Whole-cell patch-clamp recordings from identified spinal neurons in the zebrafish embryo

We describe a preparation for obtaining patch-clamp recordings from identified embryonic spinal cord interneurons, motoneurons and sensory neurons in an in vivo zebrafish preparation. This preparation is used to study the spatial and temporal patterns of spontaneous and touch-evoked electrical activity during the initial development of circuitry in the spinal cord. The combination of these physiological techniques with the powerful genetic and molecular tools available in the zebrafish has the potential to increase our understanding of the complex interactions between genes and electrical activity during the development of the vertebrate nervous system.     

Whole-cell recordings from sympathetic preganglionic neurons in rat spinal cord slices

Whole-cell patch-clamp recordings (WCR) were made from sympathetic preganglionic neurons (SPN) in neonate rat spinal cord slices. SPN were identified histologically by filling them with the fluorescent dye Lucifer Yellow contained within the patch pipette solution. Current clamp recordings were obtained from SPN with a potassium based pipette solution. The cells exhibited many of the characteristic properties of SPN seen previously with intracellular recordings in both the rat and the cat. However, we found an order of magnitude increase in both cell input resistance (950 MΩ) and time constant (118 ms) over those seen with conventional recordings. We believe these values approximate better the situation in intact cells, and will have a vital bearing upon how SPN integrate inputs. We conclude that WCR in spinal cord slices provides a powerful tool for investigating the cellular properties of SPN.     

Whole-cell and single-channel recordings of GABA-gated currents in cultured chick cerebral neurons

1. gamma-Aminobutyric acid (GABA) (10-500 microM) was applied to cultured chick cerebral neurons by pressure ejection, and the resulting currents (IGABA) were recorded using standard whole-cell voltage-clamp techniques. Plots of the peak IGABA as a function of membrane potential were nonlinear with an outwardly rectifying appearance. 2. IGABA decayed during a prolonged application of GABA. This decay was associated with a decline in the conductance of the cell, suggesting that the decline in IGABA was principally due to receptor desensitization. 3. After 5-7 days in culture, whole-cell recordings revealed the presence of spontaneous synaptic currents. These currents were presumed to be GABA-gated inhibitory postsynaptic currents (IPSCs) because they reversed at the Cl- equilibrium potential (ECl-), were blocked by picrotoxin (25 microM), and were prolonged by pentobarbital (50 microM). 4. Synaptic currents were analyzed by fitting exponential functions to their decay. In normal recording saline, 68% of the decays analyzed could be adequately described by a single exponential function. Two exponentials were necessary to describe the decay of the other 32%. The time constant of the decay (for those adequately fitted by a single exponential) increased with depolarization, from an average value of 15 ms at -80 mV to 60 ms at +40 mV. 5. A relationship was noted between IPSC amplitude and decay time constant; IPSCs with larger peak amplitudes had a slower decay. One possible explanation considered for this finding was that transmitter persists in the synaptic cleft and rebinds to the receptors, thus prolonging the decay of the IPSC. 6. Consistent with the above hypothesis was the observation that the decays of miniature IPCSs (examined under conditions of reduced transmitter release) were faster, showed less variability, and were all adequately described by a single exponential function. Furthermore, the decay times were independent of the membrane potential, suggesting that the kinetic parameters of the GABA channel which shape the decay of these miniature IPSCs are independent of voltage. 7. Single-channel activity underlying whole-cell GABA responses could be recorded in isolated outside-out and inside-out patches of membrane. In isotonic choline chloride, single-channel amplitudes were linearly related to voltage and reversed at -1.8 +/- 11.0 mV (n = 12). Under these conditions, the channel had a main conductance state of 20.8 +/- 3.4 pS (n = 12). Transitions were observed from this main conductance state to other conductance states, e.g., two subconductance states of 6 and 12 pS and one supraconductance state of 30 pS.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lithium distribution across the membrane of motoneurons in the isolated frog spinal cord

Lithium sensitive microelectrodes were used to investigate the transmembrane distribution of lithium ions (Li⁺) in motoneurons of the isolated frog spinal cord. After addition of 5 mmol·l^–1 LiCl to the bathing solution the extracellular diffusion of Li⁺ was measured. At a depth of 500 m, about 60 min elapsed before the extracellular Li⁺ concentration approached that of the bathing solution. Intracellular measurements revealed that Li⁺ started to enter the cells soon after reaching the motoneuron pool and after up to 120 min superfusion, an intra — to extracellular concentration ratio of about 0.7 was obtained. The resting membrane potential and height of antidromically evoked action potentials were not altered by 5 mmol·l^–1 Li⁺.     

