An almost completely shielded microelectrode

We present a new method of shielding microelectrodes to within 20 μm of the tip. Stray capacity is reduced to less than 50 fF. Ordinary microelectrodes are covered with silver in a vacuum evaporator. Silver is removed from the tip by contact with a ball of mercury. The microelectrode is then insulated with a glass barrel which is sealed by dipping the tip in diluted polystyrene in amyl acetate, or by dipping the electrode in melted wax. The latter method is quick, easy and reliable.     

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.     

A microelectrode for delivery of defined charge densities

A procedure is described for fabricating microelectrodes of platinum-30% iridium, insulated with Epoxylite varnish and having beveled ellipsoidal tips obtained by truncating a conical tip at an angle of 30–50°. The geometric surface area of the ellipsoidal facet is reproducible and easily measured. Using these electrodes, neurons in the cerebral cortex of cats have been activated without damage throughout 24 h or more of continuous stimulation at 15–30 μA and charge densities of 150–300 μC/cm²-phase.     

A high voltage electrometer for recording and iontophoresis with fine-tipped,high resistance microelectrodes

A high voltage electrometer is described which incorporates a controlled current source, direct current monitoring, balance bridge, electrode resistance, and capacitance compensation test circuits. This device is suitable for making biophysical measurements and iontophoresing dyes or enzymes through extremely fine micropipettes with impedances an order of magnitude higher than conventional micropipettes. Such electrodes are useful for recording from the small neurons of the central nervous system in vivo.     

An automatic voltage adjuster for a single microelectrode recording of the membrane potential and resistance

An automatic voltage adjuster was designed in order to apply an intracellular rectangular pulse of current and record the membrane potential simultaneously using a single intracellular microelectrode. The original voltage record consists of a rectangular signal (Va) due to the resistance of the microelectrode and a slowly rising signal (Vb) which is the proper value of the membrane potential shift produced by the application of the intracellular rectangular pulse of current. The instrument described in this paper samples the original signal (Va + Vb) at 0.5 ～ 1.0 ms after the application of the rectangular pulse of current, holds this voltage level (Va) and subtracts it from the original signal (Va + Vb) to obtain (Vb).     

微电极引导偏侧帕金森病猴脑深部电极植入方法的研究

目的 微电极记录(MER)联合MRI建立丘脑底核偏侧帕金森病猴脑深部电刺激(DBS)模型.方法 4只恒河猴在DSA下经一侧颈内动脉注射MPTP制作偏侧帕金森病模型,采用Kurlan Scale量表及阿扑吗啡实验评估模型效果.3.0T MRI扫描数据输入Leksell系统预定靶点坐标值,MER记录双侧丘脑底核电信号,确定靶点,植入电极并固定.术后MRI查看电极位置.结果 Kurlan Scale量表评分和阿扑吗啡实验表明:偏侧帕金森病模型建立成功.MER记录到两侧丘脑底核放电,且给药侧放电频率明显高于给药对侧(P＜0.01);其修正MRI预定坐标值,术后MRI复查电极末端均位于丘脑底核内.结论 MER是MRI显影不清时丘脑底核定位的必要补充,MER联合MRI可以成功建立定位精确的丘脑底核偏侧帕金森病猴DBS模型.     

Corticosteroid-mediated modulation of carbachol responsiveness in CA1 pyramidal neurons: A voltage clamp analysis

