. We have mutated the residues at homologous positions in the beta-, gamma-, and delta-subunits and measured the resulting change in channel conductance. For all subunits the conductance is inversely related to the volume of the amino acid residue, suggesting that they form part of the channel narrow region. Exchanges of residues between subunits do not alter the conductance, suggesting a ring-like structure formed by homologous amino acids. To investigate the relative contribution of amino acid residues at these positions in determining the channel conductance, receptors carrying the same amino acid in each subunit in the narrow region were constructed. They form functional channels in which the conductance is inversely related to the volume of the amino acids in the narrow region. Channels in which the narrow region is formed by four serines and one valine have the same conductance if the valine is located in the alpha-, beta-, or gamma-subunits, but it is smaller if the valine is located in the delta-subunit. The results suggest a structural asymmetry of the AChR channel in its narrow region formed by the hydroxylated amino acids of alpha-, gamma- and delta-subunits, where the delta-subunit serine is a main determinant of the channel conductance.

Amino Acid Sequence↗
PubMed1992

Control by asparagine residues of calcium permeability and magnesium blockade in the NMDA receptor.

The N-methyl-D-aspartate (NMDA) receptor forms a cation-selective channel with a high calcium permeability and sensitivity to channel block by extracellular magnesium. These properties, which are believed to be important for the induction of long-term changes in synaptic strength, are imparted by asparagine residues in a putative channel-forming segment of the protein, transmembrane 2 (TM2). In the NR1 subunit, replacement of this asparagine by a glutamine residue decreases calcium permeability of the channel and slightly reduces magnesium block. The same substitution in NR2 subunits strongly reduces magnesium block and increases the magnesium permeability but barely affects calcium permeability. These asparagines are in a position homologous to the site in the TM2 region (Q/R site) of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that is occupied by either glutamine (Q) or arginine (R) and that controls divalent cation permeability of the AMPA receptor channel. Hence AMPA and NMDA receptor channels contain common structural motifs in their TM2 segments that are responsible for some of their ion selectivity and conductance properties.

Amino Acid Sequence↗
PubMed1992

Calcium-permeable AMPA-kainate receptors in fusiform cerebellar glial cells.

Glutamate-operated ion channels (GluR channels) of the L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-kainate subtype are found in both neurons and glial cells of the central nervous system. These channels are assembled from the GluR-A, -B, -C, and -D subunits; channels containing a GluR-B subunit show an outwardly rectifying current-voltage relation and low calcium permeability, whereas channels lacking the GluR-B subunit are characterized by a doubly rectifying current-voltage relation and high calcium permeability. Most cell types in the central nervous system coexpress several subunits, including GluR-B. However, Bergmann glia in rat cerebellum do not express GluR-B subunit genes. In a subset of cultured cerebellar glial cells, likely derived from Bergmann glial cells. GluR channels exhibit doubly rectifying current-voltage relations and high calcium permeability, whereas GluR channels of cerebellar neurons have low calcium permeability. Thus, differential expression of the GluR-B subunit gene in neurons and glia is one mechanism by which functional properties of native GluR channels are regulated.

Animals↗
PubMed1992

Heteromeric NMDA receptors: molecular and functional distinction of subtypes.

The N-methyl D-aspartate (NMDA) receptor subtype of glutamate-gated ion channels possesses high calcium permeability and unique voltage-dependent sensitivity to magnesium and is modulated by glycine. Molecular cloning identified three complementary DNA species of rat brain, encoding NMDA receptor subunits NMDAR2A (NR2A), NR2B, and NR2C, which are 55 to 70% identical in sequence. These are structurally related, with less than 20% sequence identity, to other excitatory amino acid receptor subunits, including the NMDA receptor subunit NMDAR1 (NR1). Upon expression in cultured cells, the new subunits yielded prominent, typical glutamate- and NMDA-activated currents only when they were in heteromeric configurations with NR1. NR1-NR2A and NR1-NR2C channels differed in gating behavior and magnesium sensitivity. Such heteromeric NMDA receptor subtypes may exist in neurons, since NR1 messenger RNA is synthesized throughout the mature rat brain, while NR2 messenger RNA show a differential distribution.

Amino Acid Sequence↗
PubMed1992

A glutamate receptor channel with high affinity for domoate and kainate.

The non-NMDA family of glutamate receptors comprises a growing number of structurally related subunits (GluR-A to -D or -1 to -4; GluR-5, -6; KA-1). GluR-A to -D appear to constitute the major AMPA receptor subtypes but the functional and pharmacological characteristics of the other subunits are unresolved. Using a mammalian expression system we demonstrate here that homomeric GluR-5 receptors exhibit properties of a high affinity domoate (KD approximately 2 nM) and kainate (KD approximately 70 nM) binding site. For these receptors, the rank order of ligands competing with [3H]kainate binding was domoate much greater than quisqualate approximately glutamate much greater than AMPA approximately CNQX. The respective receptor channels were gated in decreasing order of sensitivity by domoate, kainate, glutamate and AMPA. In contrast to recombinantly expressed GluR-A to -D channels, currents elicited at GluR-5 receptor desensitize channels to all agonists. This property is characteristic of currents in peripheral neurons on sensory ganglia. These findings suggest the existence of at least two distinct types of non-NMDA receptor channels, both gated by AMPA and kainate, but differing in pharmacology and current properties.

Amino Acid Sequence↗
PubMed1992

The KA-2 subunit of excitatory amino acid receptors shows widespread expression in brain and forms ion channels with distantly related subunits.

A new ionotropic glutamate receptor subunit termed KA-2, cloned from rat brain cDNA, exhibits high affinity for [3H]kainate (KD approximately 15 nM). KA-2 mRNA is widely expressed in embryonic and adult brain. Homomeric KA-2 expression does not generate agonist-sensitive channels, but currents are observed when KA-2 is coexpressed with GluR5 or GluR6 subunits. Specifically, coexpression of GluR5(R) and KA-2 produces channel activity, whereas homomeric expression of either subunit does not. Currents through heteromeric GluR5(Q)/KA-2 channels show more rapid desensitization and different current-voltage relations when compared with GluR5(Q) currents. GluR6/KA-2 channels are gated by AMPA, which fails to gate homomeric GluR6 receptor channels. These results suggest possible in vivo partnership relations for high affinity kainate receptors.

Amino Acid Sequence↗