TY - JOUR AB - To examine possible interactions between fast depression and modulation of inhibitory synaptic transmission in the hippocampus, we recorded from pairs of synaptically connected basket cells (BCs) and granule cells (GCs) in the dentate gyrus of rat brain slices at 34 degrees C. Multiple-pulse depression (MPD) was examined in trains of 5 or 10 inhibitory postsynaptic currents (IPSCs) evoked at frequencies of 10-100 Hz under several conditions that inhibit transmitter release: block of voltage-dependent Ca2+ channels by Cd2+ (10 microM), activation of gamma-amino-butyric acid type B receptors (GABA(B)Rs) by baclofen (10 microM) and activation of muscarinic acetylcholine receptors (mAchRs) by carbachol (2 microM). All manipulations led to a substantial inhibition of synaptic transmission, reducing the amplitude of the first IPSC in the train (IPSC1) by 72%, 61% and 29%, respectively. However, MPD was largely preserved under these conditions (0.34 in control versus 0.31, 0.50 and 0.47 in the respective conditions at 50 Hz). Similarly, a theta burst stimulation (TBS) protocol reduced IPSC1 by 54%, but left MPD unchanged (0.40 in control and 0.39 during TBS). Analysis of both fractions of transmission failures and coefficients of variation (CV) of IPSC peak amplitudes suggested that MPD had a presynaptic expression site, independent of release probability. In conclusion, different types of presynaptic modulation of inhibitory synaptic transmission converge on a reduction of synaptic strength, while short-term dynamics are largely unchanged. AU - Hefft, Stefan AU - Kraushaar, Udo AU - Geiger, Jörg AU - Jonas, Peter M ID - 3801 IS - Pt 1 JF - Journal of Physiology SN - 0022-3751 TI - Presynaptic short-term depression is maintained during regulation of transmitter release at a GABAergic synapse in rat hippocampus VL - 539 ER - TY - JOUR AB - GABAergic interneurones are diverse in their morphological and functional properties. Perisomatic inhibitory cells show fast spiking during sustained current injection, whereas dendritic inhibitory cells fire action potentials with lower frequency. We examined functional and molecular properties of K(+) channels in interneurones with horizontal dendrites in stratum oriens-alveus (OA) of the hippocampal CA1 region, which mainly comprise somatostatin-positive dendritic inhibitory cells. Voltage-gated K(+) currents in nucleated patches isolated from OA interneurones consisted of three major components: a fast delayed rectifier K(+) current component that was highly sensitive to external 4-aminopyridine (4-AP) and tetraethylammonium (TEA) (half-maximal inhibitory concentrations < 0.1 mM for both blockers), a slow delayed rectifier K(+) current component that was sensitive to high concentrations of TEA, but insensitive to 4-AP, and a rapidly inactivating A-type K(+) current component that was blocked by high concentrations of 4-AP, but resistant to TEA. The relative contributions of these components to the macroscopic K(+) current were estimated as 57 +/- 5, 25 +/- 6, and 19 +/- 2 %, respectively. Dendrotoxin, a selective blocker of Kv1 channels had only minimal effects on K(+) currents in nucleated patches. Coapplication of the membrane-permeant cAMP analogue 8-(4-chlorophenylthio)-adenosine 3':5'-cyclic monophosphate (cpt-cAMP) and the phosphodiesterase blocker isobutyl-methylxanthine (IBMX) resulted in a selective inhibition of the fast delayed rectifier K(+) current component. This inhibition was absent in the presence of the protein kinase A (PKA) inhibitor H-89, implying the involvement of PKA-mediated phosphorylation. Single-cell reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed a high abundance of Kv3.2 mRNA in OA interneurones, whereas the expression level of Kv3.1 mRNA was markedly lower. Similarly, RT-PCR analysis showed a high abundance of Kv4.3 mRNA, whereas Kv4.2 mRNA was undetectable. This suggests that the fast delayed rectifier K(+) current and the A-type K(+) current component are mediated predominantly by homomeric Kv3.2 and Kv4.3 channels. Selective modulation of Kv3.2 channels in OA interneurones by cAMP is likely to be an important factor regulating the activity of dendritic inhibitory cells in principal neurone-interneurone microcircuits. AU - Lien, Cheng AU - Martina, Marco AU - Schultz, Jobst AU - Ehmke, Heimo AU - Jonas, Peter M ID - 3799 IS - Pt 2 JF - Journal of Physiology SN - 0022-3751 TI - Gating, modulation and subunit composition of voltage-gated K(+) channels in dendritic inhibitory interneurones of rat hippocampus VL - 538 ER - TY - JOUR AB - 1. GABAergic interneurones differ from glutamatergic principal neurones in their ability to discharge high-frequency trains of action potentials without adaptation. To examine whether Na+ channel gating contributed to these differences, Na+ currents were recorded in nucleated patches from interneurones (dentate gyrus basket cells, BCs) and principal neurones (CA1 pyramidal cells, PCs) of rat hippocampal slices. 2. The voltage dependence of Na+ channel activation in BCs and PCs was similar. The slope factors of the activation curves, fitted with Boltzmann functions raised to the third power, were 11.5 and 11.8 mV, and the mid-point potentials were -25.1 and -23.9 mV, respectively. 3. Whereas the time course of Na+ channel activation (-30 to +40 mV) was similar, the deactivation kinetics (-100 to -40 mV) were faster in BCs than in PCs (tail current decay time constants, 0.13 and 0.20 ms, respectively, at -40 mV). 4. Na+ channels in BCs and PCs differed in the voltage dependence of inactivation. The slope factors of the steady-state inactivation curves fitted with Boltzmann functions were 6.7 and 10.7 mV, and the mid-point potentials were -58.3 and -62.9 mV, respectively. 5. The onset of Na+ channel inactivation at -55 mV was slower in BC's than in PCs; the inactivation time constants were 18.6 and 9.3 ms, respectively. At more positive potentials the differences in inactivation onset were smaller. 6. The time course of recovery of Na+ channels from inactivation induced by a 30 ms pulse was fast and mono-exponential (τ = 2.0 ms at -120 mV) in BCs, whereas it was slower and biexponential in PCs (τ1 = 2.0 ms and τ2 = 133 ms; amplitude contribution of the slow component, 15%). 7. We conclude that Na+ channels of BCs and PCs differ in gating properties that contribute to the characteristic action potential patterns of the two types of neurones. AU - Martina, Marco AU - Jonas, Peter M ID - 3485 IS - 3 JF - Journal of Physiology SN - 0022-3751 TI - Functional differences in Na+ channel gating between fast-spiking interneurones and principal neurones in rat hippocampus VL - 505 ER - TY - JOUR AB - 1. Dendritic patch-clamp recordings were obtained from mitral cells in rat olfactory bulb slices, up to 350 μm from the soma. Simultaneous dendritic and somatic whole-cell recordings indicated that action potentials (APs) evoked by somatic or dendritic current injection were initiated near the soma. Both the large amplitude (100.7 ± 1.1 mV) and the short duration (1.38 ± 0.07 ms) of the AP were maintained as the AP propagated back into the primary mitral cell dendrites. 2. Outside-out patches isolated from mitral cell dendrites contained voltage-gated Na+ channels (peak conductance density, 90 pS μm-2 at -10 mV). When an AP was used as a somatic voltage-clamp command in the presence of 1 μM tetrodotoxin (TTX), the amplitude of the dendritic potential was attenuated to 48 ± 14 mV. This shows that dendritic Na+ channels support the active back-propagation of APs. 3. Dendritic patches contained voltage-gated K+ channels with high density (conductance density, 513 pS μm-2 at 30 mV. Dendritic K+ currents were reduced to 35% by 1 mM external tetraethylammonium chloride (TEACl). When an AP was used as a somatic voltage clamp command in the presence of TEACl, the dendritic potential was markedly prolonged. This indicates that dendritic K+ channels mediate the fast repolarization of dendritic APs. 4. We conclude that voltage gated Na+ and K+ channels support dendritic APs with large amplitudes and short durations that may trigger fast transmitter release at dendrodendritic synapses in the olfactory bulb. AU - Bischofberger, Joseph AU - Jonas, Peter M ID - 3486 IS - Pt 2 JF - Journal of Physiology SN - 0022-3751 TI - Action potential propagation into the presynaptic dendrites of rat mitral cells VL - 504 ER - TY - JOUR AB - 1. Properties of dendritic glutamate receptor (GluR) channels were investigated using fast application of glutamate to outside-out membrane patches isolated from the apical dendrites of CA3 and CA1 pyramidal neurons in rat hippocampal slices. CA3 patches were formed (15-76 μm from the soma) in the region of messy fibre (MF) synapses, and CA1 patches (25-174 μm from the soma) in the region of Schaffer collateral (SC) innervation. 