Pyramidal neurons in the rat hippocampal CA1 area contain mineralocorticoid (MRs) and glucocorticoid (GRs) receptors for corticosterone. Previous current clamp experiments showed that depolarizations evoked by carbachol(1–3 μM) depend on relative MR/GR occupation: carbachol responses are small with predominant MR-activation and larger when both receptor types are occupied. Multiple K-conductances underlie the carbachol-induced depolarization. In the present study we used the single electrode voltage clamp technique to examine which K-conductances modulated by carbachol are sensitive to corticosteroid treatment in vitro. We observed that 1 μM carbachol significantly reduced the I_{K, Leak} while the I_M was hardly affected; carbachol effects on the I_{K, Leak} were significantly reduced under conditions of predominant MR activation compared to simultaneous activation of MRs and GRs. With a higher (10 μM) carbachol dose, steroid modulation of the I_{K, Leak} showed a similar tendency. The amplitude of the I_M was largely reduced by 10 μM carbachol but appeared to be not affected by steroid treatment. We conclude that the previously described suppression of the carbachol-induced depolarization with predominant activation MRs is caused by an attenuation of the carbachol action on the I_{K, Leak}. © 1995 Wiley-Liss, Inc.     

Development of the fast, transient outward K current in embryonic sympathetic neurones

Voltage-activated outward potassium (K+) currents in developing sympathetic neurones, dissociated from the rat superior cervical ganglion (SCG), were studied using the whole-cell patch clamp recording technique. In voltage-clamped neonatal SCG cells, two voltage-dependent K+ currents were measured: the fast, transient K+ current, IA; and, the slower activating, non-inactivating delayed rectifier, IK. Only IK, however, appeared to be present in SCG neurones isolated from early embryonic (E14.5-16.5) rat pups; IA was not observed in these cells. When these embryonic neurones were maintained in cell culture, IA developed over a time course (approximately 4-6 days) similar to that seen in vivo. IA, therefore, which appears to facilitate the fast repolarization phase of the action potential in rat SCG neurones, is the last voltage-activated current to develop in these cells.     

Effects of 4-aminopyridine on calcium action potentials and calcium current under voltage clamp in spinal neurons

We examined the effects of 4-aminopyridine (4-AP) on calcium conductance mechanisms in cultured mouse spinal cord neurons. At low concentrations ( 1mM), 4-AP enhanced Ca²⁺-dependent transmitter release and prolonged the duration of Ca²⁺-dependent action potentials. Voltage clamp studies indicated that 4-AP directly facilitates Ca²⁺ entry through voltage sensitive channels apart from an effect on K⁺ currents. These results may help to explain why the drug promotes Ca²⁺-dependent transmitter release at peripheral and central synapses.     

Use of the single electrode voltage clamp to perform noise and relaxation studies of acetylcholine-activated channels in Aplysia neurons

The single electrode voltage clamp has been used to perform fluctuation analysis ("noise" analysis) and relaxation experiments in order to study the average lifetime and conductance of ACh-activated sodium channels in Aplysia neurons. Measured values of average channel lifetime, which is approximately 20 msec at --80 mV and 11 degrees C, and elementary conductance, which is approximately 8 pS, are consistent with previously published results using two electrode clamping. The frequency response of the clamp was evaluated to determine its capabilities and limitations for the study of membrane currents. Sinusoidal currents injected into a voltage-clamped model membrane to simulate the frequency components of membrane noise are accurately reproduced at frequencies up to 500 Hz. Following a voltage clamp command, the new membrane potential is established in less than 2 msec, and current relaxations recorded after that time can be used to determine average channel lifetime. Since the frequency response of the clamp is much greater than the average lifetime of ACh-activated channels in Aplysia neurons, the single electrode voltage clamp is comparable to conventional two-electrode systems for investigating the properties of these channels, and may also be useful in other systems in which the time course of membrane currents is much slower than the frequency response of the clamp.     

The identification of brainstem neurones projecting to thoracic respiratory motoneurones in the cat as demonstrated by retrograde transport of HRP

