Stephen Redman

Theory and operation of a single microelectrode voltage clamp

The theory of operation of a discontinuous single-electrode voltage clamp using an ideal microelectrode (infinite response speed) and fixed (current-passing) duty cycle has been previously described. In this paper, the theory is extended by considering a microelectrode which has a finite response speed, by allowing the duty cycle to be variable, and by considering the clamp noise. Formulate are derived for the relationships between the step response, the steady-state error, the steady-state ripple, and the stability, in terms of the cycling frequency, the duty cycle, the open loop gain, and the electrical resistance and capacitance of the microelectrode and the cell membrane. In addition, the amplification of the microelectrode noise by aliasing is analysed, the error due to incomplete decay of the microelectrode voltage is described, and the accuracy of averaging the peak current measurement is established. To achieve the fastest dynamic response and the smallest steady-state error, the cycling period should be made as small as possible, and the open-loop gain should be as large as possible, consistent with stability. Incomplete decay of the microelectrode voltage destabilizes the clamp, and can introduce a significant clamp error. The choice of duty cycle is a compromise between reducing the noise and the step response time while avoiding design problems in the current output circuit. The output noise is amplified by aliasing. It can be minimized for a given output filter cutoff frequency by keeping the cycling frequency as high as possible, and by the use of an anti-aliasing filter whose cutoff frequency must be set for each microelectrode.

Cable properties of cat spinal motoneurones measured by combining voltage clamp, current clamp and intracellular staining.

1. Spinal alpha‐motoneurones were injected with horseradish peroxidase after measuring their voltage response to a brief current pulse and their current response to a small voltage step. 2. The morphology of each motoneurone was reconstructed from serial sections. The diameters and lengths of dendritic segments were used to build a compartmental model of each neurones electrotonic structure. The specific resistivity of the membrane (Rm) was assumed to be constant throughout the dendrites, but it was lowered for the somatic membrane by the introduction of a somatic shunt resistance. 3. The specific resistances of the somatic and dendritic membrane were adjusted in the compartmental model until the responses of the model to the same current and voltage steps as those used in the experiment gave the best fits to the recorded transients. Satisfactory fits were obtained for six out of seven motoneurones. Dendritic Rm varied from 7 to 35 k omega cm2 and somatic Rm varied from 100 to 420 omega cm2. The dendritic Rm was 100‐300 times the somatic Rm for different neurones. 4. The calculated dendritic Rm was used to determine the geometric profile of the equivalent dendritic cable. This was found to be an approximately uniform cylinder for about 0.5 lambda and thereafter to taper rapidly to a final termination at 2‐3 lambda from the soma. 5. The results indicate that motoneurone dendrites are more electrically compact than was hitherto believed. The different Rm values for somatic and dendritic membrane, and the tapering of the dendritic cable, means that the cable model developed by Rall (1959, 1964) must be revised to take account of these spatial and electrical non‐uniformities.

Spatial segregation of neuronal calcium signals encodes different forms of LTP in rat hippocampus

Calcium regulates numerous processes in the brain. How one signal can coordinate so many diverse actions, even within the same neurone, is the subject of intense investigation. Here we have used two‐photon calcium imaging to determine the mechanism that enables calcium to selectively and appropriately induce different forms of long‐term potentiation (LTP) in rat hippocampus. Short‐lasting LTP (LTP 1) required activation of ryanodine receptors (RyRs), which selectively increased calcium in synaptic spines. LTP of intermediate duration (LTP 2) was dependent on activation of inositol 1,4,5‐trisphosphate (IP3) receptors (IP3Rs) and subsequent calcium release specifically in dendrites. Long‐lasting LTP (LTP 3) was selectively dependent on L‐type voltage‐dependent calcium channels (L‐VDCCs), which generated somatic calcium influx. Activation of NMDA receptors was necessary, but not sufficient, for the generation of appropriate calcium signals in spines and dendrites, and the induction of LTP 1 and LTP 2. These results suggest that the selective induction of different forms of LTP is achieved via spatial segregation of functionally distinct calcium signals.

Statistical analysis of amplitude fluctuations in EPSCs evoked in rat CA1 pyramidal neurones in vitro.

