Pedro Freire Costa

The kinetic parameters of sodium currents in maturing acutely isolated rat hippocampal CA1 neurones

Whole-cell voltage clamp techniques were used to characterize the kinetics of INa in immature (P3-5) and older (P > 25) acutely isolated rat CA1 hippocampal neurones. Fast-rising and fast-inactivating currents were recorded at all stages of maturation, evocable from Vm values of -55 to -50 mV. Currents were sensitive to TTX (1 microM) and to sodium removal from the perfusate. Current density and maximum slope conductance increased with maturation. Current decay was described by two exponentials, the faster component dominating at -35 mV or more depolarized Vm values; the ratio fast/slow inactivating component decreased with maturation. The voltage-dependence of conductance was taken as an approximation of m infinity. In younger cells, V1/2 values of the steady-state inactivation (h infinity) and activation curves (m infinity) were depolarized. Shifts of h infinity and m infinity curves were accompanied by shifts in the corresponding tau h and tau m voltage-dependence curves. In younger cells, activation curves had comparatively higher slope factors (Vs), which is an indication of a lower voltage sensitivity of activation. m infinity, tau m, h infinity, and tau h parameters were used to calculate the forward and backward activation and inactivation rate constants (alpha m, beta m, alpha h and beta h). P3-5 cells had relatively higher beta m values accounting for the lower voltage sensitivity of activation. The findings are an indication of a dominant channel variety in the younger cells with a closed state higher probability. The results are consistent with lower depolarization rates previously reported in CA1 cells at early stages of maturation. Faster inactivation due to poor expression of the slower inactivating component may compensate for poorer repolarization mechanisms due to the immaturity of outward currents previously reported at early stages of maturation.

Potassium currents in acutely isolated maturing rat hippocampal CA1 neurones

Whole-cell voltage clamp techniques were used to characterize the postnatal development of current amplitude and inactivation and activation kinetics of two potassium currents in acutely isolated CA1 cells from rats P4 to P52: the A-current (IA) and a slow-rising, slow inactivating current (IK). In the course of maturation, changes in the relative proportion of IA and IK currents were observed, the latter becoming a dominant current in older cells. The half-maximal point (Vh) of steady-state inactivation and activation of IA and IK shifted in the course of the first and second postnatal weeks. The shifts were hyperpolarizing in the case of IK, whereas IA shifted to less negative values. The shifts in steady-state inactivation Vh were accompanied by a change in the slope factor (Vs), which is an indication of a modification in the voltage sensitivity of the steady-state inactivation. The kinetics of IK evolve after birth in a fashion that matches the changes in action potential parameters previously reported.

Afterpotential characteristics and firing patterns in maturing rat hippocampal CA1 neurones in in vitro slices.

The postnatal evolution of depolarizing after-potentials (DAPs) and after-hyperpolarizations (AHPs) was studied in rat CA1 hippocampal neurones (5-68 days of age) using in vitro slices. Results were pooled into 4 age groups: P5-9, P10-16, P17-24 and P26-68. In P5-9 cells, DAPs were seen as passive signals, with a time constant similar to the time constant of the membrane. The evolution of the DAP was characterized by a decrease in amplitude, an increase in duration and a change in contour. In P10-16 and P17-24 cells, the DAPs often had a plateau or a hump-like shape which increased the probability of firing and the occurrence of spike doublets. The firing pattern and bursting behaviour of P10-16 CA1 neurones differed from the pattern typical of the adult. P5-9 and P10-16 cells had post-burst AHPs with a smaller amplitude and a more prolonged early phase than at late stages of development.

Galantamine inhibits slowly inactivating K+ currents with a dual dose-response relationship in differentiated N1E-115 cells and in CA1 neurones.

Galantamine, one of the major drugs used in Alzheimers disease therapy, is a relatively weak acetylcholinesterase inhibitor and an allosteric potentiating ligand of nicotinic acetylcholine receptors. However, a role in the control of excitability has also been attributed to galantamine via modulation of K+ currents in central neurones. To further investigate the effect of galantamine on voltage-activated K+ currents, we performed whole-cell voltage-clamp recordings in differentiated neuroblastoma N1E-115 cells and in dissociated rat CA1 neurones. In both cell models, one can identify two main voltage-activated K+ current components: a relatively fast inactivating component (Ifast; time constant approximately hundred milliseconds) and a slowly inactivating one (Islow; time constant approximately 1 s). We show that galantamine (1 pM-300 microM) inhibits selectively Islow, exhibiting a dual dose-response relationship, in both differentiated N1E-115 cells and CA1 neurones. We also demonstrate that, in contrast with what was previously reported, galantamine-induced inhibition is not due to a shift on the steady-state inactivation and activation curves. Additionally, we characterized a methodological artefact that affects voltage-dependence as a function of time in whole-cell configuration, observed in both cell models. By resolving an inhibitory role on K+ currents in a non-central neuronal system and in hippocampal neurones, we are attributing a widespread role of galantamine on the modulation of cell excitability. The present results are relevant in the clinical context, since the effects at low dosages suggest that galantamine-induced K+ current inhibition may contribute to the efficiency of galantamine in the treatment of Alzheimers disease.

Sustained potassium currents in maturing CA1 hippocampal neurones

Whole-cell voltage clamp techniques were used to characterize sustained outward currents in maturing (P4 to P48) acutely isolated rat CA1 hippocampal neurones. Sodium removal and signal subtraction were used to isolate a sodium dependent sustained potassium current (IKNa). Calcium blockade (Co2+), sensitivity to a low TEA dose (0.5 mM) and sensitivity to Charibdotoxin (CTX 25 nM) and Iberiotoxin (IbTX 25 nM), in conjunction with signal subtraction, were used to isolate a sustained current with the characteristics of IC (IKCa). IKNa was found in both immature (P4-5) and older (P > 21) cells; this corresponded, respectively, to 56 +/- 5% and 36 +/- 6% of the outward current in younger and older cells. In the course of maturation, the voltage dependence of activation of IKNa shifted to more hyperpolarized values by approximately 20 mV. In the younger cells (P5-18) there was no evidence for sensitivity to CTX or IbTX. In 55 out of 77 older cells we found a component sensitive to CTX, IbTX, 0.5 mM TEA and Co2+.

