Developmental sensitivity of hippocampal interneurons to ethanol: involvement of the hyperpolarization-activated current, Ih

Abstract

Ethanol (EtOH) has powerful effects on GABA(A) receptor-mediated neurotransmission, and we have previously shown that EtOH-induced enhancement of GABA(A) receptor-mediated synaptic transmission in the hippocampus is developmentally regulated. Because synaptic inhibition is determined in part by the firing properties of interneurons, we have investigated the mechanisms whereby EtOH influences the spontaneous firing characteristics and hyperpolarization-activated cation current (Ih) of hippocampal interneurons located in the near to the border of stratum lacunosum moleculare and s. radiatum of adolescent and adult rats. EtOH did not affect current injection-induced action potentials of interneurons that do not exhibit spontaneous firing. However, in neurons that fire spontaneously, EtOH enhanced the frequency of spontaneous action potentials (sAPs) in a concentration-dependent manner, an effect that was more pronounced in interneurons from adolescent rats, compared with adult rats. EtOH also modulated the afterhyperpolarization (AHP) that follows sAPs by shortening the tau(slow) decay time constant, and this effect was more pronounced in slices from adolescent rats. EtOH increased Ih amplitudes, accelerated Ih activation kinetics, and increased the maximal Ih conductance in interneurons from animals in both age groups. These effects were also more pronounced in interneurons from adolescents and persisted in the presence of glutamatergic and GABAergic blockers. However, EtOH failed to affect sAP firing in the presence of ZD7288 or cesium chloride. These results suggest that Ih may be of mechanistic significance in the effect of EtOH on interneuron spontaneous firing.

FIG. 1.

Identification of interneurons and pyramidal neurons by morphological and electrophysiological properties in rat hippocampal CA1 near to the border of stratum radiatum (SR) and s. lacunosum moleculare (S-LM) area. A: recorded interneuron near the border of SL-M and SR. B: recorded pyramidal cell from s. pyramidale area. a1 and b1: example images of whole cell pipettes attached to bodies of an interneuron and a pyramidal neuron under DIC. a2 and b2: images of same cells revealed by intracellular dialysis with Alexa Fluor 568 and 488 hydrazide 3–5 min after cell rupture. a3 and b3: fluorescence images of same cells (red, interneuron; green, pyramidal neuron) were taken with confocal microscopy. Scale bars = 20 μm. a4 and b4: current-clamp recordings in the same cells show voltage responses to depolarizing (600 ms, +100 pA) and hyperpolarizing (600 ms, −200 pA) current injection. Typical responses of an interneuron and a pyramidal cell (a4 and b4) respectively, were observed after current injection. Spontaneous action potentials (sAPs) and afterhyperpolarizations (AHPs) recorded from an interneuron are shown in a5.

FIG. 2.

Ethanol (EtOH) did not affect evoked action potential (eAP) firing in CA1 interneurons from adolescent and adult rats. A: interneurons identified by basic morphological and physiological properties from an adolescent (a) and an adult (b) rat. Photomicrography is fluorescence image of interneurons (scale bar = 20 μm), the traces were evoked by injecting a depolarizing current (100 pA for 600-ms duration). eAPs were obtained before (control) and after application of 50 mM EtOH. B: summary graph of the effects of EtOH on depolarization-induced eAP firing frequency (a), amplitude (b), 10–90% rise time (c), half-width (d), eAP AHP decay time τ_slow (e), and τ_fast (f) in the neurons from adolescent (n = 12) and adult (n = 7) rats.

FIG. 3.

EtOH increased sAP firing rate in CA1 interneurons from adolescent and adult rats. A, a: an interneuron identified by basic morphological and physiological properties from an adolescent rat. Photomicrography is fluorescence image of an interneuron (scale bar = 20 μm), the current trace was evoked by injection of depolarizing (100 pA) and hyperpolarizing (−200 pA) current for 600 ms. b: sAP firings were recorded from the same interneuron after addition of 30 mM EtOH. c: time course of sAP firing was recorded continuously after addition of 3, 10, 30, and 50 mM EtOH. EtOH caused a concentration-dependent increase in the number of sAP firings. B: following same experimental protocol, the results were obtained in an interneuron from an adult animal. C: EtOH concentration-response curves for changes in frequency of sAPs (%) from adolescent and adult rats. The individual data points show mean percentage change in sAP frequency plotted against log of the EtOH concentration, suggesting that EtOH enhanced sAP firing more powerfully in neurons from adolescent rats than adult rats at each concentration level (2-way ANOVA, P < 0.01). Averaged EtOH concentration-response curves were fitted by the Hill equation (adolescent, n = 18; adult, n = 16, - - -). The concentration-response curve for adult rats was shifted to the right compared with the curves from adolescent rats.