1. EPSCs were evoked in CA1 pyramidal neurones of young rats in vitro by extracellular stimulation of axons in a restricted stratum radiatum field, and were recorded using the whole‐cell technique. 2. Quantal fluctuations in EPSC amplitude could be demonstrated for nineteen of fifty EPSCs analysed. Quantal currents (at the soma) ranged from 2.6 to 9.5 pA (after correction for the access resistance) with a mean of 4.0 +/‐ 2.0 pA. 3. Quantal variance was negligible for the majority (13/19) of the EPSCs. However, a large quantal variance (with a coefficient of variation > 0.4) is one possible reason why a large number of the EPSCs (29/50) could not be shown to have quantal fluctuations. 4. The statistical pattern of fluctuations in the amplitude of the majority of the quantal EPSCs (18/19) could not be described by conventional models of transmitter release. 5. The time course of the EPSC and a compartmental model of CA1 pyramidal neurones were used to calculate synaptic location. The quantal current (at the soma) was independent of the electrotonic location of the synapse at which it was evoked. The peak quantal conductance generating each quantal current ranged from 0.5 to 6.8 nS (mean 1.3 +/‐ 1.4 nS), its magnitude increasing with distance from the soma. The mean peak conductance is likely to be generated by the opening of at least 60‐160 AMPA channels.

The role of GABAA and GABAB receptors in presynaptic inhibition of Ia EPSPs in cat spinal motoneurones.

1. The role of GABAA and GABAB receptors in presynaptic inhibition was studied by examining the effect of local application of antagonists by ionophoresis during intracellular recording of presynaptic inhibition of compound and unitary group Ia afferent excitatory postsynaptic potentials (EPSPs) in gastrocnemius motoneurones. 2. Ionophoresis of the GABAA antagonist bicuculline methochloride (BMC) was found to block presynaptic inhibition of both compound and unitary EPSPs by up to 85%. BMC also substantially reduced, and occasionally abolished, the late part of the inhibitory postsynaptic potential (IPSP) evoked in motoneurones by the conditioning stimulation. The early part of this IPSP was found to be sensitive to ionophoresis of strychnine hydrochloride. 3. Ionophoresis of 2‐OH‐saclofen caused a reduction in presynaptic inhibition of compound EPSPs by 5‐25% but had no effect on the IPSP evoked in motoneurones by the conditioning stimulation. 4. Ionophoresis of the GABAB antagonist (‐)‐baclofen reduced the amplitude of unconditioned EPSPs; however it had little effect on presynaptic inhibition. 5. It was concluded that at the Ia afferent‐motoneurone synapse presynaptic inhibition is mediated primarily through the activation of GABAA receptors. The activation of GABAB receptors appears to play only a minor role in presynaptic inhibition at this synapse. This contrasts with the relative ease with which (‐)‐baclofen can reduce transmitter release from Ia afferent terminals and suggests that the receptors activated by (‐)‐baclofen are predominantly extrasynaptic.

CHANGES IN QUANTAL PARAMETERS OF EPSCS IN RAT CA1 NEURONES IN VITRO AFTER THE INDUCTION OF LONG-TERM POTENTIATION

1. Long‐term potentiation (LTP) was induced in EPSCs evoked in CA1 pyramidal neurones of young rats in vitro by extracellular stimulation of stratum radiatum. Low frequency stimulation was paired with postsynaptic depolarization to induce LTP, using whole‐cell recording techniques. 2. Sufficient control and potentiated records were obtained under stable recording conditions to allow a quantal analysis of eleven EPSCs. The fluctuations in amplitude of all eleven EPSCs were quantized before conditioning stimulation, and they remained quantized after LTP induction, usually with an increased quantal variance. 3. Quantal current was increased by conditioning for nine out of eleven EPSCs. The increase in quantal current was correlated with the percentage increase in the EPSC. For only two EPSCs could the entire potentiation be attributed to an increase in quantal current. 4. The amplitude fluctuations of five control EPSCs could be described by binomial statistics, but after conditioning the binomial description held for only one of these EPSCs. For this EPSC, conditioning caused the release probability to increase from 0.39 +/‐ 0.05 to 0.47 +/‐ 0.02. 5. Quantal content was increased by conditioning stimulation for ten out of eleven EPSCs. The increase in quantal content was correlated with the percentage increase in the EPSC. However, for only four EPSCs could the entire potentiation be attributed to an increase in quantal content. 6. Most EPSCs were evoked with a high proportion of response failures. The probability of response failures decreased in eight out of eleven EPSCs following the induction of LTP. There was a negative correlation between the change in the probability of response failures and the amount of LTP. 7. The minimal number of sites at which transmission occurred increased for ten out of eleven EPSCs following LTP induction. Increases in the minimal number of active sites following conditioning were associated with decreases in the probability of response failures for seven out of eleven EPSCs. 8. The induction of LTP usually resulted in changes in the time course of the EPSCs. Cable analysis using a passive compartmental model of a CA1 pyramidal cell suggested that these time course changes were associated with shifts in the average electrotonic location of the active sites following LTP induction, rather than being caused by an increased duration of synaptic current. 9. LTP expression involves postsynaptic modifications to enhance the synaptic current at active sites. New sites are recruited, and our data cannot be used to determine if this is a result of a pre‐ or a postsynaptic change. Evidence for an increase in release probability was found for one EPSC.

