Whole-cell patch-clamp analysis of voltage-dependent calcium conductances in cultured embryonic rat hippocampal neurons

1. Voltage-dependent calcium currents in embryonic (E18) hippocampal neurons cultured for 1-14 days were investigated using the whole-cell patch-clamp technique. 2. Calcium currents were isolated by removing K+ from both the internal and external solutions. In most recordings the external solution contained tetrodotoxin, tetraethylammonium ions, and low concentrations of Na+, whereas the internal solution contained the large cations and anions, N-methyl-D-glucamine and methanesulphonate, and an adenosine 5'-triphosphate (ATP) regenerating system (Forscher and Oxford, 1985) to retard "run-down" of Ca currents. 3. Under these conditions, the sustained inward current triggered during depolarizing steps was enhanced when extracellular [Ca2+] ([Ca2+]0) was raised from 2 to 10 mM and abolished when [Ca2+]0 was lowered to 0.1 mM or by addition of Co2+ ions. These results indicate that the inward current was carried primarily by Ca2+ ions and was designated ICa. This current may be comparable to the "high-voltage-activated" Ca current described in other preparations. 4. In cells cultured for 1-3 days, ICa was small or absent (less than 20 pA for cells 1 day in culture and less than 80 pA for cells 3 days in culture). Although ICa decayed considerably during depolarizing steps, there was little evidence of the transient calcium current (T current) that was recorded in approximately 40% of cells cultured longer than 6 days. Maximal (i.e., the largest) ICa increased from 20 to 80 pA in 1- to 3-day cells to 150-450 pA in cells cultured for longer than 6 days. 5. The decay of ICa elicited by depolarizations from holding potentials of -60 mV or more negative was usually greatest for the maximal ICa. Replacement of extracellular Ca2+ (4 mM) with Ba2+ (2 mM) resulted in a substantial decrease in the extent of decay of ICa and a shift of the I-V relation in the hyperpolarizing direction. 6. Qualitative data obtained from experiments in which different levels of internal Ca2+ buffering were employed demonstrated that, on average, the decay of ICa was reduced as the capacity and/or rate of buffering was increased. The mean decay of ICa in cells buffered with 5 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was 9 +/- 7 (SD) %, (n = 12) and 25 +/- 12%, (n = 12) for cells buffered with the same concentration of ethyleneglycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).(ABSTRACT TRUNCATED AT 400 WORDS)     

The membrane response of hippocampal CA3b pyramidal neurons near rest: Heterogeneity of passive properties and the contribution of hyperpolarization-activated currents

Pyramidal neurons in the CA3 region of the hippocampal formation integrate synaptic information arriving in the dendrites within discrete laminar regions. At potentials near or below the resting potential integration of synaptic signals is most affected by the passive properties of the cell and hyperpolarization-activated currents (I_h). Here we focused specifically on a subset of neurons within the CA3b subregion of the rat hippocampus in order to better understand their membrane response within subthreshold voltage ranges. Using a combined experimental and computational approach we found that the passive properties of these neurons varied up to fivefold between cells. Likewise, there was a large variance in the expression of I_h channels. However, the contribution of I_h was minimal at resting potentials endowing the membrane with an apparent linear response to somatic current injection within ±10 mV. Unlike in CA1 pyramidal neurons, however, I_h activation was not potentiated in an activity-dependent manner. Computer modeling, based on a combination of voltage- and current-clamp data, suggested that an increasing density of these channels with distance from the soma, compared with a uniform distribution, would have no significant effect on the general properties of the cell because of their relatively lower expression. Nonetheless, temporal summation of excitatory inputs was affected by the presence of I_h in the dendrites in a frequency- and distance-dependent fashion.     

The induction of corticosteroid actions on membrane properties of hippocampal CA1 neurons requires protein synthesis.