Brainstem neurones which project to the immediate vicinity of the spinal motoneurones which supply the intercostal and abdominal respiratory muscles were identified by means of the retrograde transport of horseradish peroxidase (HRP). A combined electrophysiological and histological technique was used in which recording of phasic inspiratory or expiratory motoneurone activity within upper (T3-T4) or lower (T8-T9) thoracic segments was followed by the ion-tophoretic injection of HRP at these recording sites. HRP labelled cells were concentrated in those brainstem regions known to contain phasic respiratory neurones, namely the ventrolateral nucleus of the solitary tract (vl-NTS) or dorsal respiratory group (DRG), the ambiguus complex or ventral respiratory group (VRG) and the parabrachial pontine (PB) nuclei. In 18 cats, 248 cells were labelled in these three respiratory regions of the brainstem while 668 were much more diffusely distributed in other regions of the medulla and pons. The ipsilateral and contralateral contributions within the respiratory regions were respectively; 23%:77% (DRG), 33%:67% (VRG), 95%:5% (PB). These results are considered in the general context of previous electrophysiological and histological findings, but also with particular reference to a related study of the projections from brainstem neurones to the phrenic nucleus [32].     

Membrane Currents Influencing Action Potential Latency in Granule Neurons of the Rat Cochlear Nucleus

Granule cells are the most numerous neurons in the cochlear nucleus, but, because of their small size, little information on their membrane properties and ionic currents is available. We used an in vitro slice preparation of the rat ventral cochlear nucleus to make whole-cell recordings from these cells. Under current clamp, some granule neurons fired spontaneous action potentials and all generated a train of action potentials on depolarization (threshold current, 10–35 pA). Hyperpolarization increased the latency to the first action potential evoked during a subsequent depolarization. We examined which voltage-gated currents might underlie this latency shift. In addition to a fast inward Na⁺ current, depolarization activated two outward potassium currents. A transient current was rapidly inactivated by membrane potentials positive to -60 mV, while a second, more slowly inactivating current was observed following the decay of the transient current. No hyperpolarization-activated conductances were observed in these cells. Modelling of the currents suggests that removal of inactivation on hyperpolarization accounts for the increased action potential latency in granule cells. Such a mechanism could account for the 'pauser'-type firing patterns of the fusiform cells which receive a prominent projection from the granule cells in the dorsal cochlear nucleus.     

Differential arithmetic of shunting inhibition for voltage and spike rate in neocortical pyramidal cells

The main inhibitory neurotransmitter in the mammalian forebrain is gamma-amino butyric acid (GABA), which acts through A and B type receptors. GABAA receptors mediate inhibition via an increase in membrane conductance (shunting) and/or membrane potential hyperpolarization. Shunting inhibition is thought to decrease the gain between neural input and output, and thus to act as a divisor, but may do so only below the spike threshold. To investigate the role of shunting inhibition in neocortical neurons, whole-cell patch-clamp recordings were obtained from layer V pyramidal cells of somatosensory cortex in juvenile rats. Sub- and suprathreshold voltage responses were elicited by somatic step current injections and a shunting conductance was generated via a dynamic clamp. Increasing the dynamic clamp shunting conductance led to a parallel shift of the current-discharge curves and a reduced slope of the current-voltage relationship, i.e. a decrease of neural gain. Selective activation of GABAAA receptors with the competitive agonist isoguvacine or rises of endogenous GABA with the specific reuptake blocker nipecotic acid led to a proportional decrease of subthreshold membrane voltage, but a constant offset of discharge rates, thus acting like a shunting conductance. Similarly, isoguvacine and nipecotic acid decreased the gain of excitatory postsynaptic potentials. In all three experimental conditions, shunting inhibition divisively affected subthreshold voltages, while the time-averaged suprathreshold membrane potential was offset by a constant amount. I conclude that shunting inhibition in pyramidal cells has a dual impact on neural output: it is divisive for subthreshold voltages but subtractive for spike frequencies.     

