Gramicidin Perforated-patch Technique Research Articles

Ion Channels Generating Complex Spikes in Cartwheel Cells of the Dorsal Cochlear Nucleus

Cartwheel cells are glycinergic interneurons that modify somatosensory input to the dorsal cochlear nucleus. They are characterized by firing of mixtures of both simple and complex action potentials. To understand what ion channels determine the generation of these two types of spike waveforms, we recorded from cartwheel cells using the gramicidin perforated-patch technique in brain slices of mouse dorsal cochlear nucleus and applied channel-selective blockers. Complex spikes were distinguished by whether they arose directly from a negative membrane potential or later during a long depolarization. Ca(2+) channels and Ca(2+)-dependent K(+) channels were major determinants of complex spikes. Onset complex spikes required T-type and possibly R-type Ca(2+) channels and were shaped by BK and SK K(+) channels. Complex spikes arising later in a depolarization were dependent on P/Q- and L-type Ca(2+) channels as well as BK and SK channels. BK channels also contributed to fast repolarization of simple spikes. Simple spikes featured an afterdepolarization that is probably the trigger for complex spiking and is shaped by T/R-type Ca(2+) and SK channels. Fast spikes were dependent on Na(+) channels; a large persistent Na(+) current may provide a depolarizing drive for spontaneous activity in cartwheel cells. Thus the diverse electrical behavior of cartwheel cells is determined by the interaction of a wide variety of ion channels with a prominent role played by Ca(2+).

Gramicidin-perforated patch analysis on HCO3- secretion through a forskolin-activated anion channel in rat parotid intralobular duct cells.

Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO3- secretion in the intralobular duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K+, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 microM), a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. The anion current was markedly suppressed by addition of 1 mM methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO3-. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO3- significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl- channel located in luminal membranes) and production of cytosolic HCO3- induce the inward anion current and resulting depolarization. Inhibition of the Na(+)-K(+)-2Cl- cotransporter and the Cl(-)-HCO3- exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl- via the Na(+)-K(+)-2Cl- cotransporter or the Cl(-)-HCO3- exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO3- produced intracellularly, but not of Cl- due to lack of active Cl- transport in parotid duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport.

The reversal potential of Ca 2+-activated Cl − currents indicates that chick sensory neurons accumulate intracellular Cl −

In order to establish the physiological role of the Ca 2+-activated Cl − current ( I Cl(Ca)) of chick primary afferent neurons, I measured the reversal potential of this current using either the amphotericin perforated patch technique (that alters intracellular Cl −) or the gramicidin perforated patch technique (that does not perturb intracellular Cl −). In the amphotericin experiments at 35°C, I Cl(Ca) reversed at the Cl − equilibrium potential ( E Cl=−24 mV) set by the superfusate (147 mM Cl −) and the pipette solution (60 mM Cl −). In contrast, in the gramicidin experiments at 35°C, I Cl(Ca) reversed at −42±2 mV, midway between E Cl of the solutions and E Cl expected if Cl − were passively distributed. Thus the gramicidin perforated patch technique monitors Cl − currents without perturbing intracellular Cl −. Further, the data imply that chick dorsal root ganglia (DRG) neurons actively accumulate Cl −. I Cl(Ca) reversed at the same potential (−46±3 mV) at 20°C indicating that the non-equilibrium distribution of Cl − is maintained at the lower temperature. Thus, I Cl(Ca) is a depolarizing current that can contribute to the after-depolarization in chick DRG neurons and thereby alter Ca 2+ influx.

Reversibility and cation selectivity of the K(+)-Cl(-) cotransport in rat central neurons.

The reversibility and cation selectivity of the K(+)-Cl(-) cotransporter (KCC), which normally extrudes Cl(-) out of neurons, was investigated in dissociated lateral superior olive neurons of rats using the gramicidin perforated patch technique. Intracellular Cl(-) activity (alpha[Cl(-)](i)) was maintained well below electrochemical equilibrium as determined from the extracellular Cl(-) activity and the holding potential, where the pipette and external solutions contained 150 mM K(+) ([K(+)](pipette)) and 5 mM K(+) ([K(+)](o)), respectively. Extracellular application of 1 mM furosemide or elevated [K(+)](o) increased alpha[Cl(-)](i). When the pipette solution contained 150 mM Cs(+) ([Cs(+)](pipette)), alpha[Cl(-)](i) increased to a value higher than the passive alpha[Cl(-)](i). An increase of alpha[Cl(-)](i) with the [Cs(+)](pipette) was not due to the simple blockade of net KCC by the intracellular Cs(+) since alpha[Cl(-)](i), with the pipette solution containing 75 mM Cs(+) and 75 mM K(+), reached a value between those obtained using the [K(+)](pipette) and the [Cs(+)](pipette). The higher-than-passive alpha[Cl(-)](i) with the [Cs(+)](pipette) was reduced by 1 mM furosemide, but not by 20 microM bumetanide or Na(+)-free external solution, indicating that the accumulation of [Cl(-)](i) in the [Cs(+)](pipette) was mediated by a KCC operating in a reversed mode rather than by Na(+)-dependent, bumetanide-sensitive mechanisms. Replacement of K(+) in the pipette solution with either Li(+) or Na(+) mimicked the effect of Cs(+) on alpha[Cl(-)](i). On the other hand, Rb(+) mimicked K(+) in the pipette solution. These results indicate that K(+) and Rb(+), but not Cs(+), Li(+), or Na(+), can act as substrates of KCC in LSO neurons.