Sodium channel currents in maturing acutely isolated rat hippocampal CA1 neurones.

Sodium channel currents were recorded in excised inside-out patches from immature (P(4-10)) and older (P(20-46)) rat CA1 neurones. Channel conductance was 16.6+/-0.013 pS (P(20-46)) and 19.0+/-0.031 pS (P(4-10)). Opening patterns varied with step voltage and with age. In some patches bursting was apparent at voltages positive to -30 mV. Non-bursting behaviour was more dominant in patches from younger animals. In older animals mean open time (m.o.t.) was best described by two exponentials especially in the older cells; in the immature, there were fewer cases with two exponentials. The time constant of inactivation (tau(h)) estimated in ensemble averages was best described by two exponentials (tau(hf) and tau(hs)) in most patches from older cells. tau(hf) decreased with depolarization; tau(hs) increased in the range -30 to 0 mV. The voltage dependence of tau(hf) in the older cells is identical to that of the single tau(h) found in the younger; the results indicate a dominance of tau(hf) in the younger. Patches from younger cells more often showed one apparent active channel; in such cases, m.o.t. was described by a single exponential. However, in two cases, channels showed bursting behaviour with one of these channels showing a shift between bursting and non-bursting modes. Our findings are consistent with a heterogeneous channel population and with changes in the population in the course of maturation.

Slow and medium afterhyperpolarizations in maturing rat hippocampal CA1 neurones.

The postnatal evolution of the signals underlying the afterhyperpolarization (slow-AHP and medium-AHP) was studied in rat CA1 hippocampal neurones (P10-16, P17-23 and P greater than 26) using in vitro slices. Noradrenaline (NA) and signal subtraction were used to decompose the AHP (m-AHP and s-AHP). The amplitude of the s-AHP was found significant between P10-16 and P17-23 cells; the m-AHP showed no significant change. The time to peak of the s-AHP of the P greater than 26 cells was found to be significantly different from the remaining groups; the m-AHP showed no significant change. The changes in waveform and amplitude of the AHP at this stage were found to be mainly due to the modifications of the slow-AHP.

Insulin increases excitability via a dose-dependent dual inhibition of voltage-activated K+ currents in differentiated N1E-115 neuroblastoma cells

A role in the control of excitability has been attributed to insulin via modulation of potassium (K+) currents. To investigate insulin modulatory effects on voltage‐activated potassium currents in a neuronal cell line with origin in the sympathetic system, we performed whole‐cell voltage‐clamp recordings in differentiated N1E‐115 neuroblastoma cells. Two main voltage‐activated K+ currents were identified: (a) a relatively fast inactivating current (Ifast − time constant 50–300 ms); (b) a slow delayed rectifying K+ current (Islow − time constant 1–4 s). The kinetics of inactivation of Ifast, rather than Islow, showed clear voltage dependence. Ifast and Islow exhibited different activation and inactivation dependence for voltage, and have different but nevertheless high sensitivities to tetraethylammonium, 4‐aminopyridine and quinidine. In differentiated cells − rather than in non‐differentiated cells − application of up to 300 nm insulin reduced Islow only (IC50 = 6.7 nm), whereas at higher concentrations Ifast was also affected (IC50 = 7.7 µm). The insulin inhibitory effect is not due to a change in the activation or inactivation current–voltage profiles, and the time‐dependent inactivation is also not altered; this is not likely to be a result of activation of the insulin‐growth‐factor‐1 (IGF1) receptors, as application of IGF1 did not result in significant current alteration. Results suggest that the current sensitive to low concentrations of insulin is mediated by erg‐like channels. Similar observations concerning the insulin inhibitory effect on slow voltage‐activated K+ currents were also made in isolated rat hippocampal pyramidal neurons, suggesting a widespread neuromodulator role of insulin on K+ channels.

Kinetic parameters of calcium currents in maturing acutely isolated CA1 cells

Calcium currents were recorded in CA1 hippocampal cells from immature (P(4-10)) and older (P(22-55)) rats, using whole-cell voltage clamp techniques. Parameters defining the voltage-dependence of activation (tau(m)) and inactivation (tau(h)), steady-state inactivation and activation were determined at both stages of maturation. Current density increased with maturation. A transient low voltage activated (l.v.a.) current was found in P(4-10) cells, but not in the older cells. At voltages less negative than -30 mV, current inactivation was best described by two exponentials (tau(hf), tau(hs)); the ratio of the amplitudes of the two components changed with maturation, with a dominance of the faster component (tau(hf)) in the younger cells. The voltage dependence of tau(hf) followed a simple dependence model, decreased with increasing depolarization, in all cells at both stages of maturation. In P(4-10) cells, tau(hs) was voltage insensitive (range -25 to +30 mV); in P(22-55) cells, the voltage dependence of tau(hs) was found to be complex. Two current components were identified from the voltage dependence of the conductance in both groups. The first, more hyperpolarized component, the l.v.a. current found in P(4-10) cells; this was absent in the older cells, in which we found a component with a different voltage dependence. The voltage dependence of the conductance of the second, more depolarized component did not differ in younger and older cells. In the course of maturation, the steady-state inactivation of the second component underwent a hyperpolarizing shift and a decrease in voltage sensitivity.