FIG. 4.

Effects of EtOH on AHPs following each sAP, in interneurons from adolescent and adult rats. A, a: an interneuron identified by basic morphological and physiological properties from an adolescent rat. Photomicrography is fluorescence image of an interneuron (scale bar = 20 μm); the current trace is evoked by injection of depolarizing (100 pA) and hyperpolarizing (−200 pA) current for 600 ms. b: in this interneuron, the traces of sAPs were obtained before (control) and after application of EtOH 50 mM. c: the traces of single sAP AHP before and during 50 mM EtOH application were superimposed and show that EtOH decreased in AHP decay time τ_slow (1 and 2), the dashed lines represent fits to a 2-exponential function (see methods). B: same experimental protocol as A, the results were observed in a neuron from an adult rat. C: the summarized data shows mean percentage change in AHP decay time τ_slow after the application of EtOH 30 and 50 mM to slices from adolescent and adult rats. EtOH reduced AHP decay time, and its effect is greater in interneurons from adolescent animals compared with adult animals (*P < 0.05, **P < 0.01, unpaired t-test).

FIG. 5.

Effects of CsCl on sAP firing frequency and I_h in hippocampal interneurons from adolescent rats. A, a: the time course of sAP firing following bath application of 2 separate applications of EtOH (30 mM) at an interval of 15 min. The data are from an interneuron identified by basic morphological (fluorescence image, scale bar = 20 μm) and physiological properties (injected currents 100 pA, −200 pA, 600 ms). b: averaged data illustrating that there were no significant changes in the frequency of sAP firing between the 1st and 2nd application of EtOH (n = 6, paired t-test, P > 0.05). B, a: the time course of responses of sAP firing after bath application of EtOH (30 mM), then CsCl (2 mM) followed by 30 mM EtOH. CsCl decreased sAP firing frequency and blocked the effects of EtOH. b: bar graph summarizing the effects of EtOH, CsCl, and the subsequent addition of EtOH after CsCl, on the frequency of sAP firing (n = 8, 1-way ANOVA, *P < 0.05). C,a: traces of AHPs under control conditions, in the presence of CsCl, and after the subsequent addition of EtOH. - - -, fits with a 2-exponential function. b: averaged data (+SE) show the effects of CsCl alone, and after subsequent application of EtOH, on the slow component of AHP decay time. Note that CsCl (2 mM) significantly extended AHP τ_slow, and AHP τ_slow was subsequently unchanged by the addition of EtOH (30 mM; n = 8, 1-way ANOVA *P < 0.05). D: time course of the effect of CsCl on total current in an interneuron from an adolescent rat. Total currents were evoked by a 1.2-s hyperpolarizing voltage step from a holding potential of −50 to −130 mV and were plotted against time at the end of test pulse (under - - -). Inset: representative traces under control conditions and after application of CsCl. b: CsCl significantly reduced I_h amplitude (n = 6, paired t-test, **P < 0.01).

FIG. 6.

I_h is involved in EtOH enhancement of sAP firing. A, a: time course of the effect of ZD7288 (30 μM) on total current in an interneuron identified by basic morphological (fluorescence image, scale bar = 20 μm) and physiological properties (injected currents 100 pA, −200 pA, 600 ms) from an adolescent rat. Inset: representative traces under control conditions and after application of ZD7288. b: ZD7288 significantly reduced I_h amplitude (n = 7, **P < 0.01, paired t-test). B, a: the time course of responses of sAP firing after bath application of EtOH (30 mM), then ZD7288 (30 μM) followed by 30 mM EtOH in an interneuron from an adolescent rat. ZD7288 (30 μM) irreversibly decreased sAP firing frequency and blocked the effects of EtOH. b: the averaged time course graph for normalized sAP frequency (sAPs/min ±S, n = 5) from 5 cells. c: bar graph summarizing the effects of EtOH alone, ZD7288 alone, and application of EtOH after ZD7288 on the frequency of sAP firing (n = 5, 1-way ANOVA *P < 0.05). C, a: sAP traces were obtained under control, ZD7288 (30 μM), and EtOH (30 mM) after ZD7288. b: the traces of a single AHP in control (1), ZD7288 (2), and EtOH after ZD7288 (3) were superimposed. - - -, fits with a 2-exponential function. c: the summarized data show the effects of ZD7288 alone and application of EtOH after ZD7288 treatment on the slow component of AHP decay time. Note that ZD7288 significantly increased AHP τ_slow, and AHP τ_slow was unchanged by EtOH 30 mM after application of ZD7288 (n = 5, 1-way ANOVA *P < 0.05).