2. Dual-component responses consisting of a rapidly rising and decaying component followed by a second, substantially slower, component were elicited by 1 ms pulses of 1 mM glutamate in the presence of 10 μM glycine and absence of external Mg2+. The fast component was selectively blocked by 2-5 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the slow component by 30 μM D-2-amino-5-phosphonopentanoic acid (D-AP5), suggesting that the fast and slow components were mediated by the GluR channels of the L-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and NMDA type, respectively. The peak amplitude ratio of the NMDA to AMPA receptor-mediated components varied between 0.03 and 0.62 in patches from both CA3 and CA1 dendrites. Patches lacking either component were rarely observed. 3. The peak current-voltage (I-V) relationship of the fast component was almost linear, whereas the I-V relationship of the slow component showed a region of negative slope in the presence of 1 mM external Mg2+. The reversal potential for both components was close to 0 mV. 4. Kainate-preferring GluR channels did not contribute appreciably to the response to glutamate. The responses to 100 ms pulses of 1 mM glutamate were mimicked by application of 1 mM AMPA, whereas 1 mM kainate produced much smaller, weakly desensitizing currents. This suggests that the fast component is primarily mediated by the action of glutamate on AMPA-preferring receptors. 5. The mean elementary conductance of AMPA receptor channels was about 10 pS, as estimated by non-stationary fluctuation analysis. The permeability of these channels to Ca2+ was low (~5% of the permeability to Cs+). 6. The elementary conductance of NMDA receptor channels was larger, with a main conductance state of about 45 pS. These channels were 3.6 times more permeable to Ca2+ than to Cs+. 7. AMPA receptor-mediated currents activated rapidly in response to 1 ms pulses of 1 mM glutamate and deactivated with a predominant, fast time constant and a smaller, slower component (τ1≃2 ms, τ2≃8 ms, contributing ~80 and ~20% to the total decay amplitude, respectively). Desensitization of the current during a 100 ms pulse was best fitted by two time constants (τ1≃10 ms, ~60%; τ2≃34 ms, ~40%). 8. NMDA receptor-mediated currents in response to 1 ms pulses of 1 mM glutamate activated and deactivated much more slowly than AMPA receptor-mediated currents. The time course could be described by a single exponential rising phase (τ≃7 ms) followed by a double exponential decay (τ1≃200 ms, ~80%; τ2≃1-3 s, ~20%). 9. Mg2+ blocked the NMDA component in a voltage-dependent manner, with a half-maximal inhibitory concentration (IC50) of 21 μM at -80 mV. At physiological Mg2+ concentrations, block of the NMDA component could be rapidly relieved with voltage jumps from negative to positive potentials. Block of the current upon return to negative potentials occurred almost instantaneously. 10. Zn2+ also selectively-blocked the NMDA receptor-mediated current with an IC50 of 22 μM, but this block differed from that of Mg2+ in that it showed little voltage dependence. Rapid application of Zn2+ together with glutamate produced partial block of the current. More block was observed if Zn2+ and glutamate were co-applied when NMDA receptor channels were already open. 11. The functional properties of dendritic GluRs were similar to those found at the soma. Knowledge of these properties facilitated simulations investigating the contribution of coactivated AMPA and NMDA receptors to synaptic depolarization and Ca2+ entry into dendritic spines. Because of its slow deactivation, the NMDA receptor-mediated current contributes substantially to depolarization and Ca2+ entry and is susceptible to modulation over a period of seconds, either by backpropagating action potentials or by the release of Zn2+ from presynaptic boutons. AU - Spruston, Nelson AU - Jonas, Peter M AU - Sakmann, Bert ID - 3478 IS - Pt 2 JF - Journal of Physiology SN - 0022-3751 TI - Dendritic glutamate receptor channels in rat hippocampal CA3 and CA1 pyramidal neurons VL - 482 ER - TY - JOUR AB - 1. Glutamate receptor (GluR) channels were studied in basket cells in the dentate gyrus of rat hippocampal slices. Basket cells were identified by their location, dendritic morphology and high frequency of action potentials generated during sustained current injection. 