Intracellular recording from spinal cord motoneurons in the chronic cat

This report describes chronic intracellular techniques for identifying spinal cord motoneurons and recording from them for prolonged periods of time. The preparation is the intact, unanesthetized, undrugged, normally respiring cat. Comparisons are made between data obtained in the chronic state with that reported in the “acute” preparation, and standards are suggested for intracellular recording in the chronic preparation. Various traditionally used tests of neuronal functioning are outlined in the context of determining basic electrophysiological properties of motoneurons as a function of state-dependent influences.     

Long-duration perforated patch recordings from spinal interneurons of adult mice

It has been very difficult to record from interneurons in acute slices of the lumbar spinal cord from mice >3 wk of age. The low success rate and short recording times limit in vitro experimentation on mouse spinal networks to neonatal and early postnatal periods when locomotor networks are still developmentally immature. To overcome this limitation and enable investigation of mature locomotor network neurons, we have established a reliable procedure to record from spinal cord neurons in slices from adult, behaviorally mature mice of any age. Two key changes to the established neonate procedure were implemented. First, we remove the cord by a dorsal laminectomy from a deeply anesthetized animal. This enables respiration and other vital functions to continue up to the moment the maximally oxygenated lumbar spinal cord is removed, improving the health of the slices. Second, since adult spinal cord interneurons appear more sensitive to the intracellular dialysis that occurs during whole cell recordings, we introduced perforated patch recordings to the procedure. Stable recordings up to 12 h in duration were obtained with our new method. This will allow investigation of changes in mature neuronal properties in disease states or after spinal cord injury and allow prolonged recordings of responses to drug application that were previously impossible.     

Modifications in the morphogenesis of the chick embryo spinal cord induced by self-innervating grafts

Summary In 3.5-day chick embryos the tail region or the whole postumbilical part of a donor was transplanted on the lower right thoracic somites of the host. Both grafts are self-innervating and the contribution of the host seems negligible in this respect. The modifications of the host spinal cord and ganglia were studied in the segments corresponding to the implantation base of the graft.In the dorsal half of the cord's primordium mitotic activity is homolaterally augmented during the 6th, 7th and 8th day of incubation: this pattern coincides with the time and site of formation of the dorsal horn nerve cells. Limb bud transplantations are known to elicit similar responses. The present results confirm the possibility of a control of the non-nervous periphery upon the proliferative activity in the neural tube, but suggest that the stimulating activity of the transplant does not depend on its receptivity for the host's nerves. As a corollary it follows that, in early stages, morphogenetic information from periphery to centers does not travel along nerve paths.In mature embryos (14 to 18-day) no volume modification was observed in spinal ganglia and dorsal column. On the contrary, both structures exhibited a distinct hypertrophy following transplantation of a limb bud. In the present experiments, failure of ganglion hypertrophy confirms that the host does not contribute to graft innervation, while lack of final size modifications in the dorsal column is probably due to secondary quantitative adjustment of the nerve centers to the real extent of the innervation field.Dedicated with great respect to Prof. Dr. E. Blechschmidt, on the occasion of his 65th birthday.     

Excitatory amino acids and intracellular pH in motoneurons of the isolated frog spinal cord

Double-barrelled pH-sensitive micro-electrodes were used to measure changes of intracellular and extracellular pH in and around motoneurons of the isolated frog spinal cord during application of excitatory amino acids. It was found that N-methyl-D-aspartate, quisqualate and kainate produced a concentration-dependent intracellular acidification. Extracellularly, triphasic pH changes (acid-alkaline-acid going pH transients) were observed during the action of these amino acids. The possible significance of such pH changes for the physiological and pathophysiological effects of excitatory amino acids are discussed.     

