allow the time course of both endocytosis and exocytosis to be determined at individually visuali... more allow the time course of both endocytosis and exocytosis to be determined at individually visualized boutons. What makes this possible is that FM dyes are water soluble but partition preferentially into lipid membranes, and once there fluoresce intensely. In the presence of
Understanding the communication between nerve cells in the brain is key to understanding how the ... more Understanding the communication between nerve cells in the brain is key to understanding how the brain works. Communication between nerve cells involves chemical messages sent from one cell that get translated into electrical activity in the receiving cell. This electrical activity is the core language of nerve cells and of the entire brain. How does a chemical message released in one cell results in electrical activity in another nerve cell, and how did we discovery this? Let us dive together into the electrifying world of nerve cell communication. I will tell you about our experiments, which enabled us to find the most basic component of electrical activity in the brain-ion channels. The discovery of ion channels paved the way to understanding the origin of electrical activity in the brain and other organs like the heart. This discovery provided new insights into the development of drugs for treating various electrical-related diseases, such as epilepsy and heart-rate disorders.
The characterization of individual neurons by Golgi and Cajal has been the basis of neuroanatomy ... more The characterization of individual neurons by Golgi and Cajal has been the basis of neuroanatomy for a century. A new challenge is to anatomically describe, at cellular resolution, complete local circuits that can drive behavior. In this essay, we review the possibilities to obtain a model cortical column by using in vitro and in vivo pair recordings, followed by anatomical reconstructions of the projecting and target cells. These pairs establish connection modules that eventually may be useful to synthesize an average cortical column in silico. Together with data on sensory evoked neuronal activity measured in vivo, this will allow to model the anatomical and functional cellular basis of behavior based on more realistic assumptions than previously attempted.
Proceedings of the National Academy of Sciences of the United States of America, Jun 1, 1981
Planar lipid bilayers were formed with the mixed chain phospholipid I-stearoyl-3-myristoylglycero... more Planar lipid bilayers were formed with the mixed chain phospholipid I-stearoyl-3-myristoylglycero-2-phosphocholine. Acetylcholine receptor membrane fragments or the purified receptor protein was incorporated into these bilayers by fusing receptor-containing vesicles with the planar membranes a few degrees below the lipid phase transition temperature. Singlechannel currents activated by nicotinic agonists in the reconstituted system resembled those observed in intact rat and frog muscle membrane as measured by the patch clamp technique. The observed channel characteristics did not depend on the degree of receptor purification. Thus, the receptor-enriched fragments and those depleted of nonreceptor peripheral peptides, the purified receptor monomer/dimer mixtures, and the isolated receptor monomer as defined by gel electrophoresis all shared similar electrochemical properties in the synthetic lipid bilayer. The agonistactivated ionic channel seems, therefore, to be contained within the receptor monomer.
1. Calcium dynamics associated with a single action potential (AP) were studied in single boutons... more 1. Calcium dynamics associated with a single action potential (AP) were studied in single boutons of the axonal arbor of layer 2Ï3 pyramidal cells in the neocortex of young (P14-16) rats. We used fluorescence imaging with two-photon excitation and Ca¥-selective fluorescence indicators to measure volume-averaged Ca¥ signals. These rapidly reached a peak (in about 1 ms) and then decayed more slowly (tens to hundreds of milliseconds). 2. Single APs and trains of APs reliably evoked Ca¥ transients in en passant boutons located on axon collaterals in cortical layers 2Ï3, 4 and 5, indicating that APs propagate actively and reliably throughout the axonal arbor. Branch point failures are unlikely to contribute to differences in synaptic efficacy and reliability in the connections made by layer 2Ï3 pyramidal cells. 3. AP-evoked Ca¥ transients in boutons were mediated by voltage-dependent Ca¥ channels (VDCCs), predominantly by the PÏQ-and N-subtypes. 4. Ca¥ transients were, on average, of significantly larger amplitude in boutons than in the flanking segments of the axon collateral. Large amplitude Ca¥ transients in boutons were spatially restricted to within û 3 ìm of axonal length. 5. Single AP-evoked Ca¥ transients varied up to 10-fold across different boutons even if they were located on the same axon collateral. In contrast, variation of Ca¥ transients evoked by successive APs in a given single bouton was small (coefficient of variation, c.v. û 0•21). 6. Amplitudes of AP-evoked Ca¥ signals did not correlate with the distance of boutons from the soma. In contrast, AP-evoked Ca¥ signals in spines of basal dendrites decreased slightly (correlation coefficient, r 2 = −0•27) with distance from the soma. 7. Measurements with the low-affinity Ca¥ indicator Magnesium Green suggest that the volume-averaged residual free [Ca¥]é in a bouton increases on average by 500 nÒ following a single AP. Higher concentrations of indicator caused, on average, a decrease in the amplitude and an increase in the decay time constant of Ca¥ transients. Assuming a singlecompartment model the concentration dependence of decay time constants suggests a low endogenous Ca¥ binding ratio close to 140, indicating that of the total Ca¥ influx (•2 fC) less than 1 % remained free. 8. The indicator concentration dependence of decay time constants further suggests that the residual free Ä[Ca¥]i associated with an AP decays with a time constant of about 60 ms (35°C) reflecting a high Ca¥ extrusion rate of about 2600 s¢. 9. The results show that AP-evoked volume-averaged Ca¥ transients in single boutons are evoked reliably and, on average, have larger amplitudes than Ca¥ transients in other subcellular compartments of layer 2Ï3 pyramidal cells. A major functional signature is the large variation in the amplitude of Ca¥ transients between different boutons. This could indicate that local interactions between boutons and different target cells modify the spatiotemporal Ca¥ dynamics in boutons and cause target cell-specific differences in their transmitter release properties.
