A hybrid patch clamp amplifier

The current-to-voltage convertor used in patch clamping is analyzed for noise generation and the major noise sources determined. A hybrid patch clamp amplifier design is theoretically analyzed. Here it is shown that by differentiation and recombining of signals the original input signal can be reconstructed. Several circuits of this design are described and their performance compared. The optimal signal detection obtained for a 1 ms current pulse width with a signal-to-noise ratio of 1 is 0.025 pA. In these circuits, high frequency attenuation is readily accomplished with a single control. In addition, compensation for the transients which occur with step control voltages is effectively accomplished. With this circuitry, it is shown that for most patch clamp situations, the minimum pulse width and current which can be detected is determined by the patch clamp seal resistance.     

In vivo patch clamp recording technique in the study of neurophysiology

The patch clamp recording technique in vivois a blind patch clamp recording methods to record the current of the spinal or cereral neurons of anaes：hesia （ or awake） animals. This technique can be used to study the synaptic function and plasticity in central nervous system in vivoin order to understand the physiological properties of the ion channels from an integrated point of view. The advantage of this technique have already presented itself in the study of the synaptic transmission and nervous network. Nowadays, in vivo patch whole-cell recording technique in combination with other techniques is becoming a common method in the research fields.     

In vivo patch clamp recording technique in the study of neurophysiology

The patch clamp recording technique in vivois a blind patch clamp recording methods to record the current of the spinal or cereral neurons of anaesthesia (or awake) animals. This technique can be used to study the synaptic function and plasticity in central nervous system in vivoin order to understand the physiological properties of the ion channels from an integrated point of view. The advantage of this technique have already presented itself in the study of the synaptic transmission and nervous network. Nowadays, in vivo patch whole-cell recording technique in combination with other techniques is becoming a common method in the research fields.     

Dissociation and culture of mechanosensory neurons for patch clamp analysis

Single mechanosensory neurons were isolated from chordotonal organs of adult cockroach antennae. Transmission and scanning electron microscopy showed that the soma and part of the mechanosensory ending survived the dissociation. In culture, outgrowth occurred from the ending. Regions of cell membrane were accessible for patch clamp analysis and channels were recorded. The ability to record channel activity in isolated mechanosensory neurons will allow the study of mechanotransduction mechanisms at the membrane level.     

A preparation for patch clamp studies of labelled, identified neurones from guinea pig spinal cord

Details are described of techniques which allow the isolation of labelled, identified neurones suitable for patch clamp recording from the guinea pig spinal cord. Fluorescent labels, injected into either the hindlimb muscles or the cerebellum, are retrogradely transported to motoneurones or dorsal spinocerebellar tract neurones respectively. Cells are then enzymatically dissociated from spinal cord slices and identified using fluorescence microscopy. Patch clamp or whole cell recordings are then made.     

Agonist potency determination by patch clamp analysis of single glutamate receptors

Agonists (L-quisqualate, L-glutamate and L-cysteine sulphinate) of the locust muscle glutamate receptor differ in potency. Patch clamp analysis of single glutamate receptors reveals that the relative potencies of agonists are determined both by the life-times of the channels that they gate and their probability of activating the glutamate receptor-ionophore complex. Furthermore, agonists which most readily activate the receptor channel complex gate channels with longer life-times than agonists which are less efficient in this respect.     

A system for applying rapid warming or cooling stimuli to cells during patch clamp recording or ion imaging

We describe a system for superfusing small groups of cells at a precisely controlled and rapidly adjustable local temperature. Before being applied to the cell or cells under study, solutions are heated or cooled in a chamber of small volume ( approximately 150 microl) and large surface area, sandwiched between four small Peltier elements. The current through the Peltier elements is controlled by a microprocessor using a PID (proportional-integral-derivative) feedback algorithm. The chamber can be heated to at least 60 degrees C and cooled to 0 degrees C, changing its temperature at a maximum rate of about 7 degrees C per second; temperature ramps can be followed under feedback control at up to 4 degrees C per second. Temperature commands can be applied from the digital-to-analogue converter of any laboratory interface or generated digitally by the microprocessor. The peak-to-peak noise contributed by the system does not exceed that contributed by a patch pipette, holder and headstage, making it suitable for single channel as well as whole cell recordings.     

Loose patch clamp recording of ionic currents in demyelinated frog nerve fibers

Ionic currents in demyelinated segments of frog sciatic nerves have been detected by means of a patch clamp technique. Axons were examined 6-13 days after lysolecithin-induced demyelination. Both voltage-gated sodium channels and voltage-gated potassium channels could be demonstrated in the demyelinated axolemma.     

Firing pattern of rat hippocampal neurons: a perforated patch clamp study

To test whether the slow afterhyperpolarization (sAHP) underlies the filter function of hippocampal granule cells (GCs), we compared the sAHP and spike frequency adaptation between granule cells and CA3 pyramidal cells (PCs) in hippocampal slices employing gramicidin perforated patch clamp recordings to best preserve the physiological cytoplasmic constitution. sAHPs were evoked in GCs and PCs with trains of action potentials in current clamp mode and showed comparable kinetics in both types of cells. The threshold frequency (500 ms firing) triggering a detectable sAHP was approximately 10 Hz and approximately 3 Hz in GCs and PCs, respectively. Half maximal sAHPs were reached at 30 Hz and 8 Hz in GCs and PCs, respectively. Maximal amplitude of sAHPs in GCs amounted to approximately 3.5 mV, was approximately 2-fold smaller than in PCs and could not be further increased with higher firing frequencies. The time course of sAHP activation was investigated with 50 Hz trains of action potentials applied for increasing durations. In both types of cells, the sAHP amplitude increased with a time constant of approximately 400 ms. Nevertheless, sAHP never exceeded 4 mV in GCs but rose to approximately 12 mV in PCs when cells fired for 3 s. The repetitive firing pattern of GCs and PCs was compared by injecting current amplitudes adjusted to provoke an initial firing frequency of 50 Hz. In GCs firing frequency declined slower (tau = 229 ms) and leveled off at a higher tonic firing frequency (28 Hz) when compared to PCs (tau = 126 ms, 18 Hz). We conclude that the intrinsic excitability of GCs cannot be primarily regulated by the sAHP. The sAHP in GCs is minimal most likely due to a small sAHP-channel density as well as to a more rigid control of intracellular Ca(2+) levels.     