Calcitonin gene-related peptide, a peptide present in spinal cord motoneurons, increases the number of acetylcholine receptors in primary cultures of chick embryo myotubes

Using immunohistochemical methods we show that calcitonin gene-related peptide (CGRP, a peptide encoded by the calcitonin gene) is present in chick spinal cord motoneurons. When added to cultured chicken myotubes, CGRP caused an average 1.5-fold increase in levels of surface acetylcholine receptors. Moreover, the effect of CGRP was independent of the one produced by tetrodotoxin, a blocker of membrane electrical activity but not of that caused by cholera toxin, an activator of adenylate cyclase.     

A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system

(1) A preparation is described which allows patch clamp recordings to be made on mammalian central nervous system (CNS) neurones in situ. (2) A vibrating tissue slicer was used to cut thin slices in which individual neurones could be identified visually. Localized cleaning of cell somata with physiological saline freed the cell membrane, allowing the formation of a high resistance seal between the membrane and the patch pipette. (3) The various configurations of the patch clamp technique were used to demonstrate recording of membrane potential, whole cell currents and single channel currents from neurones and isolated patches. (4) The patch clamp technique was used to record from neurones filled with fluorescent dyes. Staining was achieved by filling cells during recording or by previous retrograde labelling. (5) Thin slice cleaning and patch clamp techniques were shown to be applicable to the spinal cord and almost any brain region and to various species. These techniques are also applicable to animals of a wide variety of postnatal ages, from newborn to adult.     

Whole-cell voltage-clamp recordings in granule cells acutely isolated from hippocampal slices of adult or aged rats

Whole-cell voltage-clamp recordings were undertaken in granule cells acutely dissociated by a simple enzymatic procedure from the dentate gyri of hippocampal slices obtained from adult or aged rats. This dissociation procedure also allowed the concomitant isolation of other neuron types from diverse hippocampal subfields for the study of ionic currents through voltage- and neurotransmitter-gated channels. For example, in isolated granule cells of the dentate gyrus both transient and sustained Ca2+ currents could be observed in the presence of extracellular tetrodotoxin (TTX) and intracellular Cs+. In addition, ionic currents mediated by activation of excitatory amino acid receptors of the N-methyl-D-aspartate (NMDA) type were present. A technique for the rapid dissociation of neurons from brain slices of adult or aged rats is described in detail.     

Continous electrophysiological measurements of changes in cell volume of motoneurons in the isolated frog spinal cord

One type of ion-sensitive micro-electrode (K⁺ ligand Corning 477317) is sensitive to large quaternary ammonium ions such as choline or tetramethylammonium (TMA⁺). We have now used such electrodes for continuous electrophysiological measurements of changes in cell volume of motoneurons in the isolated frog spinal cord. The electrodes were double-barrelled with tip diameters of 1 m. The reference barrel was filled with 100 mM choline or 100 mM TMA⁺ in 1 M Mg²⁺-acetate, the sensitive barrel contained the Corning K⁺ ligand. After the impalement of a motoneuron, choline or TMA⁺ diffused into the cell and about 1 h later, a steady-state concentration of these ions in the range of 10–20 mM was reached. Following this period, the motoneurons were activated by repetitive electrical stimulation or by application of amino acids via the bathing solution. All these stimuli led to a transient rise of the intracellular concentrations of choline or TMA⁺ (indicating a cell shrinkage of 3–10% difference to control volume).     

Involvement of retinoic acid and its receptor beta in differentiation of motoneurons in chick spinal cord.

Retinoic acid is known to play an important role during development of central nervous system. In order to clarify function of retinoic acid during the development, we investigated expression pattern of the chick retinoic acid receptor subtype beta gene by an in situ hybridization method. We found that expression of the beta gene is localized in neural tube at stages 16-20, then is turned to be restricted to developing motoneurons at stages 23-29. These results suggested that retinoic acid and its receptor beta are involved in differentiation of the motoneurons in spinal cord.     