Kv4 (A-type) potassium currents in the mouse medial nucleus of the trapezoid body

Principal neurones of the mouse medial nucleus of the trapezoid body (MNTB) possess multiple voltage-gated potassium currents, including a transient outward current (or A-current), which is characterized here. The A-current exhibited rapid voltage-dependent inactivation and was half inactivated at resting membrane potentials. Following a hyperpolarizing pre-pulse to remove inactivation, the peak transient current was 1.07 nA at −17 mV. The pharmacological characteristics of this A-current were consistent with Kv4 subunits in expression studies; the A-current was resistant to block by tetraethylammonium and dendrotoxin-I but sensitive to millimolar concentrations of 4-aminopyridine and 5 µ m hanatoxin. Immunohistochemistry confirmed that Kv4.3 sub-units are present in the MNTB. In a single-compartment model of an MNTB neurone, the A-current served to accelerate the decay of the initial action potentials in a stimulus train and suggested that removal of A-current steady-state inactivation could raise firing threshold for non-calyceal synaptic inputs. This A-type current was not observed in the rat.     

Voltage clamp analysis of the effect of excitatory amino acids and derivatives on Purkinje cell dendrites in rat cerebellar slices maintained in vitro

A voltage clamp analysis of the effects ofl-aspartate,l-glutamate and related derivatives on Purkinje cell dendrites was performed in rat cerebellar slices maintained in vitro. Short iontophoretic pulse applications ofl-aspartate andl-glutamate in the dendritic field of Purkinje cells induced dose-dependent inward currents with fast onset and recovery. Quisqualate application also gave rise to well developed inward currents with fast onset and slow recovery, whereas N-methyl-d,l-aspartate had no or little effect on Purkinje cell membranes unless prolonged (several seconds) applications were used. Steady applications of low doses of N-methyl-d,l-aspartate much more severely depressedl-aspartate thanl-glutamate mediated responses, whereas inward currents due to quisqualate were unaffected. Inward currents due to quisqualate were often more reduced than those due tol-aspartate by steady applications of 2-amino-5-phosphonovalerate, and the antagonistic action of this drug on responses due tol-glutamate was very weak. These results suggest that receptors of Purkinje cells for glutamate and aspartate are different, and are also different from N-methyl-d-aspartate and quisqualate receptors.     

Receptive Field Properties of Starburst Cholinergic Amacrine Cells in the Rabbit Retina

Patch-clamp recordings were made from ON starburst cholinergic amacrine cells with somas located in the ganglion cell layer of an isolated, dark-adapted rabbit retina preparation. Light responses were analysed and cell identity was confirmed anatomically. The centre light response had a linear current-voltage relation with a reversal potential close to 0 mV. The receptive field size was similar to the dendritic field size. Cholinergic amacrine cells displayed significant surround inhibition. The receptive field profile consisted of a central excitatory region flanked by an inhibitory surround. The surround attenuated the central response to 36% of the maximum. The surround was probably mediated by a combination of presynaptic and postsynaptic inhibition. Starburst amacrine cells did not display action potentials and the presence of a large, voltage-dependent outward current limited depolarizing responses to a maximum potential of about -40 mV. Light responses were completely suppressed during application of 100 μM D, L-2-amino-4-phosphonobutyric acid (APB), consistent with activation exclusively through rod bipolar cells (on) and ON-cone bipolar cells. In darkness the cells displayed a tonic inward current that could be blocked by 100 μM APB and 2 μM CNQX.     

Ionic channels in the node of Ranvier are not modulated by cyclic adenosine monophosphate

The effect of a cyclic adenosine monophosphate (cAMP) analogue on myelinated axons was studied. The lipophilic dibutyryl cAMP (dbcAMP) was applied to isolated frog axons under voltage clamp conditions. No effect on the ionic currents was observed for either external or internal application of dbcAMP. Nor was any effect of the phosphodiesterase inhibitor theophylline found. These results suggest that cAMP does not play an essential role for nerve impulse conduction in axons. This conclusion deviates from those of other studies.     