The ion-selective and ion transport properties of glycine receptor (GlyR) and y-aminobutyric acid... more The ion-selective and ion transport properties of glycine receptor (GlyR) and y-aminobutyric acid receptor (GABAR) channels in the soma membrane of mouse spinal cord neurones were investigated using the whole-cell, cell-attached and outside-out patch versions of the patch-clamp technique. 2. Current-voltage (I-V) relations oftransmitter-activated currents obtained from whole-cell measurements with 145 mM-Cl-intracellularly and extracellularly, showed outward rectification. In voltage-jump experiments, the instantaneous I-V relations were linear, and the steady-state I-V relations were rectifying outwardly indicating that the gating of GlyR and GABAR channels is voltage sensitive. 3. The reversal potential of whole-cell currents shifted 56 mV per tenfold change in internal Clactivity indicating activation of Cl-selective channels. The permeability ratio of K+ to Cl-(PK/PC1) was smaller than 0 05 for both channels. 4. The permeability sequence for large polyatomic anions was formate > bicarbonate > acetate > phosphate > propionate for GABAR channels; phosphate and propionate were not measurably permeant in GlyR channels. This indicates that open GlyR and GABAR channels have effective pore diameters of 5-2 and 5-6 A, respectively. The sequence of relative permeabilities for small anions was SCN-> 1-> Br-> Cl-> F-for both channels. 5. GlyR and GABAR channels are multi-conductance-state channels. In cellattached patches the single-channel slope conductances close to 0 mV membrane potential were 29, 18 and 10 pS for glycine, and 28, 17 and 10 pS for GABA-activated channels. The most frequently observed (main) conductance states were 29 and 17 pS for the GlyR and GABAR channel, respectively. 6. In outside-out patches with equal extracellular and intracellular concentrations of 145 mM-Cl-, the conductance states were 46, 30, 20 and 12 pS for GlyR channels and 44, 30, 19 and 12 pS for GABAR channels. The most frequently occurring main state was 46 pS for the GlyR and 30 pS for the GABAR channel. 7. Single-channel conductances measured in equal 140 mm concentrations of small anions on both membrane faces revealed a conductance sequence of Cl-> Br->
1. Simultaneous fluorescence and whole-cell current measurements using the calcium indicator dye ... more 1. Simultaneous fluorescence and whole-cell current measurements using the calcium indicator dye fura-2 were made in HEK 293 cells expressing recombinant glutamate receptor (GluR) channels, and fractional Ca2+ currents (the proportion of whole-cell current carried by CP2+ Pf) were determined. 2. Cells expressing N-methyl-D-aspartate receptor (NMDAR) channels showed glutamateactivated Ca2+ inflow in the voltage range-60 to 40 mV in normal extracellular solution. Ca2+ inflow decreased in a voltage-dependent manner at membrane potentials more negative than-30 mV due to Mg2+ block. Voltage dependence of block at negative potentials was stronger in cells expressing the NR1-NR2A as compared with cells expressing NR1-NR2C subunits. 3. Fractional Ca2+ currents through NMDARs were independent of extracellular Mg2+ and varied between 8 2 % (NR1-NR2C subunits) and 11 % (NR1-NR2A subunits) in normal extracellular solution (1 8 mM Ca2+) at-60 mV membrane potential. Pf values increased with increasing [Ca2+]o in the range of 0 5-10 mm [Ca2+]. in a saturating fashion. 4. In cells expressing a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) subunits which were unedited at the Q/R site of the putative transmembrane segment TM2 (Q-form), or in cells coexpressing unedited and edited subunits (R-form), the glutamateevoked Ca2+ inflow increased from 20 to-80 mV in an almost linear way. 5. Fractional Ca2+ currents through AMPAR channels depended on subunit composition. Pf values of Q-form homomeric channels at-60 mV and 1X8 mm [Ca2+]o were between 3-2 and 3 9%. They were slightly voltage dependent and increased with [Ca2+]o in the range 1 8-10 mm. Pf values in cells co-expressing Qand R-form subunits were almost one order of magnitude smaller (0 54 %). 6. Relative concentrations of Q-form and R-form GluR-B subunit-specific cDNAs used for cell transfection determined the expression of functionally different heteromeric AMPARS. Pf decreased with increasing relative concentration of R-form encoding cDNAs from 3-4 to 1-4 %, demonstrating that editing of the Q/R site of GluR-B subunits decreases Ca2P inflow through heteromeric AMPARs. 7. Cells expressing the GluR-6 subunit of the kainate receptor (KAR) family were characterized by Pf values which depended on the editing in the TM1 and TM2 segments. Pf values were largest for the Q-form (1-55-2 0 %) and lowest for R-form channels (< 0 2 %), suggesting that Q/R site editing also decreases Ca2P inflow through KAR channels. Cells coexpressing both subunit forms showed an intermediate value (0 58 %).