The phosphoinositide 3-kinase and p70 S6 kinase regulate long-term potentiation in hippocampal neurons

The mechanisms by which long-term changes in synaptic efficacy (e.g., long-term potentiation) are maintained are not well understood. There is evidence that reorganization of the neuronal actin cytoskeleton is important for consolidation of long-term potentiation. In non-neuronal cells, phosphoinositide 3-kinase and p70 S6 kinase have been shown to regulate actin polymerization. We have investigated the subcellular localization of these enzymes in cultured hippocampal pyramidal neurons and their possible role in hippocampal long-term potentiation. Immunohistochemical analysis revealed enrichment of both enzymes in the growth cones and filopodia of extending neurites, whereas p70 S6 kinase was also present at the soma. Antibodies to the phosphorylated form of p70 S6 kinase confirmed its activity in these locations. Interestingly, both enzymes displayed strong colocalization with F-actin in discrete regions of developing neurites. In hippocampal slices, the maintenance of long-term potentiation was attenuated by either rapamycin or 2-(4-morpholinyl)-8-phenyl-1(4H)-1-benzopyran-4-one, inhibitors of p70 S6 kinase and phosphoinositide 3-kinase, respectively. Our findings provide evidence for a novel biochemical pathway involving phosphoinositide 3-kinase and p70 S6 kinase that is important for the maintenance of hippocampal long-term potentiation, possibly via regulation of actin dynamics.

Reduction by general anaesthetics of group Ia excitatory postsynaptic potentials and currents in the cat spinal cord.

1. The effects of thiopentone and halothane on excitatory synaptic transmission at group Ia afferent synapses on lumbosacral motoneurones were studied in the anaesthetized or decerebrate cat. 2. Thiopentone (10 mg kg‐1) infused on a background of light pentobarbitone anaesthesia caused a decrease in single‐fibre monosynaptic group Ia excitatory postsynaptic potentials (EPSPs) of between 0 and 24%. A step increase in inspired halothane concentration in the range 0.7‐0.9% produced a decrease in EPSP amplitude of between 0 and 31%. These effects were reversible when the anaesthetic level was reduced. 3. Fluctuation analysis of selected single‐fibre group Ia EPSPs revealed that these effects could be accounted for by a decrease in the probability of occurrence of EPSPs of larger amplitude, and an increase in the probability of occurrence of EPSPs of smaller amplitude. The mean separation between discrete amplitudes was not altered by either anaesthetic agent. 4. EPSPs whose time course indicated a somatic site of origin were voltage clamped to study the effect of the anaesthetics on the time course of the synaptic currents. Neither thiopentone nor halothane produced a consistent effect on the time constant of decay of the current, although they both depressed its peak amplitude. 5. The results are interpreted as indicating a presynaptic site of action of both anaesthetics at the concentrations studied: the probability of release of neurotransmitter is reduced, without any detectable change in the mean duration of the postsynaptic conductance increase. These findings are discussed in relation to the mechanisms of action of anaesthetics on exocytosis and presynaptic inhibition.

Voltage dependence of Ia reciprocal inhibitory currents in cat spinal motoneurones.

