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1Departments of Psychiatry, 2Pharmacology, 5Surgery, and 6Neurobiology, Duke University Medical Center, Durham; 3Neurobiology Research Laboratory, Veterans Affairs Medical Center, Durham; and 4Research Service of the Veterans Affairs Medical Center, Durham, North Carolina
Submitted 13 May 2008; accepted in final form 21 October 2008
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ABSTRACT |
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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. |
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INTRODUCTION |
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). The interneurons located at the border of stratum radiatum (SR) and s. lacunosum moleculare (S-LM) of the CA1 region of hippocampus exert inhibition on other CA1 neurons. Thus they have a powerful inhibitory influence on hippocampal circuit function and sculpt cortico-hippocampal interactions (Freund and Buzsaki 1996; Vida et al. 1998). The net effect of synaptic inhibition is determined, in part, by the firing properties of inhibitory cells as well as by the dynamics of GABA release and the location of inhibitory synapses.
EtOH increases the frequency of GABAA receptor-mediated spontaneous and miniature inhibitory postsynaptic currents in hippocampal CA1 (Ariwodola and Weiner 2004; Li et al. 2003, 2006, Sanna et al. 2004) or CA3 pyramidal cells (Galindo et al. 2005). In addition, EtOH inhibits hippocampal interneuron firing induced by kainate receptor activation (Carta et al. 2003). Although the effect of EtOH on the function of interneurons is clearly one mediator of its inhibitory effects, neither the parameters nor the mechanisms of these effects are known.
While initially observed in cardiac pacemaker cells (Brown and Difrancesco 1980), the slowly developing, hyperpolarization-activated cation current, Ih, is also observed in GABAergic hippocampal interneurons (Aponte et al. 2006; Lupica et al. 2001; Maccaferri and McBain 1996) and has become the subject of intensive investigation in neuroscience (see Pape 1996 for review). The physiological function of Ih is believed to contribute to the neuronal resting membrane potential, the generation of spontaneous firing discharges, and GABA release in hippocampal interneurons (Aponte et al. 2006; Lupica et al. 2001; Maccaferri and McBain 1996). Several reports (Brodie and Appel 1998; Okamoto et al. 2006) have suggested that enhancement of Ih current could be an important cellular mechanism whereby EtOH increases the firing rates of dopaminergic (DA) neurons in the ventral tegmental area (VTA) and that this effect can be blocked by the Ih antagonists, ZD7288 and cesium. However, another study (Appel at al. 2003) indicates that several types of delayed rectifier potassium currents may be responsible for EtOH-induced increases in VTA DA neuron excitability because it was blocked by quinidine but not cesium or ZD 7288. In the present study, we addressed this question by examining the effects of EtOH on spontaneous firing and Ih in hippocampal GABAergic interneurons.
It appears that the effects of EtOH on both behavioral sedation (Little et al. 1996; White et al. 2000; White and Swartzwelder 2004) and GABAA receptor-mediated activity in hippocampal CA1 pyramidal cells (Li et al. 2003, 2006) are developmentally dependent. Furthermore, EtOH promotes extrasynaptic GABAA receptor-mediated tonic currents more efficaciously in dentate granule cells from adolescent rats compared with those from adults (Fleming et al. 2007). Thus it seems that the developmental modulation of the effects of EtOH on GABA receptor-mediated activity is not simply a matter of developing tissue being less sensitive to the effects of EtOH. Therefore to begin a systematic assessment of the effects of EtOH on identified interneurons, we focused primarily on a subgroup of hippocampal interneurons with intrinsic spontaneous firing activity to assess the effect of EtOH on action potential firing and to determine if Ih contributes to the EtOH regulation of firing properties.
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METHODS |
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Hippocampal slices were prepared from male, Sprague-Dawley, periadolescent [postnatal day (PD) 30–40] and adult (PD70-80) rats. Although the periadolescent period of development in the rat has been the subject of some controversy, in recent years, based on an accumulating literature, the period between PD 30 and 50 has become an accepted norm for this period in the male rat (see Spear 2000). The animals were handled and housed according to the guidelines of the National Institutes of Health Committee on Laboratory Animal Resources. All experimental procedures or protocols were approved by the Animal Care and Use Committee of the Duke University and Durham Veterans Affairs Medical Center. The rats were deeply anesthetized with halothane (Sigma, St. Louis, MO) and decapitated. The brains were quickly removed from the skulls and placed in ice-cold (<4°C), modified artificial cerebrospinal fluid (ACSF) containing (in mM) 120 NaCl, 3.3 KCl, 1.23 NaH2PO4, 1 MgSO4, 0.2 CaCl2, 25 NaHCO3, and 10 D-glucose with pH 7.3, previously saturated with 95%O2-5%CO2. The tissue was completely submerged into ice-cold modified ACSF and sectioned in 300- to 350-µm-thick slices using a Vibratome series 1000 sectioning system (Vibratome, St. Louis, MO). The brain slices were first transferred to ACSF containing 0.5 mM Ca2+ and incubated at room temperature for 20 min to allow gradual adaptation to the slight elevation of extracellular Ca2+, then the slices were allowed to equilibrate for 
1 h, at 35°C, in normal ACSF containing (in mM) 120 NaCl, 3.3 KCl, 1.23 NaH2PO4, 1 MgSO4, 2.0 CaCl2, 25 NaHCO3, and 10 D-glucose, during which time they were continuously bubbled with a mixture of 95% O2-5% CO2 gas. After this period, the brain slices were maintained at room temperature (22–24°C) until recordings were initiated. This procedure was developed and utilized in our laboratory to facilitate the production of physiologically viable slices from adult rats (Li et al. 2003, 2006).
Whole cell electrophysiology
After incubation, one hippocampal slice was transferred to the recording chamber that was connected to a Masterflex C/l pump superfusion system (Cole-Parmer Instrument, Vernon Hills, IL). The slice was held to the bottom of the chamber with silver wires and superfused at a constant rate of 2 ml/min with ACSF, which was bubbled with a mixture of 95% O2-5% CO2 gas. The recording chamber temperature was kept at 29–30°C by Chamber System Temperature Controllers (TC-344B, Warner Instruments, Hamden, CT). The slice was visualized with infrared differential interference contrast (DIC, Zeiss Axioskope), using an upright microscope, with a x40 water-immersion objective, and displayed on a monitor. Pyramidal cells and interneurons located in different hippocampal layers were easily distinguishable on visual inspection and were then selected for whole cell recording.
