European Journal of Neuroscience, Vol. 40, pp. 2241–2254, 2014

doi:10.1111/ejn.12581

MOLECULAR AND SYNAPTIC MECHANISMS

Tonic current through GABAA receptors and hyperpolarization-activated cyclic nucleotide-gated channels modulate resonance properties of rat subicular pyramidal neurons Nirnath Sah and Sujit K. Sikdar Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India Keywords: dynamic clamp, GABAA receptor current rectification, hippocampus, hyperpolarization-activated cyclic nucleotidegated channels, theta frequency resonance

Abstract The subiculum, considered to be the output structure of the hippocampus, modulates information flow from the hippocampus to various cortical and sub-cortical areas such as the nucleus accumbens, lateral septal region, thalamus, nucleus gelatinosus, medial nucleus and mammillary nuclei. Tonic inhibitory current plays an important role in neuronal physiology and pathophysiology by modulating the electrophysiological properties of neurons. While the alterations of various electrical properties due to tonic inhibition have been studied in neurons from different regions, its influence on intrinsic subthreshold resonance in pyramidal excitatory neurons expressing hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is not known. Using pharmacological agents, we show the involvement of a5bc GABAA receptors in the picrotoxin-sensitive tonic current in subicular pyramidal neurons. We further investigated the contribution of tonic conductance in regulating subthreshold electrophysiological properties using current clamp and dynamic clamp experiments. We demonstrate that tonic GABAergic inhibition can actively modulate subthreshold properties, including resonance due to HCN channels, which can potentially alter the response dynamics of subicular pyramidal neurons in an oscillating neuronal network.

Introduction Extrasynaptic GABAA receptors composed of heterogeneous subunits are observed in a cell-type-specific manner throughout various brain structures that mediate tonic inhibition (Brickley & Mody, 2012) or tonic excitation (Song et al., 2011). The extrasynaptic highaffinity GABAA receptors generally include a d subunit (Stell et al., 2003; Mtchedlishvili & Kapur, 2006) or a5 subunit in hippocampal neurons (Caraiscos et al., 2004; Glykys & Mody, 2006). Somatic whole-cell patch-clamp recordings from subicular excitatory and inhibitory neurons revealed the presence of phasic and tonic inhibition (Biagini et al., 2010; Panuccio et al., 2012). Semi-quantitative polymerase chain reaction and western blot experiments using whole subiculum showed the presence of a5 and d subunits (Curia et al., 2009). Furthermore, a qualitative and semi-quantitative evaluation of the immunocytochemical distribution of the GABAA receptor subunits suggest the presence of both a5 and d subunits in the subiculum (Pirker et al., 2000). These experiments used the complete subicular region containing both excitatory and inhibitory neurons along with astrocytes that express GABAA receptors (Yoon et al., 2012). Hence,

Correspondence: Dr S. K. Sikdar, as above. E-mail: [email protected] Received 10 February 2014, accepted 5 March 2014

it is not clear whether subicular pyramidal excitatory neurons express a5 and/or d subunit-containing GABAA receptors. The subicular neurons in the hippocampus have been implicated in learning and memory and processing spatial information (Sharp & Green, 1994; O’Mara et al., 2001; Kim et al., 2012) as well as pathophysiologies such as epilepsy and Fragile-X syndrome (Cohen et al., 2002; Curia et al., 2009). Oscillations with characteristic frequencies are associated with different physiological and pathophysiological activities (Jefferys et al., 2012). Theta oscillation (4– 12 Hz), a prominent network pattern in the limbic system, is suggested to arise from network interaction between pyramidal neurons and oriens lacunosum-moleculare inhibitory neurons (Buzsaki, 2002). Intrinsic membrane potential oscillation and resonance properties due to certain voltage-gated ion channels, such as hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, play an important role in theta oscillations (Buzsaki, 2002; Wang et al., 2006; Boehlen et al., 2009; Yeung et al., 2013). Although tonic inhibition is known to modulate characteristic offset in CA1 hippocampal pyramidal neurons (Pavlov et al., 2009), its influence on subthreshold oscillations has not been studied, including those of the subicular pyramidal neurons. In an in vitro hippocampal brain slice preparation, we tested whether subicular pyramidal neurons exhibit GABAA receptor-mediated tonic inhibition. We also examined the pharmacological nature

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd

2242 N. Sah and S. K. Sikdar of these receptors and how their activation alters cellular intrinsic properties. Our observations demonstrate the presence of the a5 subunit in extrasynaptic GABAA receptors of subicular pyramidal neurons. The extrasynaptic GABAA receptors along with HCN channels actively fine-tune resonance strength with minor alteration of the characteristic resonance frequency. These results suggest that subicular pyramidal neurons can sense ambient GABA depending upon the inhibitory neuronal activity that can act as an adaptive filter for distinct synaptic inputs.