Methods for measuring whole-cell and unitary Cl- currents in the soma membrane of cultured spinal... more Methods for measuring whole-cell and unitary Cl- currents in the soma membrane of cultured spinal cord neurons by the patch-clamp technique are described. To separate single channel Cl- currents activated by the two putative inhibitory transmitters GABA and glycine from other membrane currents, isotonic KCl pipette solutions are used for measurements on "cell-attached" membrane patches. To isolate unitary Cl- currents in "cell-free" membrane patches, symmetrical, isotonic Tris Cl- solutions are used in both the pipette and the bath. Single Cl- channels are opened following binding of two agonist molecules to their respective receptors. The gating behavior of single receptor-channel complexes is described in terms of the activation and desensitization mechanism suggested originally by del Castillo and Katz (1957) and Katz and Thesleff (1957).
allow the time course of both endocytosis and exocytosis to be determined at individually visuali... more allow the time course of both endocytosis and exocytosis to be determined at individually visualized boutons. What makes this possible is that FM dyes are water soluble but partition preferentially into lipid membranes, and once there fluoresce intensely. In the presence of
Understanding the communication between nerve cells in the brain is key to understanding how the ... more Understanding the communication between nerve cells in the brain is key to understanding how the brain works. Communication between nerve cells involves chemical messages sent from one cell that get translated into electrical activity in the receiving cell. This electrical activity is the core language of nerve cells and of the entire brain. How does a chemical message released in one cell results in electrical activity in another nerve cell, and how did we discovery this? Let us dive together into the electrifying world of nerve cell communication. I will tell you about our experiments, which enabled us to find the most basic component of electrical activity in the brain-ion channels. The discovery of ion channels paved the way to understanding the origin of electrical activity in the brain and other organs like the heart. This discovery provided new insights into the development of drugs for treating various electrical-related diseases, such as epilepsy and heart-rate disorders.
The characterization of individual neurons by Golgi and Cajal has been the basis of neuroanatomy ... more The characterization of individual neurons by Golgi and Cajal has been the basis of neuroanatomy for a century. A new challenge is to anatomically describe, at cellular resolution, complete local circuits that can drive behavior. In this essay, we review the possibilities to obtain a model cortical column by using in vitro and in vivo pair recordings, followed by anatomical reconstructions of the projecting and target cells. These pairs establish connection modules that eventually may be useful to synthesize an average cortical column in silico. Together with data on sensory evoked neuronal activity measured in vivo, this will allow to model the anatomical and functional cellular basis of behavior based on more realistic assumptions than previously attempted.
Proceedings of the National Academy of Sciences of the United States of America, Jun 1, 1981
Planar lipid bilayers were formed with the mixed chain phospholipid I-stearoyl-3-myristoylglycero... more Planar lipid bilayers were formed with the mixed chain phospholipid I-stearoyl-3-myristoylglycero-2-phosphocholine. Acetylcholine receptor membrane fragments or the purified receptor protein was incorporated into these bilayers by fusing receptor-containing vesicles with the planar membranes a few degrees below the lipid phase transition temperature. Singlechannel currents activated by nicotinic agonists in the reconstituted system resembled those observed in intact rat and frog muscle membrane as measured by the patch clamp technique. The observed channel characteristics did not depend on the degree of receptor purification. Thus, the receptor-enriched fragments and those depleted of nonreceptor peripheral peptides, the purified receptor monomer/dimer mixtures, and the isolated receptor monomer as defined by gel electrophoresis all shared similar electrochemical properties in the synthetic lipid bilayer. The agonistactivated ionic channel seems, therefore, to be contained within the receptor monomer.