Recordings were made by using standard whole cell patch recording techniques. Patch pipettes were borosilicate glass capillaries (1.5 mm OD, 0.86 mm ID, Sutter Instrument, Novato, CA), pulled on a Flaming/Brown Micropipette Puller (Sutter Instrument, Model P-97) to produce electrodes with 3–5 M
resistance. The pipette solution for current-clamp experiments consisted of (in mM) 130 K-gluconate, 5 KCl, 1 MgCl2, 0.5 EGTA, 10 HEPES, 4 Mg-ATP, 0.5 Tris-GTP, and 10 phosphocreatine (pH = 7.3, 290 mosM). For voltage-clamp experiments (Ih), the patch pipettes were filled with (in mM) 125 KMeSO4, 5 KCl, 5 NaCl, 1 MgCl2, 11 HEPES, 0.02 EGTA, 4 Mg-ATP, 0.5 Na-GTP, and 10 phosphocreatine (pH = 7.3, 290 mosM). Tight seals (>1G) were formed on cell bodies, and whole cell recordings were made by rupturing the cell membrane with negative pressure. Either an Axopatch 1D amplifier or an Axopatch 200B (Axon Instruments Inc., Union City, CA) was used for current- and voltage-clamp recordings which were low-pass filtered (2 kHz or 5 kHz, Bessel filter). Output signals were DC coupled to a digital oscilloscope (Model 410, Nicolet, Madison, WI), and data acquisition was performed using Strathclyde Electrophysiology Software, Whole Cell Program (WINWCP) (Courtesy of Dr. John Dempster) or pCLAMP 10 (Axon Instruments, Union City, CA), with an interface (BNC-2090, National Instruments, Austin, TX) or a DigiData 1440A (Axon Instruments), coupled with a PC computer. Resting membrane potential was directly measured in current-clamp mode after membrane rupture (range: –78 to –53 mV), and only cells with a resting membranes potential more negative than –58 mV were studied. The liquid junction potential was estimated to be 15.9 mV for the current-clamp solution and was not corrected. Input resistance was calculated from membrane voltage deflection, evoked by 600-ms hyperpolarizing current injections (0 to –300 pA in steps of 50 pA). To measure cell capacitance, interneurons were depolarized by applying 5 mV at a holding potential of –70 mV, and cell capacitance was measured from the change in membrane charge, determined from the integrated capacity transients. Series resistance was 
15 M and was monitored by small (depolarizing 5 mV, 150 ms) voltage steps during voltage-clamp recording or current steps (hyperpolarizing 25 pA, 50 ms) during current-clamp recording. Cells were rejected from analysis if the series resistance changed by >15%.
Visualization of recorded cells
We initially visualized and selected interneurons for recording based on somatic shape and electrophysiological properties as described elsewhere (Christie et al. 2000; Gulyas et al. 1998). We employed several electrophysiological criteria, including the response to depolarizing current injections, and the observation of short-duration and fast spike action potentials that were followed by large afterhyperpolarizing potentials (AHPs) (Lacaille and Schwartzkroin 1988; Schwartzkroin and Mathers 1978). In contrast, CA1 pyramidal neurons generated action potentials that accommodated during maintained depolarization (Madison and Nicoll 1984). The recorded cells were also filled with Alexa Fluor 568 hydrazide or Alexa Fluor 488 hydrazide (50–80 µM, Invitrogen, Carlsbad, CA) to reveal their morphological characteristics for post hoc analysis. At the end of recordings, the florescence filled slices were fixed with 4% paraformaldehyde for 20–30 min and rinsed three times using a 0.2 M phosphate buffer. The slices were then mounted on gelatin-coated slides with Prolong Gold Antifade (Invitrogen, Molecular Probes, Carlsbad, CA), and visualized with a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems, Exton, PA).
Pharmacology
To isolate Ih, tetraethyl-ammonium chloride (TEA-Cl, 2 mM), tetrodotoxin (TTX, 1 µM), and BaCl2 (1 mM) were substituted for equimolar NaCl to block unwanted potassium, sodium, and inward rectifier potassium currents, respectively. TTX, ZD7288, and D-2-amino-5-phosphonovaleric acid (APV) were purchased from Tocris Cookson (Ellisville, MO). (–)-bicuculline methiodide (BMI), 6,7-dinitroquinoxaline-2,3-dione (DNQX), and the other chemicals were obtained from Sigma-Aldrich. The drugs were dissolved in distilled water or dimethyl sulfoxide (DMSO) to make stock solutions. The stock solutions were stored, frozen, in 1 ml aliquots, and before each experiment were diluted in ACSF to their final concentrations. The drugs were infused into the recording chamber using a standard perfusion system. After the establishment of stable baseline recordings, EtOH was added to the bath solution in incremental concentrations of 3, 10, 30, and 50 mM, and each concentration was maintained for 5–10 min followed by a washout period of 10–20 min.
Data analysis
The stored data signals were processed using either the Clampfit 10 (Axon Instruments) or Mini Analysis Program (Synaptosoft, Decatur, GA). Numerical data are presented as means ± SE, and n represents the number of cells tested per condition. Action potential amplitude, frequency, rise time (10–90%), half-width, and afterhyperpolarization (AHP) decay time were analyzed with the Mini Analysis Program (Synaptosoft). Paired or unpaired t-test, and one- or two-way ANOVAs followed by Tukey post hoc tests, when appropriate, were used to test statistical inferences related to grouped data. Calculated P values of 
0.05 were accepted as evidence of statistically significant differences.
Ih was evoked by 1.2-s hyperpolarizing steps to –130 or –140 mV from –50 mV. Ih amplitude was measured as the difference between the current level at the end of a 1.2-s hyperpolarizing step command and at the beginning, after the capacitive transient had subsided. The EtOH concentration response curves were fitted by the Hill equation: Rexp = Rmax/{1 + [EC50/(A)]n}, where Rexp is the expected response, Rmax is the maximal response, EC50 is the concentration of EtOH that induced the half-maximal response, (A) is the concentration of the EtOH, and n is the Hill coefficient. AHP decay time constants were obtained by fitting a two-exponential function, I(t) = If exp(–t/fast) + Is exp (–-t/slow). If and Is are the amplitudes of fast and slow components in the AHP, respectively. fast and slow are decay time constants. Ih activation time constants were fitting with a single-exponential function of the equation: I(t) = Ih exp(–t/) + ISS, where I(t) is the amplitude of the current at time t, ISS is the steady-state current during a single voltage step, Ih is instantaneous current subtracted from ISS, and is the time constant of activation. The Ih activation curve was obtained from the Ih current amplitude at each test potential and was converted into conductance (G) using the relation G = I/(V – Er), where Er is the reversal potential for Ih. In the present experiment, the value of Er is –30.5 ± 3.02 mV (n = 7) calculated by the method of Mayer and Westbrook (1983). The conductance values (G) for each cell were fitted with Boltzmann relations of the form G = Gmax/[1 + exp(V – V1/2)/k], where Gmax is the extrapolated maximum conductance, V is the test voltage, V1/2 is the half-activation voltage, and k is the slope factor. In each cell, sAPs and AHPs were assessed using data from 2-min periods before and during each treatment.