2. Dual-component currents were activated by fast application of glutamate to outside-out membrane patches isolated from basket cell somata (10 μM glycine, no external Mg2+). The fast component was selectively blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the slow component by D-2-amino-5-phosphonopentanoic acid (D-AP5). This suggests that the two components were mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR)/kainate receptor and N-methyl-D-aspartate receptor (NMDAR) channels, respectively. The mean ratio of the peak current of the NMDAR component to that of the AMPAR/kainate receptor component was 0.22 (1 ms pulses of 10 mM glutamate). 3. The AMPAR/kainate receptor component, which was studied in isolation in the presence of D-AP5, was identified as AMPAR mediated on the basis of the preferential activation by AMPA as compared with kainate, the weak desensitization of kainate-activated currents, the cross-desensitization between AMPA and kainate, and the reduction of desensitization by cyclothiazide. 4. Deactivation of basket cell AMPARs following 1 ms pulses of glutamate occurred with a time constant (τ) of 1.2 ± 0.1 ms (mean ± S.E.M.). During 100 ms glutamate pulses, AMPARs desensitized with a τ of 3.7 ± 0.2 ms. 5. The peak current-voltage (I-V) relation of AMPAR-mediated currents in Na+-rich extracellular solution showed a reversal potential of -4.0 ± 2.6 mV and was characterized by a doubly rectifying shape. The conductance of single AMPAR channels was estimated as 22.6 ± 1.6 pS using non-stationary fluctuation analysis. AMPARs expressed in hippocampal basket cells mere highly Ca2+ permeable (P(Ca)/P(K) = 1.79). 6. NMDARs in hippocampal basket cells were studied in isolation in the presence of CNQX. Deactivation of NMDARs activated by glutamate pulses occurred bi-exponentially with mean τ values of 266 ± 23 ms (76%) and 2620 ± 383 ms (24%). 7. The peak I-V relation of the NMDAR-mediated component in Na+-rich extracellular solution showed a reversal potential of 1.5 ± 0.6 mV and a region of negative slope at negative membrane potentials in the presence of external Mg2+, due to voltage-dependent block by these ions. The conductance of single NMDAR channels in the main open state was 50.2 ± 1.8 pS. NMDARs in hippocampal basket cells were highly permeable to Ca2+ (P(Ca)/P(K) = 6.68). 8. AMPARs in hippocampal basket cells are characterized by about threefold faster kinetics and twentyfold higher Ca2+ permeability than AMPARs in hippocampal granule or pyramidal cells. Simulations show that the Ca2+ influx through basket cell AMPARs is comparable to that through NMDARs at negative membrane potentials with physiological concentrations of Ca2+ and Mg2+. This suggests a dual pathway of synaptically mediated Ca2+ entry into interneurones. AU - Koh, Duk AU - Geiger, Jörg AU - Jonas, Peter M AU - Sakmann, Bert ID - 3479 IS - Pt 2 JF - Journal of Physiology SN - 0022-3751 TI - Ca(2+)-permeable AMPA and NMDA receptor channels in basket cells of rat hippocampal dentate gyrus VL - 485 ER - TY - JOUR AB - 1. The influence of intracellular factors on current rectification of different subtypes of native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) was studied in rat brain slices by combining fast application of glutamate with patch pipette perfusion. 2. The peak current-voltage (I-V) relation of the AMPARs expressed in Bergmann glial cells of cerebellum and dentate gyrus (DG) basket cells of hippocampus was weakly rectifying in outside-out patches and nystatin-perforated vesicles, but showed a doubly rectifying shape with a region of reduced slope between 0 and +40 mV in nucleated patches. The I-V relation of AMPARs expressed in hippocampal CA3 pyramidal neurones was linear in all recording configurations. 3. Intracellular application of 2.5 μM spermine, a naturally occurring polyamine, blocked outward currents in outside-oat patches from Bergmann glial cells and DG basket cells in a voltage-dependent manner, generating I-V relations with a doubly rectifying shape which were similar to those recorded in nucleated patches. AMPARs in CA3 pyramidal cell patches were unaffected by 25 μM spermine. 