Corticosterone can affect electrical properties of CA1 pyramidal neurons via binding to two corticoid receptor types, the mineralocorticoid (MR) and glucocorticoid receptor (GR). Previously we have shown that MR-activation leads to attenuation of serotonin (5-HT)-induced membrane hyperpolarization, while GR-activation induces an increase in the amplitude of the afterhyperpolarization (AHP) following a short current pulse. In this study we show that the MR- and GR-mediated changes of the membrane properties are prevented in the presence of the protein synthesis inhibitor cycloheximide, thus suggesting a genomic action of the steroids.     

Two classes of spontaneous GABA-mediated miniature synaptic currents in cultured rat hippocampal neurons.

Amplitude and time course of spontaneous gamma-aminobutyric acid (GABA)-mediated miniature postsynaptic currents (MPSCs), recorded in cultured embryonic hippocampal neurons in presence of either tetrodotoxin (TTX) or increased external [Mg2+/Ca2+] ratio, revealed that they form two classes. The distribution of the most commonly recorded MPSCs was skewed both in terms of peak amplitude and rise-time (skew-MPSCs, mode: 70-120 pS). Another, less frequent class (mode: 1-3 nS) formed bell-shaped (bell-MPSCs) amplitude and rise-time distributions. MPSC initial slope did not correlate with rise time, indicating that smaller MPSCs were not electrotonically attenuated. Bell-MPSCs did not result from the integration of skew-MPSCs and both classes appeared to be composed of subunits.     

A patch-clamp analysis of membrane currents in salamander olfactory receptor cells

Isolated olfactory receptor cells were obtained from salamander olfactory epithelium and kept in short term culture conditions. They were studied by means of the whole cell patch-clamp technique associated with ionic substitutions and channel blockers. Under physiological ionic gradients, these cells had a resting potential of –39±10 mV and an input resistance above 2 G.Using different channel blockers and ion substitutions, we could separate several distinct components in the overall whole cell current. In most cells, inward current reflected the activation of a TTX resistant conductance which was blocked by cobalt ions. This inward current lasted only for about 5 min of whole cell recording. In a minority of cells, a TTX sensitive sodium current was also observed.The outward K⁺ current was blocked when the cells were loaded with cesium and tetraethylammonium. It inactivated slowly and incompletely and could act as a depolarization limiter in case of intense odour stimulations. Single channel analysis from outside out patches suggested that it corresponded to the activity of 34 pS channels. In some cells a rapidly inactivating K⁺ current was also present.Single channel activities (27±6 pS) were commonly recorded with KCl-filled pipettes, at resting or hyperpolarized membrane potentials but not at depolarized potentials. Membrane hyperpolarization increased the open-state probability. A preliminary study with odorant stimulations indicated the existence of a stimulus-induced current probably corresponding to the activation of the chemoreceptive membrane.     

Long-term alteration of S-type potassium current and passive membrane properties in aplysia sensory neurons following axotomy

In many neurons, axotomy triggers long-lasting alterations in excitability as well as regenerative growth. We have investigated mechanisms contributing to the expression of axotomy-induced, long-term hyperexcitability (LTH) of mechanosensory neurons in Aplysia californica. Electrophysiological tests were applied to pleural sensory neurons 5-10 days after unilateral crush of pedal nerves. Two-electrode current-clamp experiments revealed that compared with uninjured sensory neurons on the contralateral side of the body, axotomized sensory neurons consistently displayed alterations of passive membrane properties: notably, increases in input resistance (R(in)), membrane time constant (tau), and apparent input capacitance. In some cells, axotomy also depolarized the resting membrane potential (RMP). Axotomized sensory neurons showed a lower incidence of voltage relaxation ("sag") during prolonged hyperpolarizing pulses and greater depolarizations during long (2 s) but not brief (20 ms) pulses. In addition to a reduction in spike accommodation, axotomized sensory neurons displayed a dramatic decrease in current (rheobase) required to reach spike threshold during long depolarizations. The increase in tau was associated with prolongation of responses to brief current pulses and with a large increase in the latency to spike at rheobase. Two-electrode voltage-clamp revealed an axotomy-induced decrease in a current with two components: a leakage current component and a slowly activating, noninactivating outward current component. Neither component was blocked by agents known to block other K(+) currents in these neurons. In contrast to the instantaneous leakage current seen with hyperpolarizing and depolarizing steps, the late component of the axotomy-sensitive outward current showed a relatively steep voltage dependence with pulses to V(m) > -40 mV. These features match those of the S-type ("serotonin-sensitive") K(+) current, I(K,S). The close resemblance of I(K,S) to a background current mediated by TREK-1 (KCNK2) channels in mammals, raises interesting questions about alterations of this family of channels during axotomy-induced LTH in both Aplysia and mammals. The increase in apparent C(in) may be a consequence of the extensive sprouting that has been observed in axotomized sensory neurons near their somata, and the decrease in I(K,S) probably helps to compensate for the decrease in excitability that would otherwise occur as new growth causes both cell volume and C(in) to increase. In peripheral regions of the sensory neuron, a decrease in I(K,S) might enhance the safety factor for conduction across regenerating segments that are highly susceptible to conduction block.     