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RESULTS |
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Interneurons located in or near the border of SL-M and SR (Fig. 1 A) were first identified electrophysiologically by their unique characteristic features distinguished from pyramidal neurons (Fig. 1B) and other cells as described previously (Christie et al. 2000; Gulyas et al. 1998). Typical examples of an interneuron (Fig. 1A) and a pyramidal cell (B) are shown along with their morphological (a3 and b3) and electrophysiological (a4 and b4) properties. The soma of the interneuron in Fig. 1A was situated in SL-M, near the SR border, was <20 µm in diameter, and the dendrites were smooth (Fig. 1A, a3). This cell demonstrated a typical response to depolarizing and hyperpolarizing current injections, and fired spontaneous action potentials (7 Hz) which were followed by large AHPs (a5). It also exhibited trains of minimally accommodating action potentials in response to depolarizing current injections (a4). These characteristics are consistent with interneurons as described previously (Bertrand and Lacaille 2001; Cope et al. 2002; Lacaille and Schwartzkroin 1988; Schwartzkroin and Mathers 1978; Vita et al. 1998). In contrast, CA1 pyramidal neurons typically fired action potentials of longer duration, which accommodated markedly during maintained depolarization (Fig. 1B, b4). These data are consistent with previous descriptions of the physiological characteristics of pyramidal cells (Madison and Nicoll 1984).
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We first assessed the effects of EtOH on evoked action potentials (eAP) of interneurons that exhibited no spontaneous firing. eAPs were elicited by injection of a depolarizing pulse (100 pA, 600-ms duration) in interneurons from both age groups. The basal amplitude and firing frequency of eAPs were not significantly different between interneurons from adolescent (68.68 ± 3.79 mV and 22.94 ± 1.68 Hz, n = 12) and adult rats (64.16 ± 4.50 mV and 22.41 ± 1.11 Hz, n = 7). There were no significant developmental differences in resting membrane potentials (adolescent, –65.67 ± 1.09 mV, n = 12; adult, –65.28 ± 1.15 mV, n = 7). Typical voltage traces are illustrated in Fig. 2. Data from an interneuron from an adolescent rat is shown in Fig. 2Aa, this neuron fired 16 APs per evoked response under control conditions and 17 APs after bath application of EtOH (50 mM). Figure 2Ab shows an interneuron from an adult rat, EtOH (50 mM) did not change eAP firing (14 APs to 14 APs per evoked response). The averaged data for repetitive eAP firing frequency (a), amplitude (b), 10–90% rise time (c), and half-width (d) are shown in Fig. 2B. The results indicate that EtOH did not affect the repetitive firing frequency in response to depolarizing current injection in interneurons from adolescent (from 22.94 ± 1.68 to 23.47 ± 1.58 Hz, n = 12) and adult (from 22.41 ± 1.11 to 22.91 ± 1.20 Hz, n = 7) rats. EtOH had no significant effects on eAP amplitude (adolescent, from 68.68 ± 3.79 mV in control to 68.11 ± 3.56 mV, n = 12; adult, from 64.16 ± 4.50 mV in control to 64.38 ± 5.17 mV, n = 7), 10–90% rise time (adolescent, from 2.16 ± 0.2 ms in control to 2.25 ± 0.24 ms, n = 12; adult, from 2.34 ± 0.39 ms in control to 2.28 ± 0.45 ms, n = 7), or half-width (adolescent, from 6.08 ± 0.86 ms in control to 6.24 ± 0.98 ms, n = 12; adult, from 6.72 ± 0.82 ms in control to 6.56 ± 1.01 ms, n = 7). The effects of EtOH on AHP decay time after eAPs are shown in Fig. 2B, d and e. EtOH (50 mM) did not change either AHP slow or fast decay time in neurons from adolescent (slow, from 55.15 ± 3.86 to 54.29 ± 3.34 ms; fast, from 3.03 ± 0.36 to 2.96 ± 0.38 ms) and adult rats (slow, from 54.98 ± 5.47 to 52.70 ± 5.61 ms; fast, from 3.15 ± 0.50 to 3.11 ± 0.57 ms). These results are consistent with those of Carta et al. (2003), in which hippocampal CA1 interneurons were recorded in the same region as we recorded, though without a developmental comparison.