Muscle elastography: a new imaging technique for multiple sclerosis spasticity measurement

Multiple sclerosis (MS) spasticity is currently evaluated on the basis of neurological examinations such as Ashworth Scale (AS) and 0–10 NRS. Severity of spasticity is difficult to quantify. We investigated the use of real time elastography (RTHE) ultrasounds for evaluating objectively the muscle fibers status in MS spasticity patients and their changes after a new antispasticity treatment. Two studies were performed. In study A, 110 MS patients underwent a neurological evaluation based on the AS and RTHE. The RTHE images were scored with the new 1-5 muscle fibers rigidity imaging scale, here called MEMSs (Muscle Elastography Multiple Sclerosis Score). The correlation between AS and MEMSs was found to be statistically significant. In study B, 55 MS patients treated with THC:CBD oromucosal spray for their resistant spasticity were followed prospectively. MS spasticity was evaluated by the 0–10 NRS scale at baseline and after 4 weeks of treatment. MEMSs’ figures were obtained at both timepoints. Responders to THC:CBD oromucosal spray (pre-defined as an improvement ≥20% in their 0–10 NRS spasticity score vs. baseline) were 65% of sample. These patients had a mean 0-10 NRS reduction of 1.87 and a MEMSs reduction of 1.97 (P values <0.0001). The remaining 35% of patients, classified as clinically non-responders, showed still a significant mean reduction in MEMSs (0.8, P = 0.002). Our overall results showed that RTHE, operativized throughout MEMSs, could be an objective gold standard to evaluate MS muscle spasticity as well as the effectiveness of antispasticity therapy.     

A self-monitoring technique for increasing productivity in multiple media

During the baseline condition the number of lines written on a word processor and the amount of time spent on studio art were recorded. During Phase I self-recording of the number of lines written were graphed. Additional baseline information was obtained concerning the time spent on studio art. In Phase II both the number of lines written and time spent on art were recorded. Treatment effects were maintained at a 10 months follow-up.     

一种新的肿瘤干细胞球囊研究技术——蛋清包埋技术

目的 探索一种新的能长期、高效保存并研究肿瘤干细胞球囊的技术.方法 从恶性胶质瘤原代肿瘤细胞中培养出肿瘤干细胞.将第5代肿瘤干细胞球囊经蛋清石蜡两次包埋、切片后,行HE染色、免疫组织化学染色和免疫组织荧光染色,并与传统的肿瘤干细胞球囊包埋技术及球囊免疫组织荧光染色实验进行比较.结果 应用蛋清石蜡两次包埋处理的球囊切片经HE染色后,球囊分布均匀、结构完整,胞核、胞浆颜色鲜艳、对比度高.球囊切片的免疫组织化学染色和免疫组织荧光染色能准确定位球囊中阳性细胞和阳性蛋白表达位点,可进行阳性细胞及蛋白的半定量分析.结论 肿瘤干细胞球囊蛋清石蜡两次包埋切片技术能提高实验效率,减少实验误差,节省成本,优于传统球囊包埋技术及传统球囊免疫荧光技术.     

A new method for the analysis of simple and complex planar rapid movements

Recent developments in the field of simple human movement modelling provide new ways in which to view complete models for analysing and understanding complex movements. Based on a kinematic theory and a vectorial delta-lognormal model recently proposed by Plamondon (1993a); Plamondon (1995a); Plamondon (1995b); Plamondon (1995c) and Plamondon (1998), a new method for exploring and understanding the inherent mechanisms that govern planar movement generation and predict human behaviour is presented here. This paper describes an approach for analysing simple as well as complex movements such as cursive handwriting. It highlights some difficulties encountered in the analysis of complex movements. Problems such as the development of robust approaches to solve the reverse engineering problem of automatic parameter extraction of a succession of time-overlapped nonlinear functions are discussed. The analysis of natural cursive handwriting shows many interesting properties of the model and proposes new ways to study perturbed movement phenomena.     

Unified patch clamp protocol for the characterization of Pannexin 1 channels in isolated cells and acute brain slices

In the central nervous system, Pannexin 1 (Panx1) channels are implicated in a variety of physiological and pathological conditions. One of the prerequisites to enlighten the role of Panx1 is the development and standardization of reliable methods. Here, we address the applicability of voltage clamp protocols to identify Panx1 channel mediated currents in neurons of acutely dissected brain slices. We improved an established protocol and report on a modified paradigm that robustly evokes Panx1 channel currents. Crucial advances are the use of physiologic ion gradient conditions and a preconditioning step of depolarizing membrane potential ramps of long duration. This new paradigm provides significant impact on membrane current generation at hypo- and depolarized holding potential steps post voltage ramp preconditioning in heterologous expression systems and primary hippocampal CA1 neurons of mouse brain slices in vitro. Finally, we demonstrate that under these conditions the analysis of tail currents elicited by repolarization of the cells from preconditioning holding potential depolarization permits an independent method to isolate Panx1 mediated channel activity. In summary, this study provides a comprehensive methodological improvement in the biophysical analysis of Panx1 channels with a particular focus on investigations under physiological conditions in complex tissues.