Membrane properties of mesopontine cholinergic neurons studied with the whole-cell patch-clamp technique: implications for behavioral state control.

1. The whole-cell patch-clamp technique was used to study the membrane properties of identified cholinergic and noncholinergic laterodorsal tegmental neurons in slices of rat brain maintained in vitro. 2. On the basis of their expression of the transient outward potassium current IA and the transient inward calcium current IT, three classes of neurons were observed: type I neurons exhibited a large IT; type II neurons exhibited a prominent IA; and type III neurons exhibited both IA and IT. 3. Combining intracellular deposition of biocytin with NADPH diaphorase histochemistry revealed that the vast majority of type III neurons were cholinergic, whereas only a minority of type I and type II neurons were cholinergic. Thus mesopontine cholinergic neurons possess intrinsic ionic currents capable of inducing burst firing. 4. Delineation of the intrinsic membrane properties of identified mesopontine cholinergic neurons, in concert with recent results regarding the responses of these neurons to neurotransmitter agents, has led us to present a unifying and mechanistic hypothesis of brain stem cholinergic function in the control of behavioral states.     

Effect of hypoxia on membrane potential and resting conductance in rat hippocampal neurons.

The present patch-clamp study describes the effect of hypoxia at 30-31 degrees C on membrane potential and resting conductance in pyramidal cells from the hippocampal CA1 region in rat brain slices. The initial effect of hypoxia was a gradual hyperpolarization; the peak change in membrane potential measured over 15 min was -5.3 +/- 0.22 mV (P < 0.0001). After reoxygenation followed a transient hyperpolarization measuring -1.8 +/- 0.24 mV (P < 0.0001) and a subsequent normalization of the membrane potential, which after 5 min did not differ from its level prior to the hypoxic episode. Voltage-clamp analysis showed that the hypoxic hyperpolarization was related to an outward current at the holding potential (-60 mV) and an increase in resting conductance. The effect was not influenced by intracellular Cl- concentration, which indicated that it was not due to an inward flow of Cl- ions. The addition of tolbutamide, glibenclamide and dantrolene sodium did not affect the hypoxic hyperpolarization, neither did the presence of ATP in the pipette solution. The presence/absence of glucose in the perfusion medium did not influence the initial hyperpolarization during hypoxia; however, glucose seemed to prevent the subsequent depolarization under hypoxia. It was concluded that hypoxia caused an initial hyperpolarization of CA1 cells which was related to an increase in the resting conductance. The results did not suggest the involvement of ATP-sensitive K+ channels.     

Electrotonic profile and passive propagation of synaptic potentials in three subpopulations of hippocampal CA1 interneurons.