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We found that EtOH enhanced the frequency of sAPs but did not significantly affect the resting membrane potential of interneurons from the hippocampal region described in the preceding text. However, in a few cases (2 cells from adolescent rats and 1 cell from an adult rat), even 3 mM EtOH produced a reversible depolarization (>5 mV) that was accompanied by high-frequency firing (>50 Hz). Data from these cells were not included in the analyses for this study. In addition, EtOH 50 mM did not significantly affect action potential firing that was evoked by a depolarizing current (100 pA for 600 ms duration) injection into spontaneously firing interneurons from adolescent rats (from 31.67 ± 1.41 and 32.22 ± 1.70 Hz, respectively, n = 6, paired t-test t(5) = –0.79, P = 0.47). EtOH (3, 10, 30, and 50 mM) did not significantly alter the sAP amplitude of interneurons from adolescent [1-way ANOVA, F(3, 68) = 1.15, P = 0.34] or adult [1-way ANOVA, F(3, 60) = 1.54, P = 0.21] animals. However, bath application of EtOH caused a dose-dependent increase in the frequency of sAPs in cells from animals of either age group. The EtOH-induced increase in frequency of sAPs was reversed after the EtOH was washed out of the recording chamber for 15–30 min. Figure 3 Ab shows action potentials recorded from an interneuron from an adolescent rat, and an increase in sAP frequency to 12 Hz from 4.22 Hz, 10 min after 30 mM EtOH was applied. EtOH did not alter the resting membrane potential. In slices from adolescent rats, we completed the EtOH concentration protocol in 18 of 23 interneurons that had sAP firing. In those cells, EtOH increased sAP frequency in a concentration-dependent manner. A representative example of interneuron responsiveness to EtOH from 3 to 50 mM is shown in Fig. 3Ac. On average, EtOH, at concentrations of 3, 10, 30, and 50 mM, increased sAP frequency by 53.79 ± 10.44, 142.19 ± 14.98, 202.04 ± 19.74, and 216.83 ± 20.23%, respectively (Fig. 3C, 
). Among cells from adolescent rats, all EtOH concentrations resulted in increases of sAP frequency relative to control conditions [1-way ANOVA, F(3, 68) = 18.278, P = 8.38E-9]. Figure 3Bb shows that EtOH (30 mM) also significantly enhanced the frequency of sAPs of interneurons recorded from adult rats. In slices from adult rats, we completed the EtOH concentration protocol in 16 of 20 interneurons that had sAP firing. A representative example is shown in Fig. 3Bc. EtOH, at concentrations of 3, 10, 30, and 50 mM, increased firing frequency by 10.54 ± 1.96, 39.84 ± 6.14, 113.72 ± 13.91, and 131.85 ± 15.69% (n = 16), respectively (Fig. 3C, 
). Among cells from adult rats, EtOH concentrations of 10 mM resulted in increases of sAP frequency relative to control conditions [1-way ANOVA, F(3, 60) = 26.29, P = 5.57E-11]. EtOH increased sAP frequency more powerfully in interneurons from adolescent rats, compared with those from adults [2-way ANOVA, Tukey test, F(1,128) = 56.25, P = 9.41E-12, Fig. 3C]. The average concentration-response curves for the effect of EtOH on sAP firing frequency are shown Fig. 3C. The curves were fitted to averaged percentage increase in sAP frequency at each EtOH concentration by Hill equation (see METHODS). Best-fit curves, which fitted to data obtained from individual neurons revealed that EC50s for EtOH were 10.78 ± 1.38 mM (n = 18) for cells from adolescent rats and 20.54 ± 2.24 mM (n = 16) for cells from adults. The Rmax values were 238.30 ± 21.07 and 154.99 ± 16.81% for cells from adolescent rats and adult rats, respectively. These were significant differences for EC50 (unpaired t-test, t(32) = –3.68, P = 0.0085) and Rmax (t(32) = 2.95, P = 0.0059) between the two age groups. The concentration-response curve for interneurons from adolescents was shifted significantly to the left compared with the curve for interneurons from the adult group (Fig. 3C). There was no significant difference in the Hill coefficient between two age groups (adolescent, 1.56 ± 0.20; adult, 1.89 ± 0.23; unpaired t-test, t(32) = 1.05, P = 0.300).
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In CA1 hippocampal interneurons, AHPs regulate firing pattern and discharge frequency. Thus an effect of EtOH on AHPs could have substantial impact on circuit level inhibition in the hippocampus and could represent an important mechanism driving the effects of EtOH on hippocampal function. We next evaluated EtOH-induced changes in the time course of AHP decay in interneurons from animals in both age groups. AHP decay time constants were obtained by fitting a two-exponential function (see METHODS). EtOH (30 or 50 mM) did not change the fast decay time components in interneurons from adolescent or adult rats. In cells from adolescent rats, the AHP fast decay time (10–90%) was 9.73 ± 1.71 ms (n = 12), which did not change significantly after application of EtOH 30 mM (9.39 ± 1.82 ms, n = 12; t(11) = 1.02, P = 0.32). Bath application of EtOH (50 mM) also did not affect the AHP fast decay time (from 9.48 ± 1.35 to 9.12 ± 1.35 ms, n = 11; paired t-test, t(10) = 1.63, P = 0.13). In addition, neither 30 nor 50 mM EtOH significantly altered AHP fast decay time (30 mM, from 8.79 ± 0.95 to 8.39 ± 0.89 ms, n = 13, paired t-test, t(12) = 2.08, P = 0.06; 50 mM, from 8.93 ± 0.96 to 8.53 ± 1.0 ms, n = 11, paired t-test, t(10) = 1.43, P = 0.18) in adult animals. However, EtOH (30 and 50 mM) decreased the AHP slow decay time in CA1 interneurons from animals of both age groups, and the results are illustrated in Fig. 4. As demonstrated in Fig. 4A, b and c, bath application of 50 mM EtOH accelerated the frequency of sAPs recorded from an interneuron from an adolescent rat. The AHP slow decay time was 31.07 ms at baseline and 15.80 ms after bath application of 50 mM EtOH (Fig. 4Ac). In cells from adolescent rats, the average AHP slow decay time (10–90%) at baseline was 28.90 ± 2.73 ms, and it was significantly reduced to 18.8 ± 2.07 ms by 30 mM EtOH (n = 12, paired t-test, t(11) = 6.50, P = 4.44E-05), and 50 mM EtOH reduced slow from 30.61 ± 2.61 to 14.67 ± 1.66 ms (n = 11, paired t-test, t(10) = 4.27, P = 0.0016). Figure 4B shows that bath application of 50 mM EtOH also accelerated the frequency of sAPs isolated from an interneuron from an adult rat (Fig. 4Bb). Typical voltage traces are shown in Fig. 4B. b and c). In this interneuron, the AHP decay time was 29.08 ms at baseline and 21.07 ms after bath application of 50 mM EtOH (Fig. 4Bc). In cells from adults, the average AHP slow decay time was significantly reduced to 23.10 ± 3.48 ms from 29.23 ± 3.98 ms after application of 30 mM EtOH (n = 13, paired t-test, t(12) = 5.48, P = 1.41E-04), and from 31.43 ± 2.62 to 24.88 ± 2.86 ms after bath applied 50 mM EtOH (n = 11, paired t-test, t(10) = 5.18, P = 4.12E-04). We compared the effects of EtOH on the slow decay time of AHPs in interneurons from adolescent and adult rats, and the results are plotted in Fig. 4C. Both 30 and 50 mM EtOH decreased AHP slow decay time more powerfully in cells from adolescent rats compared with those from adults (unpaired t-test, t(23) = –2.12, P = 0.044 at 30 mM; t(20) = 2.86, P = 0.0095 at 50 mM), suggesting that the EtOH sensitivity of AHP decay time constants may be a mechanism underlying the developmental sensitivity of interneurons to EtOH.