4. The half-maximal blocking concentration of spermine at +40 mV was 0.3 μM in Bergmann glial cell patches and 1.5 μM in DG basket cell patches, whereas it was much higher (≥ 100 μM) for CA3 pyramidal. cell patches. Spermidine also affected current rectification, but with lower affinity. The block of outward current by polyamines following voltage jumps developed within < 0.5 ms. 5. We conclude that current rectification, rather than being an intrinsic property of the Ca2+ permeable AMPAR channel, is generated by polyamine block. AU - Koh, Duk AU - Burnashev, Nail AU - Jonas, Peter M ID - 3481 IS - Pt 2 JF - Journal of Physiology SN - 0022-3751 TI - Block of native Ca(2+)-permeable AMPA receptors in rat brain by intracellular polyamines generates double rectification VL - 486 ER - TY - JOUR AB - 1. A potassium channel activated by internal Na+ ions (K+Na channel) was identified in peripheral myelinated axons of Xenopus laevis using the cell-attached and excised configurations of the patch clamp technique. 2. The single-channel conductance for the main open state was 88 pS with [K+]o = 105 mM and pS with [K+]o = 2.5 mM ([K+]i = 105 mM). The channel was selectively permeable to K+ over Na+ ions. A characteristic feature of the K+Na channel was the frequent occurrence of subconductance states. 3. The open probability of the channel was strongly dependent on the concentration of Na+ ions at the inner side of the membrane. The half-maximal activating Na+ concentration and the Hill coefficient were 33 mM and 2.9, respectively. The open probability of the channel showed only weak potential dependence. 4. The K+Na channel was relatively insensitive to external tetraethylammonium (TEA+) in comparison with voltage-dependent axonal K+ channels; the half-maximal inhibitory concentration (IC50) was 21.3 mM (at -90 mV). In contrast, the channel was blocked by low concentrations of external Ba2+ and Cs+ ions, with IC50 values of 0.7 and 1.1 mM, respectively (at -90 mV). The block by Ba2+ and Cs+ was more pronounced at negative than at positive membrane potentials. 5. A comparison of the number of K+Na channels in nodal and paranodal patches from the same axon revealed that the channel density was about 10-fold higher at the node of Ranvier than at the paranode. Moreover, a correlation between the number of K+Na channels and voltage-dependent Na+ channels in the same patches was found, suggesting co-localization of both channel types. 6. As weakly potential-dependent ('leakage') channels, axonal K+Na channels may be involved in setting the resting potential of vertebrate axons. Simulations of Na+ ion diffusion suggest two possible mechanisms of activation of K+Na channels: the local increase of Na+ concentration in a cluster of Na+ channels during a single action potential or the accumulation in the intracellular axonal compartment during a train of action potentials. AU - Koh, Duk AU - Jonas, Peter M AU - Vogel, Werner ID - 3475 JF - Journal of Physiology SN - 0022-3751 TI - Na+-activated K+ channels localized in the nodal region of myelinated axons of Xenopus VL - 479 ER - TY - JOUR AB - 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. AU - Jonas, Peter M AU - Major, Guy AU - Sakmann, Bert ID - 3474 JF - Journal of Physiology SN - 0022-3751 TI - Quantal components of unitary EPSCs at the mossy fibre synapse on CA3 pyramidal cells of rat hippocampus VL - 472 ER - TY - JOUR AB - Currents activated by glutamate receptor (GluR) agonists were recorded from outside-out patches isolated from the soma of visually identified pyramidal neurones of the (CA3 and CA1 region of rat hippocampal slices. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). L-glutamate (L-Glu), and kainate (KA) were delivered either by bath application through perfusion of the recording chamber or by rapid application via a piezo-driven two-barrelled fast application system. 2. Bath application of each of the three agonists activated inward currents in all patches (n = 134) at holding potentials of -50 or -60 mV. The current amplitude increased in size between 3 to 30 μM-AMPA and 100 μM to 1 mM-KA. With this slow mode of bath application, the responses showed no apparent desensitization even at saturating concentrations of AMPA (30 μM) and KA (1 mM). 