1. Calcium dynamics associated with a single action potential (AP) were studied in single boutons... more 1. Calcium dynamics associated with a single action potential (AP) were studied in single boutons of the axonal arbor of layer 2Ï3 pyramidal cells in the neocortex of young (P14-16) rats. We used fluorescence imaging with two-photon excitation and Ca¥-selective fluorescence indicators to measure volume-averaged Ca¥ signals. These rapidly reached a peak (in about 1 ms) and then decayed more slowly (tens to hundreds of milliseconds). 2. Single APs and trains of APs reliably evoked Ca¥ transients in en passant boutons located on axon collaterals in cortical layers 2Ï3, 4 and 5, indicating that APs propagate actively and reliably throughout the axonal arbor. Branch point failures are unlikely to contribute to differences in synaptic efficacy and reliability in the connections made by layer 2Ï3 pyramidal cells. 3. AP-evoked Ca¥ transients in boutons were mediated by voltage-dependent Ca¥ channels (VDCCs), predominantly by the PÏQ-and N-subtypes. 4. Ca¥ transients were, on average, of significantly larger amplitude in boutons than in the flanking segments of the axon collateral. Large amplitude Ca¥ transients in boutons were spatially restricted to within û 3 ìm of axonal length. 5. Single AP-evoked Ca¥ transients varied up to 10-fold across different boutons even if they were located on the same axon collateral. In contrast, variation of Ca¥ transients evoked by successive APs in a given single bouton was small (coefficient of variation, c.v. û 0•21). 6. Amplitudes of AP-evoked Ca¥ signals did not correlate with the distance of boutons from the soma. In contrast, AP-evoked Ca¥ signals in spines of basal dendrites decreased slightly (correlation coefficient, r 2 = −0•27) with distance from the soma. 7. Measurements with the low-affinity Ca¥ indicator Magnesium Green suggest that the volume-averaged residual free [Ca¥]é in a bouton increases on average by 500 nÒ following a single AP. Higher concentrations of indicator caused, on average, a decrease in the amplitude and an increase in the decay time constant of Ca¥ transients. Assuming a singlecompartment model the concentration dependence of decay time constants suggests a low endogenous Ca¥ binding ratio close to 140, indicating that of the total Ca¥ influx (•2 fC) less than 1 % remained free. 8. The indicator concentration dependence of decay time constants further suggests that the residual free Ä[Ca¥]i associated with an AP decays with a time constant of about 60 ms (35°C) reflecting a high Ca¥ extrusion rate of about 2600 s¢. 9. The results show that AP-evoked volume-averaged Ca¥ transients in single boutons are evoked reliably and, on average, have larger amplitudes than Ca¥ transients in other subcellular compartments of layer 2Ï3 pyramidal cells. A major functional signature is the large variation in the amplitude of Ca¥ transients between different boutons. This could indicate that local interactions between boutons and different target cells modify the spatiotemporal Ca¥ dynamics in boutons and cause target cell-specific differences in their transmitter release properties.