背侧抑制性轴突导向蛋白对鸡胚脊髓背侧中间神经元发育的影响

目的:探讨在鸡胚脊髓发育早期背侧抑制性轴突导向蛋白(draxin)对脊髓背侧中间神经元(dorsal in-terneurons,dI)迁移特性的影响。方法:应用免疫组织化学的方法观察鸡胚脊髓dI的发育特性;应用电穿孔的方法在鸡胚一侧脊髓内过表达draxin,观察draxin过表达后对鸡胚脊髓内dI神经元发育特性的影响。结果:随着胚胎的发育,鸡胚脊髓内dI3中间神经元首先在脊髓背侧区形成并逐渐向腹侧迁移,而dI2、dI4和dI6中间神经元没有形成明显的腹侧迁移特性;鸡胚脊髓内分别过表达分泌型和跨膜型draxin时,dI3中间神经元的腹侧迁移延迟,且在跨膜型draxin过表达时其受影响程度较高;而dI2、dI4和dI6中间神经元的迁移未受到明显影响。结论:Draxin参与鸡胚脊髓内dI3中间神经元腹侧迁移的调节。     

Absence of histochemical immunoreactivity to calcitonin gene-related peptide (CGRP) in spinal cord motoneurons from (+)-tubocurarine-treated chick embryos

A physiological neuronal death that implicates about 50% of the motoneuron population occurs in the chick embryo between the 6th (E6) and 9th (E9) day of incubation. This natural death can be prevented by administration of neuromuscular blocking agents (e.g. (+)-tubocurarine ((+)-Tc)). In this study, calcitonin gene-related peptide-like immunoreactivity (CGRP-LIR) was studied in spinal cord motoneurons from normal and (+)-Tc-treated chick embryos. In normal embryos CGRP-LIR was found in a neuronal subpopulation of the spinal cord lateral motor column (LMC) that was maximal between the 14th (E14) and 16th (E16) embryonic days with a subsequent decrease. In LMC neurons from (+)-Tc-treated chick embryos examined at E14-16 days no histochemically detectable CGRP-LIR could be observed.     

Sexual dimorphism of the motoneurons in the human spinal cord

There have been few morphometric studies on the size of the motoneurons of the human spinal cord. The purpose of the present study is to report the differences in the size of the motoneurons between males and females in the human spinal cord. We examined numbers and transverse cell body areas of the motoneurons of the anterior horn using 16 male and 21 female human spinal cords at levels C5 and L3. The sizes of the motoneurons were larger in males than in females, but their numbers were practically the same. These results can be of great importance in setting standard values for the understanding of morphological and functional correlations.     

The fine localization of ATPases in cultures in vitro of chick embryo spinal cord.

Summary A histochemical study of the ultrastructural localization of ATPases in cultures of chick embryo spinal cord has been carried out. The localization of Ca²⁺ and Mg²⁺ activated membrane ATPases appears similar: both enzyme activities are localized on the outer surfaces of plasma membranes of all kinds of cell present in the cultures, with the exception of the membranes in direct contact with the culture medium. The results are discussed in relation to data concerning the localization and function of ATPasesin vivo and in relation to the possible establishment of mechanisms of nutrient uptake and transfer in cultures of nervous tissue.     

Two populations of tyrosine hydroxylase-positive cells occur in the spinal cord of the chick embryo and hatchling

The existence of tyrosine hydroxylase (TH)-containing neurons in the spinal cord of the chick embryo was investigated by anti-TH immunocytochemistry. Two populations of intensely immunostained cells were observed along the entire extent of the cord, beginning late in chick embryogenesis. One group of TH-positive cells was particularly numerous and found ventral to the central canal. The other group, which was smaller in number, was located along the superficial and lateral border of the dorsal horn of the spinal cord. When examined by the glyoxylic acid histofluorescence technique, cells could be visualized only very infrequently ventral to the central canal, and not at all within the dorsal horn. However, after pretreatment of hatchlings with the catecholamine synthesis precursor L-DOPA, cells ventral to the canal were readily observed by histofluorescence, while the dorsally located cells seldom visualized. Since these populations of TH-positive cells appear to only partially express the catecholaminergic phenotype, these cells may provide a model in which factors regulating the expression of neurotransmitter phenotypes can be examined in neurons of the developing CNS.