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Previous studies have shown that Ih contributes to resting membrane potential and spontaneous pace-making activity in hippocampal interneurons (Lupica et al. 2001; Maccaferri and McBain 1996). However, the role of Ih in mediating EtOH–induced enhancement of neuronal firing rates remains unclear (Appel et al. 2003; Okamoto et al. 2006; see also Lupica and Brodie 2006 for review). To better understand the potential contribution of Ih to EtOH-induced increases in interneurons firing, we investigated the effects of Ih channel antagonists on EtOH-enhanced sAP firing in hippocampal interneurons from adolescent rats.
First, to determine that repeated exposures to EtOH in our experimental protocol did not induce rapid tolerance, we applied EtOH (30 mM) to the same slices twice at an interval of 15–18 min. Typical responses are shown in Fig. 5 Aa. On average, the firing rates of the interneurons were significantly increased from 3.80 ± 0.38 to 8.74 ± 0.78 Hz (n = 6, 132.03 ± 12.08%) after the first bath application of EtOH (30 mM). The firing rates returned to baseline after the 15-min washout period and when the same concentration of EtOH was re-applied. The average sAP firing rate was again increased significantly to 8.45 ± 0.84 from 3.75 ± 0.40 Hz (n = 6, 128.99 ± 15.40%; Fig. 5Ab) following bath-applied EtOH for the second time. There was no significant difference in the increase of firing rates between the first and second EtOH applications (n = 6, paired t-test, t(5) = 0.29, P = 0.78), indicating that repeated exposures using this protocol did not result in rapid tolerance.
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slow was extended after bath application of CsCl 2 mM, and slow was not changed by subsequent application of 30 mM EtOH. In addition, CsCl had no effect on the fast component of AHP decay time (from 9.21 ± 0.86 to 9.65 ± 0.97 ms, n = 8). The averaged effect of CsCl on AHP slow and its responsiveness to EtOH are shown in Fig. 5Cb, indicating that CsCl significantly prolonged AHP slow decay time to 101.77 ± 13.77 ms from 39.46 ± 6.04 ms of control [n = 8, 1-way ANOVA, F(2, 21) = 7.28, P = 0.0039], and this prolongation was not changed by EtOH (30 mM, 94.67 ± 13.91 ms, n = 8). We also found that CsCl significantly reduced Ih amplitude. After obtaining stable Ih currents by applying hyperpolarizing voltage steps (1.2-s duration) from holding potentials of –50 to –130 mV under voltage-clamp condition, CsCl (2 mM) was applied in the bath and blocked the recorded Ih currents. Representative traces recorded from an interneuron from an adolescent rat are shown in Fig. 5Da. Each data point was obtained from plotting the total current at the end of the test pulse (- - -, inset) before and after CsCl application. The results obtained from six interneurons are illustrated in Fig. 5Db. CsCl reduced Ih amplitude from –199.17 ± 35.58 to –5.30 ± 1.19 pA (n = 6, paired t-test, t(5) = –4.32, P = 0.0042). This suggests that cesium may have blocked EtOH-induced increases in sAP firing rates due to its suppression of Ih.
In similar experiments, we replaced cesium with another Ih channel blocker, ZD7288, which is widely used although its selectivity on Ih channels has been questioned in at least one report (Chevaleyre and Castillo 2002). As shown in Fig. 6 Aa, Ih was blocked by ZD7288 (30 µM) in an interneuron from an adolescent rat, and the total current at the end of the test pulse (- - -, inset) was plotted before and during ZD7288 application. Figure 6Ab shows that ZD7288 significantly reduced Ih amplitude from –172.91 ± 33.23 to –2.35 ± 2.70 pA (n = 7, paired t-test, t(6) = –4.89, P = 0.0027), and this effect was irreversible. Bath application of ZD7288 (30 µM) for 7 min also irreversibly decreased sAP firing frequency in five of eight cells tested from adolescent rats and completely blocked sAP firing in three of eight cells (these cells were excluded from analysis). Similar to cesium, blockade of Ih by ZD7288 also significantly reduces the EtOH-induced enhancement of sAP frequency, and a typical example of EtOH (30 mM) on sAP firing is shown in Fig. 6Ba. In this interneuron, bath application of 30 mM EtOH increased sAP frequency from 5.08 to 11.78 Hz. After spontaneous activity returned to the baseline following the EtOH washout, ZD7288 (30 µM) was applied in the bath for 7 min, followed by a re-application of 30 mM EtOH. EtOH failed to increase the frequency of sAPs after the Ih was blocked by ZD7288. The time course for normalized sAP frequency is illustrated in Fig. 6Bb. The summarized data are shown in Fig. 6Bc, indicating that EtOH (30 mM) significantly increased sAP frequency to 11.65 ± 1.35 from 4.90 ± 0.47 Hz, and it was reduced to 2.44 ± 0.15 Hz after application of ZD7288 [n = 5, 1-way ANOVA, F(3, 16) = 53.99, P = 1.367E-8]. After blockade of Ih channels with ZD7288, re-application of EtOH (30 mM) to the slice did not affect frequency of sAPs (2.49 ± 0.19 Hz). ZD7288 (30 µM) also induced a small hyperpolarization of 3.42 ± 0.43 mV (n = 5, –66.8 ± 1.43 to –70.22 ± 1.32 mV). For comparative purposes, we tested the effects of ZD 7288 and CsCl on the resting membrane potential of interneurons from adolescent rats that did not fire spontaneous action potentials. There were no significant changes in the resting membrane potentials by application of CsCl (2 mM) and ZD 7288 (30 µM) [from –66.15 ± 1.76 to –67.98 ± 2.20 mV (n = 6) after application of CsCl; from –66.40 ± 1.67 to –68.20 ± 1.92 mV (n = 5) after application of ZD7288 30 µM].