3. The ratio of currents activated by 30 μM-AMPA and 300 μM-KA showed a characteristic difference between CA3 and CA1 neurones. The ratio was 0.242 ± 0.028 (mean ± S.E.M., n = 16) for CA3 cell patches and 0.097 ± 0.012 (n = 8) for CA1 cell patches indicating that GluRs in the two cell populations are different. 4. The steady-state current-voltage relations (I-Vs) for AMPA- and KA-activated currents showed pronounced outward rectification for both cell types (when the main cations are Na+ in the bath and Cs+ in the pipette solution). The current reversed close to 0 mV and the ratio of chord conductances 80 mV on either side of the reversal potential was 2.66 for KA-activated currents in CA3 cell patches and 2.60 in CA1 cell patches. AMPA-activated currents showed a time-dependent increase after steps to positive membrane potentials and a decrease after steps to negative voltages, indicating that a gating process is responsible for outward rectification of the steady-state I-IV. 5. The permeability (P) of GluR channels was high for Na+ as compared to Cs+ for both cell types (P(Na)/P(Cs) = 0.88 and 0.84). The permeability was low for N-methyl-D-glucamine+ (P(NMG)/P(Cs) ≤ 0.03) and Ca2+ (P(Ca)/P(Cs) ≤0.05). 6. The current noise level increased during application of AMPA or KA. Apparent single-channel conductances obtained from fluctuation analysis were higher for AMPA than for KA, but similar for both cell types. In CA3 cell patches, AMPA activated channels with an apparent chord conductance of 7.2 pS, KA of 3.0 pS conductance. 7. Fast agonist application revealed desensitization of GluR channels which was dependent on the type of agonist, currents activated by AMPA and L-Glu rose rapidly to a peak and then desensitized to a steady-state current. In contrast, currents activated by fast application of KA rose to a plateau and did not desensitize. The steady state current expressed as a percentage of the peak current was higher for L-Glu than for AMPA and slightly higher for CA3 than for CA1 cell patches. For CA3 cell patches, this fraction amounted to 6.2 %, with 300 μM-L-Glu and 2.8%, with 300 μM-AMPA. For CA1 cell patches, corresponding values were 3.6 and 1.9 % 8. The dose response relations for the peak current activated by AMPA and L-Glu and the steady-state current activated by KA were similar for CA3 and CA1 cell patches. The order of potency was AMPA > L-Glu ≃ KA for both cell types EC50 values 189, 342 and 344 μM for CA3 cell patches and 183, 424 and 474 μM for CA1 cell patches). In all cases, the Hill coefficients ranged between 12 and 1.7. 8. The rise of AMPA and L-Glu-activated currents became faster with increasing agonist concentration for both cell types. With L-Glu, rise times decreased from about 3 ms at 100 μM to 500 μs at 3 mM. The delay for agonist concentrations ≥ 300 μM was described by the sum of two exponential functions. The time constant of the predominant fast component was slightly concentration dependent and decreased from about 12 ms at 300 μM to 8 ms at 3 mM-L-Glu. 10. The current voltage relations of the peak currents activated by 300 μM-AMPA were linear for both cell types with a reversal potential close to OmV. 11. It is concluded that the GluR channels in pyramidal cells of hippocampal CA3 and CA1 regions are distinet but share many pharmacological and functional properties. Comparison of the properties of native and recombinant GluRs suggests that in both CA3 and CA1 regions GluR channels are hetero-oligomers containing the GluR-B subunit. AU - Jonas, Peter M AU - Sakmann, Bert ID - 3470 JF - Journal of Physiology SN - 0022-3751 TI - Glutamate receptor channels in isolated patches from CA1 and CA3 pyramidal cells of rat hippocampal slices VL - 455 ER - TY - JOUR AB - 1. Outside-out patches were isolated from granule cells of dentate gyrus and pyramidal cells of CA3 and CA1 regions of rat hippocampal slices. Patches were exposed briefly to L-glutamate using a piezo-driven double-barrelled application pipette. 2. Applications of glutamate (1 mM) of 1 ms duration activated patch currents which rose and decayed rapidly. The 20-80% rise time of these glutamate receptor (GluR)-mediated currents was usually 0.2-0.6 ms. At -50 mV the peak current varied from 10 to 500 pA in different patches. 