To elucidate the role of dendritic morphology in signal transfer, the passive propagation of somatic and dendritic potentials was compared in multi-compartment models of three interneuron subpopulations in the CA1 region. Nine calbindin-, 15 calretinin- and 10 parvalbumin-containing cells were modelled incorporating the detailed geometry, the currents of the action potentials in the soma, and the AMPA, N-methyl-D-aspartate and GABA-B receptor-mediated postsynaptic currents in the dendrites. The cable properties show characteristic differences among the subpopulations. The morphotonic length of calbindin and calretinin cell dendrites is larger than of parvalbumin cells. Thus parvalbumin cells are more compact than calbindin or calretinin cells unless the ratio of their axial and membrane resistivities exceeds the ratios of the other two cell types by more than 33%. In calbindin cells, the distal parts of the extremely long dendrites that invade the alveus are virtually isolated from the soma for passively propagating signals. The synaptic potentials evoked at a given morphotonic distance from the soma show larger differences locally on the dendrites than on the soma in all subpopulations. Both the somatic and dendritic amplitude ratios are the smallest in PV cells. In calbindin cells the somatic amplitude of synaptic potentials evoked at the same morphotonic distance from the soma is similar regardless of the number of branchpoints along their path. In calretinin and parvalbumin cells, from dendrites with long primary segments synaptic potentials reach the soma with larger amplitude than from dendrites that are branching close to the soma. The dendrites with the larger impact on somatic membrane potential are usually the dendrites that enter the stratum lacunosum-moleculare. These results indicate that dendritic morphology plays a role in changing the effectiveness of synaptic potentials evoked at different dendritic locations, and in this way is likely to be an important factor in determining the integrative properties of the different neuron populations.     

Changes in membrane currents of hippocampal neurons evoked by brief anoxia

1. Effects of anoxia (2-4 min of 95% N2-5% CO2) on membrane currents of CA1 neurons were studied by single-electrode voltage clamp in hippocampal slices (from Sprague-Dawley rats) kept in an interface-type chamber at 33.5 degree. 2. When recording with KCl electrodes at a holding potential (VH) near-70 mV, anoxia evoked a slow outward current [0.18 +/- 0.06 (SE) nA], accompanied by a conductance increase ( + 46 +/- 20%, mean +/- SE). The difference current evoked by N2 had a reversal potential near-100 mV. It was much smaller in presence of 2-4 mM extracellular Cs, and any remaining outward current was abolished by 10 mM tetraethylammonium (TEA). Only inward currents were observed when recording with CsCl electrodes. 3. Inward relaxations evoked by large hyperpolarizing pulses from VH less than or equal to - 70 mV (Q-type) were not significantly depressed by anoxia (-1.5 +/- 6.0%). 4. Some voltage-dependent outward currents (evoked by 200-ms depolarizing pulses) were depressed during anoxia: 1) a fast-inactivating (A-like) current, obtained at VH less than or equal to -70 mV and suppressed by 200 microM 4-AP, was reduced by 25.6 +/- 7.3% (n = 5); 2) a slower, noninactivating (C-like) current, suppressed by TEA, was reduced by 52 +/- 7.2% (n = 16). Neither of these currents (1 or 2) was observed when recording with 2- to 3-M CsCl electrodes; and 3) small (M-like) inward relaxations, observed at VH approximately -40 mV 5. Net inward currents could be evoked after blockage of GK with 10 mM TEA when recording with KCl electrodes or by recording with CsCl electrodes. At VH less than or equal to -70 mV, large, transient, and incompletely controlled currents were evoked by depolarizing pulses; at VH less than or equal to -50 mV, smaller and more persistent currents were evoked by depolarizing pulses (L-like), and transient currents (T-like?) were seen immediately after hyperpolarizing pulses. 6.L-type currents (at VH less than or equal to -50 mV) were nearly abolished after 1-2 min anoxia (by approximately 90%). This was equally true of the currents evoked by constant pulses or peak currents in I-V plots. After reoxygenation, recovery was biphasic, with a quick early phase (to 50-80% in 2 min) and then a much slower one (to 60-90% by 10-15 min).(ABSTRACT TRUNCATED AT 400 WORDS)     