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slow was 43.9 ms at baseline, 140 ms after bath application of ZD7288 30 µM, and 136.4 ms after bath application of EtOH (Fig. 6Cb). Furthermore, ZD7288 30 µM had no effect on the fast component of AHP decay time (from 9.72 ± 0.67 to 10.56 ± 1.23 ms, n = 5). The averaged data representing the effect of ZD7288 on AHP slow are shown in Fig. 6Cc, indicating that ZD7288 significantly prolonged AHP decay time slow components (slow) to 127.73 ± 8.55 from 40.41 ± 3.37 ms of control [n = 5, 1 way ANOVA, F(2, 12) = 18.80, P = 2.01E-04], and slow remained unchanged in the subsequent application of EtOH (30 mM, 125.57 ± 15.24 ms, n = 5). Altogether these results indicate that Ih participates in the mediation of EtOH-induced increases of sAP firing in hippocampal interneurons. EtOH effects on Ih in interneurons from adolescent and adult rats
The preceding finding, that Ih mediates EtOH-induced increases in sAP firing, prompted us to explore if it could be of developmental significance. To pursue this, we assessed the effects of ethanol on Ih currents recorded in interneurons from animals in both age groups. We evoked Ih by using 1.2-s hyperpolarizing voltage steps to –130 mV from the –50 mV holding potential. The average Ih amplitude was –180.57 ± 21.14 pA (n = 21) in interneurons from adolescent animals and –163.89 ± 17.89 pA (n = 19) in interneurons from adults. This difference, however, was not statistically significant. Bath application of EtOH (30 and 50 mM) reversibly increased Ih amplitude in cells from animals in both age groups (Fig. 7, A and B). Typical current traces of Ih in the presence of EtOH (30 and 50 mM) of interneurons recorded from adolescents and adults are shown in Fig. 7, A and B, a and b. The total currents corresponding to the - - - in Fig. 7, Aa and Ba, were calculated and plotted before and during EtOH application. EtOH (30, 50 mM) increased Ih amplitude in interneurons from both age groups (Fig. 7Ca). In interneurons from adolescent animals, EtOH (30 mM) significantly enhanced Ih amplitude from –175.61 ± 25.02 to –246.76 ± 26.67 pA (n = 13, paired t-test, t(12) = 11.28, P = 9.60E-08) and 50 mM EtOH further increased Ih amplitudes to –262.42 ± 15.38 from –169.35 ± 15.94 pA (n = 13, paired t-test, t(12) = 26.83, P = 4.41E-12). In interneurons from adult rats, EtOH also increased Ih amplitudes from –171.78 ± 19.56 and –163.15 ± 21.73 to –203.57 ± 19.99 pA (EtOH 30 mM, n = 14, paired t-test, t(13) = 7.79, P = 2.98E-06) and –212.38 ± 24.30 pA (EtOH 50 mM, n = 13, paired t-test, t(12) = 6.81, P = 1.86E-05), respectively (Fig. 7Ca). Because EtOH concentrations <30 or 50 mM had affected sAP firing, we also tested the effect of 10 mM EtOH on Ih and found that Ih amplitude was increased to –200.17 ± 16.50 pA from –170.0 ± 14.50 pA (n = 6, 30 pA) in slices from adolescent rats (t[5] = 4.6056, P = 0.0058).
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We next assessed the effect of EtOH on voltage-dependent Ih activation by constructing conductance-voltage (G-V) relation curves. Test pulses to potentials between –60 and –140 mV were applied from a holding potential of –50 mV (Fig. 8). Figure 8, A and B, shows that typical Ih voltage-dependent response traces and fitted activation time course of current traces (at –140 mV) in interneurons from adolescent (A) and adult rats (B) after application of 30 mM EtOH. The activation time courses were analyzed by fitting the activating phase of the current trace at the hyperpolarizing step pulse of –140 mV with a single-exponential function. EtOH (30 mM) significantly decreased the activation time constant of Ih from 155.55 ± 17.56 ms in control to 105.03 ± 9.80 ms (n = 12, paired t-test, t(11) = –4.54 and P = 8.50E-04) in neurons from adolescent rats (Fig. 8Ca) and from 144.75 ± 14.04 ms in control to 118.66 ± 9.65 (n = 9, paired t-test, t(8) = –2.97 and P = 0.018) in neurons from adults. This effect was more pronounced in neurons from adolescent rats (30.16 ± 3.81%, n = 12), compared with those from adults (16.74 ± 4.24%, n = 9, unpaired t-test, t(19) = 2.97, P = 0.03, Fig. 8 Cb). Voltage-dependent Ih activation curves were also analyzed before and after application of EtOH (30 mM). Each activation curve was plotted by calculating Ih amplitude at each of the test pulses, and converting into conductance (see METHODS). For each cell, the calculated conductance was normalized by the control maximal conductance in the absence of EtOH, which was estimated from the Boltzmann equation (Okamoto et al. 2006). We tested 12 neurons from adolescent and 9 neurons from adult animals. The basalV1/2 value and maximal Ih conductance were not significantly different in interneurons from adolescent animals compared with those from adult animals. Addition of EtOH (30 mM) shifted Ih activation curves to more depolarized potentials and also caused an increase in the maximal Ih conductance in interneurons from animals in both age groups. As demonstrated in Fig. 8D, in adolescent animals, fitted data show that 30 mM EtOH shifted V1/2 from –84.95 ± 2.44 to –73.20 ± 2.19 mV (n = 12, paired t-test, t(11) = –10.54, P = 4.41E-07). It also increased maximal Ih Gmax from 2.02 ± 0.23 to 2.74 ± 0.29 nS (n = 12, paired t-test, t(11) = –4.83, P = 5.24E-04) but did not change the k value. In adult animals, 30 mM EtOH also induced a depolarizing shift of V1/2 (from –83.85 ± 1.11 to –77.09 ± 1.19 mV, n = 9, paired t-test, t(8) = –4.68, P = 0.0016), enhanced Gmax (from 2.13 ± 0.18 to 2.38 ± 0.21 nS, n = 9, paired t-test, t(8) = –4.80, P = 0.0013), and failed to alter k values.