FIG. 7.

Effects of EtOH on I_h recorded in CA1 interneurons from adolescent and adult rats. A, a: representative traces of current obtained before (control) and after application of EtOH 30 and 50 mM in an interneuron identified by basic morphological (fluorescence image, scale bar = 20 μm) and physiological properties (injected currents 100 pA, −200 pA, 600 ms) from an adolescent rat. The current traces are evoked by a 1.2-s hyperpolarizing voltage step from a holding potential of −50 to −130 mV. b: time course of the effects of EtOH (30 and 50 mM) on currents of the same interneuron. Total current (see a) measured at - - - was plotted before and during EtOH application. B: the same experimental protocol as A in an interneuron from an adult rat. C, a: summarized results show that EtOH (30, 50 mM) increased I_h amplitudes in interneurons from adolescent and adult rats (**P < 0.01, paired t-test). b: summarized bar graph shows the effects of EtOH (30 and 50 mM) on I_h density, which was obtained from EtOH-induced increase in the net I_h amplitude divided by each cell is capacitance. EtOH enhanced I_h density more powerfully in neurons from adolescent animals than from adult animals (**P < 0.01, unpaired t-test).

FIG. 8.

The effect of EtOH on I_h activation curves in interneurons from adolescent and adult rats. A, a: an interneuron identified by morphological (fluorescence image, scale bar = 20 μm) and physiological properties (injected currents 100 pA, −200 pA, 600 ms) from an adolescent rat. b: the I_h current traces evoked by a series of voltage steps were obtained from the same interneuron (a) before (control) and after application of 30 mM EtOH. c: the left overlapped traces of I_h were obtained at a test potential of −140 mV and were further scaled by amplitude at the end of a 1.2-s hyperpolarizing step command (right overlapped traces) before and after application of 30 mM EtOH. ···, fits by a single-exponential function, and the activation τ was obtained from fitted results. B: the same experimental protocol as A was employed in an interneuron from adult rat. C, a: he summarized I_h activation time course (see overlapping traces, Fig. 9A,c and B,c) at a hyperpolarizing pulse of −140 mV was shown, and EtOH significantly decreased the activation τ in both age groups (**P < 0.01, paired t-test). b: the bar graph shows EtOH induced percentage changes in I_h activation τ in interneurons from adolescent and adult animals (**P < 0.01, unpaired t-test). D: the summary of the effects of EtOH on I_h activation curves in cells recorded from adolescent (n = 12) and adult animals (n = 9). I_h amplitude from each neuron was converted to conductance (G, see methods). The conductance G for the same cell was normalized to the control maximal I_h G the value of which was measured at −140-mV test potential before application of EtOH. The normalized data points were fitted with a Boltzman function. EtOH shifted the curves to the right by altering V_1/2 and increased G_max in both age groups. E: bar graphs of summarized data showing change in V_1/2 and G_max during EtOH application. EtOH shifts V_1/2 and increases maximal I_h conductance more in interneurons from adolescent compared with adult rats (**P < 0.01, unpaired t-test).

FIG. 9.

Effects of EtOH on sAP firing frequency of interneurons from adolescent rats in the presence of excitatory and inhibitory antagonists. A, a: an interneuron identified by basic morphological and physiological properties from an adolescent rat. Photomicrography is a fluorescence image of an interneuron (scale bar = 20 μm); the current trace is evoked by injection of depolarizing (100 pA) and hyperpolarizing (-200 pA) current for 600 ms. b: representative traces of firing rates are shown under control and following bath application of inhibitory and excitatory blockers (see labels for each antagonists applied) and EtOH. B, a: the bar graph shows averaged sAP firing frequency under each condition (n = 10, *P < 0.05, 1 1-way ANOVA). b: the summarized data show that EtOH increased the mean percentage of sAP frequency in the absence (control) and presence of inhibitory and excitatory antagonists, there was no significant difference between 2 conditions.