The passive electrical properties of the membrane of a molluscan neurone

1. The passive electrical properties of the membrane of the gastrooesophageal giant neurone (G cell) of the marine mollusc, Anisodoris nobilis were studied with small current steps.2. The membrane transient response can be fitted with a theoretical curve assuming as a model for the cell a sphere (soma) connected to a cable (axon). The axo-somatic conductance ratio (rho), determined by applying this model, is large (approximately 5) and the membrane time constant (tau) is long (approximately 1 sec).3. When the actual surface area of the cell, corrected for surface infoldings, and the spread of current along its axon is taken into account, the electrical measurements imply a specific resistance of the membrane of approximately 1.0 MOmega.cm(2).4. Estimates of specific membrane capacity, either from measurements of the initial portion of the membrane transient or from the ratio of the time constant to the specific membrane resistance are close to the value of 1 muF/cm(2) expected for biological membranes.5. Thus, our measurements of specific capacitance, time constant, length constant and axo-somatic conductance ratio all indicate that the value found for the specific membrane resistance of the G cell, while unexpectedly large, is valid.6. The magnitude of this value suggests that the conductance (permeability) of its membrane to ions is much smaller than that previously assumed for nerve membranes; this small conductance may be related to the larger surface-to-volume ratio of the G cell.     

应用穿孔全细胞膜片钳记录技术研究培养大鼠耳蜗螺旋神经节神经元的基本电生理特性

为了深入了解耳蜗螺旋神经节(SG)神经元的基本电生理特点,本研究首先成功培养了急性分离的大鼠SG神经元,然后采用穿孔全细胞膜片钳记录技术观察了培养SG神经元的基本电生理特性。结果表明,可从培养的SG神经元记录到动作电位,且不同的SG神经元具有异质性的放电特征。上述结果提示穿孔全细胞膜片钳记录技术可用于体外培养耳蜗SG神经元的电生理特征研究,为开展听觉信息传导和调控的功能研究开辟了新途径。     

Separate mechanisms of long-term potentiation in two input systems to CA3 pyramidal neurons of rat hippocampal slices as revealed by the whole-cell patch-clamp technique.

Excitatory synaptic transmission from two input systems to hippocampal CA3 pyramidal neurons was investigated by the whole-cell patch-clamp technique for thin slice preparation, with special reference to long-term potentiation (LTP) in these systems. Excitatory postsynaptic currents (EPSCs) evoked by fimbrial stimulation consisted of two components; one was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the other was persistent at depolarized membrane potentials and blocked by D-2-amino-5-phosphonovalerate (D-AP5). The contribution of the D-AP5-sensitive component to EPSCs evoked by stimulation of mossy fibers was much less than that to fimbrial EPSCs. High-frequency stimulation of afferent fibers, under current-clamp conditions, elicited LTP. Bath application of D-AP5 blocked the induction of LTP in the fimbrial but not in the mossy fiber synapses. Induction of fimbrial LTP was completely blocked by 10 mM BAPTA applied intracellularly. In contrast, mossy fiber LTP was not blocked by 10 mM BAPTA. Furthermore, mossy fiber LTP, but not fimbrial LTP, was elicited by high-frequency stimulation under voltage-clamp (-80 mV) conditions. These results suggest that activation of NMDA receptors, increase in postsynaptic [Ca2+]i, and postsynaptic membrane depolarization are required for the induction of fimbrial but not for mossy fiber LTP.     