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Effects of EtOH on sAPs in interneurons in the presence of glutamatergic and GABAergic blockers
By using acutely isolated VTA neurons in which all synaptic inputs were absent, it has been determined that EtOH excites VAT neurons directly (Brodie et al. 1999; Koyama et al. 2007). Although our preceding results confirmed the involvement of Ih currents in mediating EtOH-induced enhancement of firing rates, these experiments were performed in the presence of all synaptic inputs. Thus it was not clear whether the effect of EtOH on interneuron firing may have been mediated by alterations of excitatory and/or inhibitory synaptic inputs onto the recorded interneurons. We therefore assessed the effects of EtOH on spontaneous firing in 10 interneurons from PD 30–40 rats, after inhibitory and excitatory synaptic inputs were sequentially blocked by BMI (GABAA receptor antagonist), DNQX (AMPA receptor antagonist), and APV (N-methyl-D-aspartate receptor antagonist), respectively. As demonstrated in Fig. 9, the sAPs were recorded from morphologically and physiologically identified interneurons (Fig. 9Aa), and typical traces under each treatment are shown in Fig. 9Ab. In the baseline condition, EtOH (30 mM) increased sAP firing frequency to 9.18 ± 0.77 Hz from 3.97 ± 0.46 Hz (n = 10) in the absence of excitatory or inhibitory antagonists. After washout, the sAP firing frequency returned to baseline (4.23 ± 0.50 Hz, n = 10), and application of BMI (20 µM) did not change the frequency of sAP firing. Similarly, the addition of DNQX (20 µM) did not significantly change the sAP firing frequency (4.16 ± 0.55 Hz, n = 10). However, the addition of APV (50 µM) reduced sAP firing frequency to 2.62 ± 0.43 Hz (n = 10). Under these conditions, i.e., the block of excitatory and inhibitory neurotransmission, EtOH (30 mM) significantly increased sAP firing frequency [5.76 ± 0.82 Hz, n = 10, 1-way ANOVA, F(6, 63) = 11.92, P = 6.84E-9]. The summarized sAP firing frequency from 10 cells under this treatment sequence are illustrated in Fig. 9Ba. The averaged percent change in sAP firing frequency following administration of EtOH (30 mM) in the absence (139.76 ± 10.42%, n = 10) and presence (135.66 ± 14.30%, n = 10) of excitatory and inhibitory antagonists are shown in Fig. 9Bb. There was no significant difference between those two conditions (n = 10, paired t-test, t(9) = 0.33, P = 0.74). Thus it seems unlikely that the effects of EtOH on firing rates in interneurons that we have described in the preceding text are mediated indirectly, through actions on excitatory or inhibitory synaptic activity.
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DISCUSSION |
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The effect of EtOH on spontaneous firing in hippocampal CA1 interneurons recorded from periadolescent and adult rats has not been reported previously. In the present study, we chose to record from a subpopulation of spontaneously firing interneurons for this study. Thus these findings represent the first whole cell electrophysiological evidence for developmental modulation of the effects of acute EtOH on spontaneously active hippocampal CA1 interneurons. Consistent with the present findings, we have recently shown that GABAA receptor-mediated tonic currents in dentate granule cells are promoted more efficaciously in slices from periadolescent rats than in those from adults (Fleming et al. 2007). These data suggest that certain GABA receptor-mediated functions in the hippocampal formation are more sensitive to EtOH during adolescence, whereas others are more sensitive during adulthood and underscore the complex nature of the effects of EtOH on inhibitory circuits through which some of these effects may be mediated during development.
Whether acute ethanol enhances or attenuates neuronal firing activity seems to depend on the types of neurons tested. For example, EtOH reduces firing rate of pyramidal cells with regular spiking activity in somatosensory cortex (Sessler et al. 1998) and inhibits persistent activity in prefrontal cortical pyramidal cells (Tu et al. 2007). In contrast, enhancement of neuronal firing by acute EtOH has been demonstrated in dopaminergic cells in VTA (Appel et al. 2003; Brodie and Appel 1998; Koyama et al. 2007; Okamoto et al. 2006), and cerebellar Golgi cells (Carta et al. 2004). Furthermore, in slices from rats that experience withdrawal after repeated intermittent EtOH exposure, VTA dopaminergic cells display enhanced burst firing patterns (Hopf et al. 2007). Thus the present results that EtOH increases sAP firing in this subpopulation of hippocampal GABAergic interneurons is similar to those from dopaminergic cells in the VTA and Golgi cells in cerebellum and provides new evidence for a possible ionic mechanism whereby Ih mediates EtOH-induced increases in hippocampal interneurons. The developmental differences that we have observed underscore the potential importance of the present findings.
However, there is also some controversy in studies of Ih in VTA dopaminergic neurons, and the role of this current in mediating the effects of EtOH. In one recent study, EtOH-induced argumentation of Ih currents in VTA neurons from C57BL/6J mice was attenuated by ZD7288 (Okamoto et al. 2006). Those findings are consistent with the present data from hippocampal interneurons, although a separate study has shown that the stimulatory effect of EtOH on the excitability of VTA neurons from rats was blocked by quinidine but not by cesium or ZD7288 (Appel et al. 2003), suggesting that Ih could be one of many ionic currents responsible for EtOH-induced increase in firing rates VTA cells. Furthermore, it is possible that barium-sensitive currents may also be involved in the effects of EtOH on VTA neurons (McDaid et al. 2008).
The present data also indicate that EtOH-induced enhancement of sAP firing rates in interneurons from periadolescent rats is not dependent on glutamatergic or GABAergic synaptic inputs as this effect persisted even after excitatory and inhibitory synaptic activities mediated by ionotropic neurotransmitter receptors were blocked. A similar finding was also reported in VTA neurons (Okamoto et al. 2006). Given the fact that EtOH-induced increases in firing rates of interneurons were observed in the absence of this synaptic activity, the present data expand current knowledge regarding the role of Ih in the mediation of EtOH effects on nondopaminergic cells and suggest that Ih channels could serve a direct target for EtOH. It should also be noted that the concentration of EtOH that produced the measurable effects of EtOH on the firing rates in our present studies is much lower than those reported in VTA dopaminergic neurons of CB57 mice (Okamoto et al. 2006) or Fisher-334 rats (Appel et al. 2003). As shown in Fig. 3C, EtOH, at concentrations as low as 3 mM, increased in firing rates in neurons from animals of both age groups, suggesting that hippocampal interneurons are very sensitive to EtOH.