3. The peak current-voltage relation for brief pulses of 1 mM glutamate was virtually linear in normal extracellular solution for patches from the three cell types (-100 to 60 mV). 4. The permeability of GluR channels activated at the peak to Ca2+, relative to K+, was less than 0.1 for all three cell types (under bi-ionic conditions with Ca2+ on the extracellular side and K+ on the intracellular side of the membrane). 5. The offset decay time constant of the current following 1 ms pulses of 1 mM glutamate was brief, with mean values of 3.0 +/- 0.8, 2.5 +/- 0.7, and 2.3 +/- 0.7 ms for dentate, CA3 and CA1 cell patches, respectively. Offset time constants were independent of membrane potential and independent of glutamate concentration (200 microM and 1 mM) for the three cell types. 6. Applications of 1 mM glutamate of 100 ms duration showed that glutamate responses desensitized rapidly. The time constants for desensitization were 9.4 +/- 2.7, 11.3 +/- 2.8, and 9.3 +/- 2.8 ms for patches from dentate, CA3 and CA1 cells respectively. Desensitization time constants were only weakly dependent on glutamate concentration (200 microM and 1 mM) for the three cell types. Thus offset time constants are about four times faster than desensitization time constants for both glutamate concentrations. 7. Double pulse application of glutamate indicated that even a 1 ms pulse of 1 mM glutamate causes partial (about 60%) desensitization of GluR channels. The time course of recovery from desensitization was slower in dentate gyrus granule cell patches than in CA3 or CA1 pyramidal cell patches. 8. Desensitization was studied at equilibrium by exposing patches to low glutamate concentrations for at least 15 s before a 1 ms test pulse of 1 mM glutamate. AU - Colquhoun, D. AU - Jonas, Peter M AU - Sakmann, Bert ID - 3471 JF - Journal of Physiology SN - 0022-3751 TI - Action of brief pulses of glutamate on AMPA/kainate receptors in patches from different neurones of rat hippocampal slices VL - 458 ER - TY - JOUR AB - The effects of mast cell degranulating peptide (MCDP), a toxin from the honey bee, and of dendrotoxin (DTX), a toxin from the green mamba snake, were studied in voltage-clamped experiments with myelinated nerve fibres of Xenopus. MCDP and DTX blocked part of the K+ current. About 20% of the K+ current, however, was resistant to the toxins even in high concentrations. In Ringer solution half-maximal block was reached with concentrations of 33 nM MCDP and 11 nM DTX. In high-K+ solution the potency of both toxins was lower. β-Bungarotoxin (β-BuTX), another snake toxin, also blocked part of the K+ current, but was less potent than MCDP and DTX. Tail currents in high-K+ solution were analysed and three K+ current components were separated according to Dubois (1981b). Both MCDP and DTX selectively blocked a fast deactivating, slowly inactivating K+ current component which steeply activates between E = -60 mV and E = -40 mV (component f1). In concentrations around 100 nM, MCDP and DTX blocked neither the slow K+ current (component s) nor the fast deactivating, rapidly inactivating K+ current which activates between E = -40 mV and E = 20 mV (component f2). Similar results could be derived from K+ outward currents in Ringer solution. In high-K+, IC50 of MCDP for component f1 was 99 nM, whereas it was 7.6 μM for f2. Corresponding values for DTX are 68 nM and 1.8 μM. Binding studies with nerve fibre membranes of Xenopus reveal high-affinity binding sites for 125I-labelled DTX )K(D) = 22 pM in Ringer solution and 81 pM in high-K+ solution). 125I-labelled DTX can be displaced from its sites completely by unlabelled DTX, toxin I (black mamba toxin), MCDP, and partially by β-BuTX. Immunocytochemical staining demonstrates that binding sites for DTX are present in nodal and paranodal regions of the axonal membrane. The axonal membrane of motor and sensory nerve fibres is equipped with three types of well-characterized K+ channels and constitutes so far the best preparation to study MCDP- and DTX-sensitive K+ channels with electrophysiological and biochemical methods. AU - Bräu, Michael AU - Dreyer, Florian AU - Jonas, Peter M AU - Repp, Holger AU - Vogel, Werner ID - 3467 JF - Journal of Physiology SN - 0022-3751 TI - A K+ channel in Xenopus nerve fibres selectively blocked by bee and snake toxins: binding and voltage-clamp experiments VL - 420 ER -