Prostaglandin E2 mediates cellular effects of interleukin-1beta on parvocellular neurones in the paraventricular nucleus of the hypothalamus

Abstract Interleukin-1beta (IL-1beta) is involved in hypothalamic regulation of corticotrophin-releasing hormone secretion, autonomic activation and consequent downstream modulation of the neuroimmune response. Previously, we have shown that IL-1beta depolarises parvocellular neurones in the paraventricular nucleus (PVN) of the hypothalamus, and these effects are dependent on attenuation of gamma-amino butyric acid (GABA)-ergic input. In the present study, using whole-cell patch clamp recordings of rat neurones in a slice preparation of the PVN, we show that the effects of IL-1beta are abolished in the presence of a cyclooxygenase (COX)-2 inhibitor, NS-398, indicating a dependence on prostaglandin (PG) synthesis and activation. In response to 1 microM PGE2, 64% of parvocellular neurones tested exhibited a clear depolarisation, which was abolished in the presence of tetrodotoxin (TTX). Furthermore, neurones responsive to both IL-1beta and PGE2 exhibited a decrease in the frequency of inhibitory post-synaptic potentials, suggesting that effects of these modulators are mediated via a decrease in GABA-ergic input to these neurones. A proportion (44% and 40%, respectively) of putative GABA-ergic neurones in the halo region surrounding the PVN demonstrated hyperpolarising responses to 1 nM IL-1beta and 1 microM PGE2, and these effects were maintained in TTX. Furthermore, direct hyperpolarising effects of IL-1beta were blocked in the presence of NS-398. Together, these data suggest that PGE2, synthesised in response to IL-1beta-activation of COX-2 expressing cells, directly hyperpolarises putative GABA-ergic neurones in the halo zone surrounding and projecting to the PVN, resulting in a decrease in GABA-ergic input to parvocellular neurones and consequent depolarisation. These data further elucidate the cellular mechanisms by which IL-1beta exerts its neuroimmune-related actions in the PVN.     

Potassium ions play a role in the glycine-induced inhibition of rat substantia nigra zona compacta neurones

Glycine directly inhibits the firing, it hyperpolarizes and/or depolarizes the dopaminergic neurones and increases the membrane conductance. In voltage clamp experiments (near resting potential) either outward and/or inward currents were produced. These actions were present in tetrodotoxin and in 0-calcium-cobalt-treated slices and were antagonized by strychnine. The hyperpolarization depended on the extracellular concentration of potassium and was prevented by cesium diffusion into the cell. Thus potassium ions participate in the glycine mediated inhibition of the firing of substantia nigra zona compacta cells.     

Regeneration of axons into the trochlear rootlet after anterior medullary lesions in the rat is specific for ipsilateral IVth nerve motoneurones

The fibre projection from the IVth nerve nucleus to the superior oblique muscle was determined quantitatively in the normal rat by defining fibre numbers in transverse sections of the IVth nerve, and neurone numbers after retrograde labelling by horseradish peroxidase (HRP) injection into the muscle. There were 183 ± 27 (S.E.) labelled neurones in the nucleus contralateral to the injected muscle and only 2 ± 1 ipsilateral. The ipsilateral fibre number was 234 ± 7 and the cell/axon ratio 0.8 ± 0.1. Extensive analysis of all HRP retrogradely labelled material revealed no central fibre contribution to the IVth nerve other than from neurones resident in the trochlear nucleus. The central portion of the trochlear nerve tract was severed at its point of decussation in the anterior medullary velum. Ninety days after lesion, 10 ± 4 (6% of control) neurones were labelled in the ipsilateral trochlear nucleus; none were labelled in the contralateral nucleus or in any other part of the midbrain, pons, medulla, or cerebellum. The number of myelinated fibres in the IVth nerve had decreased to 21 ± 5 (9% of control) so that the cell/axon ratio was 0.4 ± 0.2, thus suggesting that a single motoneurone has more fibres after lesion. In electron micrographs of the IVth nerve, larger than normal numbers of unmyelinated fibres were seen. Many myelinated fibres displayed signs of abnormal myelination. After regeneration, the projection was exclusively ipsilateral and not crossed as in the normal. These findings establish that there is a high degree of specificity after regeneration since no myelinated central nervous system axons other than trochlear fibres select the IVth nerve root as a trajectory over which to regenerate.