Physiology and pharmacology of native glycine receptors in developing rat ventral tegmental area neurons

Properties of whole-cell glycine-activated currents ( I Gly) of neurons freshly isolated from ventral tegmental area of rats between postnatal day (P) 0 and 40 (P0–P40) were examined using the gramicidin-perforated patch technique. I Gly was present throughout the period of investigation and displayed age-related alternations in its kinetics, reversal potentials ( E Gly) and sensitivity to antagonists. During development, the time constant for desensitization of I Gly decreased about two-fold, and the E Gly shifted to more negative potentials. Specifically, E Gly was −29 mV in neonatal (P0–P7) and decreased to −50 mV in adult (P24–P40) neurons. While the neonatal glycine receptors (GlyRs) are resistant to strychnine, the adult GlyRs are sensitive to STR. The values for 50% inhibition (IC 50) of I Gly was 110 and 12 nM in P0–P3 and P24–P40 neurons, respectively. Picrotoxin, on the other hand, was more effective in blocking neonatal neurons. The IC 50 values were 17 and 231 μM for the neonatal and adult neurons, respectively. These observations are discussed in terms of developmental changes of subunit composition of GlyRs and of intracellular Cl − concentrations.

Glycine-activated currents are changed by coincident membrane depolarization in developing rat auditory brainstem neurones.

1. During early ontogeny, glycine receptors (GlyRs) exert depolarizing responses which may be of developmental relevance. We have used the gramicidin-perforated patch technique to elucidate the mechanism of glycine-activated currents in developing neurones of the rat lateral superior olive (LSO). 2. When the holding potential was set to -60 mV, perforated-patch recordings revealed glycine-induced inward currents in 59 %, outward currents in 5 % and biphasic currents in 34 % of the LSO neurones tested (n = 44). The biphasic currents were characterized by a transient outward phase which was followed by an inward phase. 3. Ion substitution experiments showed that both Cl- and HCO3- contributed to the glycine- induced biphasic current responses. 4. In the biphasic responses, the reversal potential of the glycine-induced current (Egly) depended on the response phase. A strong shift of Egly from a mean of -72 mV during the outward phase of the glycine response to a mean of -51 mV during the inward phase was observed, suggesting a shift of an ion gradient. 5. When the membrane potential was depolarized, 'tail' currents were induced in the presence of glycine. An increased duration or amplitude of the evoked depolarizations resulted in a proportional enlargement of these tail currents, indicating that they were produced by a shift of an ion gradient. Since changes of the HCO3- gradient are negligible, because of the carbonic anhydrase activity, we suggest that these tail currents were caused by a shift of the Cl- gradient. 6. We conclude that Cl- accumulates intracellularly during the activation of GlyRs and, consequently, Egly moves towards more positive values. 7. Coincident depolarizing stimuli enhanced intracellular Cl- accumulation and the shift of Egly, thereby switching hyperpolarizing to depolarizing action. This change could assist in an activity-dependent strengthening and refinement of glycinergic synapses during the maturation of inhibitory connectivity.

Gramicidin perforated patch-clamp technique reveals glycine-gated outward chloride current in dissociated nucleus solitarii neurons of the rat.

1. The inhibitory response of exogenously applied glycine was investigated in freshly dissociated rat nucleus tractus solitarii neurons under whole cell configuration using new perforated patch-clamp technique termed "gramicidin perforated patch technique," which maintains intact intracellular Cl- concentrations. 2. Using the gramicidin perforated patch technique, at a holding potential (VH) of -45 mV, glycine induced outward currents in a concentration-dependent manner with a EC50 of 4.0 x 10(-5) M and at a Hill coefficient of 1.5. In contrast, using the nystatin perforated patch technique, glycine induced inward currents at the same VH in a concentration-dependent manner with an EC50 of 4.9 x 10(-5) M and at a Hill coefficient of 1.2. 3. The glycine-induced outward currents were blocked by strychnine in a concentration dependent manner with an IC50 of 2.2 x 10(-8) M. The blockade was competitive. 4. The current-voltage relationship for the 10(-5) M glycine response showed a clear outward rectification. 5. Ten-fold change of extracellular Cl- with a large impermeable anion resulted in a 65 mV shift of the reversal potential of glycine-induced currents (EGly), indicating that the membrane behaves like a Cl- electrode in the presence of glycine. 6. The intracellular Cl- activity calculated from the EGly ranged from 7.3 to 18.2 mM, with a mean value of 13.3 mM. 7. The values of EGly in the individual neurons were significantly negative to the resting membrane potentials, suggesting the existence of active transport of Cl-.