Releasing the peri-neuronal net to patch-clamp neurons in adult CNS

The extracellular matrix of adult neural tissue contains chondroitin sulphated proteogylcans that form a dense peri-neuronal net surrounding the cell body and proximal dendrites of many neuronal classes. Development of the peri-neuronal net beyond approximately postnatal day 17 obscures visualization and often access by patch electrodes to neuronal membranes with the result that patch clamp recordings are most readily obtained from early postnatal animals. We describe a technique in which the surface tension of a sucrose-based medium promotes partial dissociation of thin tissue slices from adult tissue. Surface tension spreads the tissue and loosens the peri-neuronal net from neuronal membranes within minutes and in the absence of proteolytic enzymes. Furthermore, the extent of dissociation can be controlled so as to maintain the overall slice structure and allow identification of specific cell classes. Excellent structural preservation of neurons and dendrites can be obtained and full access by patch electrodes made possible for current- or voltage-clamp recordings in tissue well beyond the development of peri-neuronal nets. We demonstrate the feasibility of using this approach through patch recordings from neurons in the brainstem and cerebellum of adult gymnotiform fish and in deep cerebellar nuclei of rats as old as 6 months.*E. Morales and F. Fernandez contributed equally to this work     

Morphometric analysis of the differentiation process of hippocampal neurons in vitro

Neuronal cultures of the central nervous system are widely used to study the molecular mechanisms that rule the differentiation process. These cultures have also been used to evaluate drugs and to develop new therapies. From this we can infer the relevance of performing an extended characterization that involves the main aspects driving such process. To carry out such characterization in the present study we prepared primary cultures from hippocampal cells to study cell identity, development of neuronal processes (dendrites and axons), density of synaptic vesicles and development of growth cones. Using immunofluorescence techniques, specific antibodies and non-immunological probes, we studied the changes experienced by the structures under study during different temporal stages (1-21 days). We observed a major proportion of neurons over glia, normal development of neuronal networks (formed by dendrites and axons), increase in the length of dendrites and axons and establishment of synaptic connections. Synaptic vesicles also showed an increase in their densities as long as the time of the culture progressed. Finally, we studied the morphological changes of the growth cones and observed that those were mostly closed at the beginning of the culture period. As neurons matured we observed an increase in the proportion of open growth cones. This work represents an advance in the morphometric characterization of neuronal cultures, since it gathers the main aspects that outline the neuronal differentiation process. In this study, measurement of these morphological features made possible to establish quantitative markers that will allow establishing more precisely the different stages of neuronal differentiation.     

NMDA-induced calcium loads recycle across the mitochondrial inner membrane of hippocampal neurons in culture

Mitochondria sequester N-methyl-D-aspartate (NMDA)-induced Ca(2+) loads and regulate the shape of intracellular Ca(2+) concentration ([Ca(2+)](i)) responses in neurons. When isolated mitochondria are exposed to high [Ca(2+)](,) Ca(2+) enters the matrix via the uniporter and returns to the cytosol by Na(+)/Ca(2+) exchange. Released Ca(2+) may re-enter the mitochondrion recycling across the inner membrane dissipating respiratory energy. Ca(2+) recycling, the continuous uptake and release of Ca(2+) by mitochondria, has not been described in intact neurons. Here we used single-cell microfluorimetry to measure [Ca(2+)](i) and mitochondrially targeted aequorin to measure matrix Ca(2+) concentration ([Ca(2+)](mt)) to determine whether Ca(2+) recycles across the mitochondrial inner membrane in intact neurons following treatment with NMDA. We used ruthenium red and CGP 37157 to block uptake via the uniporter and release via Na(+)/Ca(2+) exchange, respectively. As predicted by the Ca(2+) recycling hypothesis, blocking the uniporter immediately following challenge with 200 microM NMDA produced a rapid and transient increase in cytosolic Ca(2+) without a corresponding increase in matrix Ca(2+). Blocking mitochondrial Ca(2+) release produced the opposite effect, depressing cytosolic Ca(2+) levels and prolonging the time for matrix Ca(2+) levels to recover. The Ca(2+) recycling hypothesis uniquely predicts these reciprocal changes in the Ca(2+) levels between the two compartments. Ca(2+) recycling was not detected following treatment with 20 microM NMDA. Thus Ca(2+) recycling across the inner membrane was more pronounced following treatment with a high relative to a low concentration of NMDA, consistent with a role in Ca(2+)-dependent neurotoxicity.