The ion-selective and ion transport properties of glycine receptor (GlyR) and y-aminobutyric acid... more The ion-selective and ion transport properties of glycine receptor (GlyR) and y-aminobutyric acid receptor (GABAR) channels in the soma membrane of mouse spinal cord neurones were investigated using the whole-cell, cell-attached and outside-out patch versions of the patch-clamp technique. 2. Current-voltage (I-V) relations oftransmitter-activated currents obtained from whole-cell measurements with 145 mM-Cl-intracellularly and extracellularly, showed outward rectification. In voltage-jump experiments, the instantaneous I-V relations were linear, and the steady-state I-V relations were rectifying outwardly indicating that the gating of GlyR and GABAR channels is voltage sensitive. 3. The reversal potential of whole-cell currents shifted 56 mV per tenfold change in internal Clactivity indicating activation of Cl-selective channels. The permeability ratio of K+ to Cl-(PK/PC1) was smaller than 0 05 for both channels. 4. The permeability sequence for large polyatomic anions was formate > bicarbonate > acetate > phosphate > propionate for GABAR channels; phosphate and propionate were not measurably permeant in GlyR channels. This indicates that open GlyR and GABAR channels have effective pore diameters of 5-2 and 5-6 A, respectively. The sequence of relative permeabilities for small anions was SCN-> 1-> Br-> Cl-> F-for both channels. 5. GlyR and GABAR channels are multi-conductance-state channels. In cellattached patches the single-channel slope conductances close to 0 mV membrane potential were 29, 18 and 10 pS for glycine, and 28, 17 and 10 pS for GABA-activated channels. The most frequently observed (main) conductance states were 29 and 17 pS for the GlyR and GABAR channel, respectively. 6. In outside-out patches with equal extracellular and intracellular concentrations of 145 mM-Cl-, the conductance states were 46, 30, 20 and 12 pS for GlyR channels and 44, 30, 19 and 12 pS for GABAR channels. The most frequently occurring main state was 46 pS for the GlyR and 30 pS for the GABAR channel. 7. Single-channel conductances measured in equal 140 mm concentrations of small anions on both membrane faces revealed a conductance sequence of Cl-> Br->
1. Simultaneous fluorescence and whole-cell current measurements using the calcium indicator dye ... more 1. Simultaneous fluorescence and whole-cell current measurements using the calcium indicator dye fura-2 were made in HEK 293 cells expressing recombinant glutamate receptor (GluR) channels, and fractional Ca2+ currents (the proportion of whole-cell current carried by CP2+ Pf) were determined. 2. Cells expressing N-methyl-D-aspartate receptor (NMDAR) channels showed glutamateactivated Ca2+ inflow in the voltage range-60 to 40 mV in normal extracellular solution. Ca2+ inflow decreased in a voltage-dependent manner at membrane potentials more negative than-30 mV due to Mg2+ block. Voltage dependence of block at negative potentials was stronger in cells expressing the NR1-NR2A as compared with cells expressing NR1-NR2C subunits. 3. Fractional Ca2+ currents through NMDARs were independent of extracellular Mg2+ and varied between 8 2 % (NR1-NR2C subunits) and 11 % (NR1-NR2A subunits) in normal extracellular solution (1 8 mM Ca2+) at-60 mV membrane potential. Pf values increased with increasing [Ca2+]o in the range of 0 5-10 mm [Ca2+]. in a saturating fashion. 4. In cells expressing a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) subunits which were unedited at the Q/R site of the putative transmembrane segment TM2 (Q-form), or in cells coexpressing unedited and edited subunits (R-form), the glutamateevoked Ca2+ inflow increased from 20 to-80 mV in an almost linear way. 5. Fractional Ca2+ currents through AMPAR channels depended on subunit composition. Pf values of Q-form homomeric channels at-60 mV and 1X8 mm [Ca2+]o were between 3-2 and 3 9%. They were slightly voltage dependent and increased with [Ca2+]o in the range 1 8-10 mm. Pf values in cells co-expressing Qand R-form subunits were almost one order of magnitude smaller (0 54 %). 6. Relative concentrations of Q-form and R-form GluR-B subunit-specific cDNAs used for cell transfection determined the expression of functionally different heteromeric AMPARS. Pf decreased with increasing relative concentration of R-form encoding cDNAs from 3-4 to 1-4 %, demonstrating that editing of the Q/R site of GluR-B subunits decreases Ca2P inflow through heteromeric AMPARs. 7. Cells expressing the GluR-6 subunit of the kainate receptor (KAR) family were characterized by Pf values which depended on the editing in the TM1 and TM2 segments. Pf values were largest for the Q-form (1-55-2 0 %) and lowest for R-form channels (< 0 2 %), suggesting that Q/R site editing also decreases Ca2P inflow through KAR channels. Cells coexpressing both subunit forms showed an intermediate value (0 58 %).
Methods for measuring whole-cell and unitary Cl- currents in the soma membrane of cultured spinal... more Methods for measuring whole-cell and unitary Cl- currents in the soma membrane of cultured spinal cord neurons by the patch-clamp technique are described. To separate single channel Cl- currents activated by the two putative inhibitory transmitters GABA and glycine from other membrane currents, isotonic KCl pipette solutions are used for measurements on "cell-attached" membrane patches. To isolate unitary Cl- currents in "cell-free" membrane patches, symmetrical, isotonic Tris Cl- solutions are used in both the pipette and the bath. Single Cl- channels are opened following binding of two agonist molecules to their respective receptors. The gating behavior of single receptor-channel complexes is described in terms of the activation and desensitization mechanism suggested originally by del Castillo and Katz (1957) and Katz and Thesleff (1957).
Uploads
Papers by bert sakmann