1. Inhibitory postsynaptic currents (IPSCs) were recorded in voltage clamped posterior biceps or semitendinosus motoneurones of the cat during reciprocal inhibition. 2. Population IPSCs, recorded following stimulation of the whole quadriceps muscle nerve, had an average time‐to‐peak of 0.51 +/‐ 0.02 ms (+/‐ S.E.M., n = 22) and decayed exponentially, with an average time constant of 0.99 +/‐ 0.04 ms (at 37 degrees C) at resting membrane potentials. 3. Unitary IPSCs, recorded following spike‐triggered averaging from an identified reciprocal inhibitory interneurone, had amplitudes of 120‐220 pA with an average time‐to‐peak of 0.40 +/‐ 0.06 ms (n = 5). The decay of these unitary currents was exponential, with an average time constant of 0.82 +/‐ 0.07 ms (at 37 degrees C) at resting membrane potentials. 4. The time course of IPSCs was unaffected by either alpha‐chloralose or pentobarbitone at concentrations necessary for deep anaesthesia. 5. The peak synaptic current varied linearly with the membrane potential over the range ‐90 to ‐30 mV, and had an average reversal potential of ‐80.7 +/‐ 1.5 mV (+/‐ S.E.M., n = 6) when measured using KCH3SO4‐filled electrodes. 6. The reversal potential for the IPSC was used to calculate [Cl‐]i. This was estimated to be 6.5 mM assuming that the inhibitory synaptic current was mediated purely by Cl‐ ions. 7. The rate at which synaptic currents decayed was exponentially dependent on the postsynaptic membrane potential, the decay time constant increasing e‐fold for a 91 mV depolarization. This result was independent of [Cl‐]i or of the magnitude of the synaptic conductance and was interpreted as a voltage dependence of the glycine channel open time. 8. The average unitary peak conductance was 9.1 +/‐ 1.7 nS (+/‐ S.E.M., n = 5), corresponding to the opening of approximately 200 glycine‐activated postsynaptic channels following neurotransmitter release from a single Ia reciprocal interneurone.

The dependence of motoneurone membrane potential on extracellular ion concentrations studied in isolated rat spinal cord.

1. Intracellular recordings from ninety‐nine motoneurones have been made in an in vitro hemisected spinal cord preparation. Their mean resting membrane potential in normal artificial cerebrospinal fluid (CSF) was ‐71 +/‐ 0.5 mV (+/‐ S.E.M.). The mean amplitude of the action potential was 84.0 +/‐ 1.4 mV (n = 50), and the mean input conductance was 101 +/‐ 7 nS (n = 49). 2. Both membrane potential and input conductance were sensitive to changes in [K+]o, [Na+]o, [Cl‐]o and [Ca2+]o. 3. Replacement of extracellular Ca2+ by Mn2+ resulted in less than 1 mV hyperpolarization and a decrease in input conductance from 102 +/‐ 7 to 93 +/‐ 6 nS (n = 15). 4. At high [K+]o (greater than 10 mM) the membrane potential followed the potential predicted by the Nernst equation for K+ ions with a slope of 58 mV per 10‐fold change in [K+]o. At low [K+]o (less than 10 mM) there was significant deviation from K+ equilibrium potential (EK). 5. [K+]i was found to be 106 mM when estimated from the reversal potential of the after‐hyperpolarization of the antidromic action potential. 6. The reversal potential of the recurrent inhibitory postsynaptic potential (IPSP) in normal CSF was used to calculate [Cl‐]i. This was 6.6 mM, which is less than would be expected if Cl‐ was passively distributed, indicating the presence of an outwardly directed Cl‐ pump. 7. Decreasing [Cl‐]o from control (134 mM) to 4 mM resulted in a depolarization of 6.9 +/‐ 0.9 mV and a decrease in input conductance from 102 +/‐ 5 to 90 +/‐ 5 nS (n = 14) in 3 mM [K+]o. 8. Decreasing [Na+]o from 156 to 26 mM by substitution with choline resulted in a 6.2 +/‐ 0.5 mV hyperpolarization and a decrease in input conductance from from 102 +/‐ 4 to 76 +/‐ 4 nS (n = 5) in 3 mM [K+]o. 9. The input conductances for Na+, Cl‐ and K+ at the resting potential were calculated. After allowing for a microelectrode leak conductance, the relative input conductances were gNa/gK = 0.13 and gCl/gK = 0.25.