It has been shown that the AHP is involved in regulating the firing of s. radiatum hippocampal interneurons (Savic et al. 2001). In the present study, we demonstrated that the decay time (slow component) of AHPs following sAPs was shortened by EtOH. As one of many important conductances governing the excitability of neurons, the hyperpolarization-activated current, Ih, modulates the AHP time course and neurotransmitter release (Aponte et al. 2006; Maccaferri et al. 1993; Stocker et al. 1999). In spontaneously firing neurons, Ih contributes to the pacemaker depolarization that generates rhythmic activity; in nonpacing cells, Ih modulates resting membrane properties and limits the extent of hyperpolarizing and depolarizing responses (Maccaferri and McBain 1996; Pape 1996). The present results show that Ih in interneurons recorded from both age groups was irreversibly blocked by ZD7288 or cesium, and these results are consistent with previous reports (Aponte et al. 2006; Maccaferri and McBain 1996; Lupica et al. 2001). Additionally, the data indicate that Ih is involved in the regulation of spontaneous firing as demonstrated by the suppression of sAP firing by cesium and ZD7288. Ih and apamin-sensitive Ca2+-activated potassium currents are known to contribute to AHPs in hippocampal interneurons and pyramidal neurons (Maccaferri and MacBain 1996; Savic et al. 2001; Stocker et al. 1999) and to regulate their firing patterns, and AHPs consist of fast (fAHP, lasting 2–5 ms), medium (mAHP, lasting 50–100 ms), and slow (sAHP, lasting >1 s) components in hippocampal cells (Storm 1989). Interestingly, we found that the decay time of the AHP slow component (slow), but not the fast component, was decreased by EtOH. This is consistent with studies of dopaminergic cells and indicates that Ih may also contribute to EtOH enhancement of sAP firing in hippocampal interneurons through limiting the AHP duration and shortening the interval between action potentials to promote multiple discharges during EtOH exposure. Ih is known to be sensitive to the intracellular concentration of Ca2+ in thalamic neurons (Lüthi and MacCormick 1998, 1999), and EtOH-induced increases in intracellular Ca2+ have been detected in neocortical neurons (Allansson et al. 2001) and in cerebellar Purkinje neurons (Gruol et al. 1997). Thus it is possible that EtOH- induced enhancement of Ih could be mediated by internal Ca2+ concentration and thereby contribute to AHP activation.
The growing literature indicates that the effects of EtOH on neurobehavioral function in general are developmentally regulated (Little et al. 1996; Markwiese et al. 1998; Swartzwelder et al. 1995a,b; White and Swartzwelder 2004; White et al. 2000), and particularly for GABAA receptor-mediated inhibitory function in the hippocampus (Li et al. 2003, 2006). We have shown previously that EtOH increases the frequency of sIPSCs in hippocampal pyramidal cells more potently in slices from adult rats than in that from adolescents in the absence of any age-dependent effect on mIPSCs frequency (Li et al. 2003, 2006). Our present data partially agree with our previous findings that the EtOH increases frequency of sIPSCs in pyramidal cells recorded from both age groups. In contrast to more potent EtOH enhancement of sIPSCs observed in CA1 pyramidal cells recorded from adult rats, EtOH enhances action potential frequency by interneurons but its effect is less potent for adults than for adolescents.
There is an apparent discrepancy between the present results from interneurons and our previous reports on data from pyramidal cells. Specifically, our earlier results showing greater enhancement of sIPSC frequency by EtOH in CA1 pyramidal cells from adult animals compared with those from adolescents (Li et al. 2006) would seem to predict that EtOH would increase interneuron firing more rather than less potently in interneurons from adults. However, in this report we found the opposite. EtOH promoted interneuron firing more potently in cells from periadolescents than in those from adults. One possible explanation would be that in the present report we recorded from interneurons that do not directly produce IPSCs in CA1 pyramidal cells. We sampled a small subset of interneurons located within SML-SR subregions. It is possible that these interneurons could have their primary synaptic influence elsewhere, while a distinct set of interneurons contact primarily the apical and basal dendrites of pyramidal cells (Buhl et al. 1994). It is well known that interneurons have diversified morphological and electrophysiological properties (Freund and Buzsaki 1996; Somogyi and Klausberger 2005), and it is likely that interneurons carrying different biomarkers may have different sensitivities to GABAA receptor modulators (Karson et al. 2008; Thomson et al. 2000). Indeed when CA1 pyramidal neurons are activated by either proximal or distal stimulation, the effect of ethanol on the resulting IPSCs is quite different. Proximally evoked IPSCs are far more sensitive to ethanol than those that are evoked by distal stimulation nearer to the region from which we recorded interneurons for the present study (Weiner et al. 1997), and this insensitivity is mediated at least in part by GABAB receptor activation (Wu et al. 2005). Thus any of a number of factors could account for the apparent discrepancy between the present data and those from CA1 pyramidal cells. In the future it will be important to conduct experiments like those reported here on interneurons more proximal to CA1 pyramidal cells.
There could also be differences associated with the Ih channel's physiological properties, developmental regulation of Ih channel subunit expression, or Ih function among special interneuronal populations. For instance, we found that Ih channel physiological function with regard to its sensitivity to EtOH is regulated developmentally as Ih current density was significantly increased by EtOH in cells from periadolescent than adult rats. EtOH (30 mM) caused V1/2 to shift toward more depolarization 12 mV in periadolescent rats, whereas there is 6 mV shifting in the adults. Furthermore, EtOH increased maximum Ih more powerfully in interneurons from periadolescents than in those from adults. Those results further support the idea that developmental regulation of the EtOH sensitivity of sAP firing could be due to its action on the Ih channel's physiological function. Four Ih channel subunits (HCN1-4) encoding Ih are expressed in axons and presynaptic terminals of GABAergic interneurons in the hippocampus (Notomi and Shigemoto 2004), and differential regulation of HCN isoform expression has been described during hippocampus development (Bender et al. 2001). The progressive expression of HCN1 is confined primarily to parvalbumin-expressing basket-type cells, whereas HCN2 is more prominent in other interneuron populations (Bender et al. 2001), although one study indicates that parvalbumin-expressing fast-spiking interneurons co-express HCN1 and HCN2 (Aponte et al. 2006). Moreover, an age-dependent spatiotemporal evolution of specific HCN expression in distinct hippocampal cell populations has been demonstrated (Brewster et al. 2007), suggesting that these channels co-expressing with unique biomarkers in hippocampal interneurons may have differing functions in mediation of EtOH sensitivity of interneurons in local network activity.
In summary, these results demonstrate for the first time that EtOH regulates spontaneous firing in a subpopulation of hippocampal interneurons, and this effect is mediated by EtOH action on Ih expressed in these interneurons and is developmentally regulated. This finding is of particular interest because it demonstrates the complex nature of the effects of EtOH on inhibitory circuits and the developmental mediation of these effects.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. S. Swartzwelder, 508 Fulton St. Bldg. 16, Rm. 27, VAMC, Durham, NC 27705 (E-mail: hss{at}duke.edu)
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