Materials and methods Preparation of brain slices Juvenile Wistar rats (17–21 days old) were anaesthetized with halothane and decapitated in accordance with European guidelines (86/ 609/EEC) and CPSCEA guidelines (India) with the approval of the Indian Institute of Science Ethical Committee (Sah et al., 2011). Brains were transferred to cooled (~4 °C) sucrose-based artificial cerebrospinal fluid (ACSF), containing (mM): 250 sucrose, 2.5 KCl, 3 MgCl2, 1.25 NaH2PO4, 2 CaCl2, 26 NaHCO3, 10 glucose and 0.4 ascorbic acid in Milli-Q water, equilibrated with 95% O2 and 5% CO2 (pH 7.4, osmolarity 300–310 mOsm). Transverse slices (300 lm thick) of dorsal hippocampus were obtained by using standard techniques using a vibratome (Leica - VT 1000S) and were transferred to incubation solution containing (mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2 CaCl2, 2 MgCl2, 25 glucose and one ascorbic acid in Milli-Q water (equilibrated with 95% O2 and 5% CO2; pH 7.4, osmolarity 300 mOsm). Following an incubation period of 10 min at 36 °C, slices were allowed to incubate at room temperature for ≥ 1 h. Experiments were performed in ACSF (in mM: 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2 CaCl2, 1 MgCl2 and 25 glucose, equilibrated with 95% O2 and 5% CO2; pH 7.4, osmolarity 300 mOsm) in a submerged-type recording chamber (flow rate 2–2.5 mL/min at 30 °C). In vitro electrophysiology The whole-cell patch-clamp configuration was used to record GABAA receptor-mediated currents in voltage clamp mode. Intrinsic electrophysiological properties and their modulation through tonic GABAergic currents were studied in current clamp mode. Proximal subicular pyramidal cells were identified using infrared Dodt gradient contrast video microscopy (Olympus BX51WI). Somatic wholecell recordings in fast current-clamp mode were performed using an Axopatch 200B amplifier (Axon Instruments, Union City, CA, USA), digitized (PCI-MIO-16E-4 and BNC 2090; National Instruments), filtered at 5 kHz and stored on disk at > 20 kHz sampling frequency. Data were acquired using the WinWCP and WinEDR software packages (Strathclyde Electrophysiology Software, Glasgow, UK). Boro-silicate glass capillaries (Harvard Instruments, USA) with outer diameter of 1.5 mm and inner diameter of 0.86 mm were used for all experiments. Patch pipettes (resistance 3–5 MΩ) were filled with an internal solution containing (in mM): 115 K-gluconate, 7 KCl, 0.2 EGTA, 10 HEPES, 5 Mg-ATP, 0.5 Na-GTP and 10 Na-phosphocreatine (pH 7.3 adjusted with KOH; osmolarity 290–300 mOsm) for current clamp experiments and has been termed ‘K+-internal solution’ throughout for convenience. ZD-7288 (20 lM) was included in the pipette solution for dynamic clamp experiments. For tonic current measurements and pharmacological studies under voltage clamp, the patch electrode solution consisted of (in mM) 133.5 CsCl, 10 HEPES, 0.2 EGTA,

2 Mg-ATP, 0.5 Na-GTP, 10 Na-phosphocreatine and 5 QX314Br (pH 7.3 adjusted with CsOH). This is referred to as ‘Cesium-high chloride internal solution’ in the text. A Cs+-based patch electrode solution containing low chloride concentration (Cesium-low chloride internal solution) was used for characterizing tonic current under voltage clamp mode that consisted of (in mM) 126.5 Cs-acetate, 7 CsCl, 10 HEPES, 0.2 EGTA, 2 Mg-ATP, 0.5 Na-GTP, 10 Na-phosphocreatine and 5 QX314Br (pH 7.3 adjusted with CsOH). A liquid junction potential of 11.3 mV was corrected off-line for the low chloride patch electrode solution. Somatic whole-cell voltage clamp used the same amplifier, digitizer and acquisition software as mentioned above. The signals were filtered at 2 kHz and stored on disk at 20 kHz sampling frequency. Series resistance was read directly from the amplifier in response to a 10-mV depolarizing pulse. A 100% series resistance (6–20 MO) was compensated for in current clamp mode, and 70% for voltage clamp mode. Recordings in which the access resistance did not change > 5 MO throughout the duration of the experiment were included for analysis. All the experiments were conducted in the presence of CNQX (10 lM), AP5 (50 lM) and CGP 55845 (1 lM) in the ACSF. Additionally, 100 lM CdCl2 was used to block voltage-gated calcium channels in voltage clamp experiments where step voltage pulses were used from
80 to +50 mV. Neuronal data analysis Synaptic events were analysed using Igor Pro (Wave Metrics Inc., Lake Oswego, OR, USA). Event detection was performed using amplitude threshold crossing. Approximately 100 s of raw trace before and after pharmacological manipulation was used for data analysis. All the events detected within 100 s of traces were analysed for peak amplitude, frequency, rise time and decay time constant in each experimental epoch, which were averaged for each cell and used for further statistical analysis. A single exponential function was fitted to decay times of individual events to evaluate the decay time constant. Peak amplitude of spontaneous synaptic events was determined relative to the baseline current value. The 20–80% rise-time was calculated between the start and the peak of a synaptic event. Tonic GABA current (Itonic) was calculated as the difference between the baseline holding current (Ihold) and Ihold in the presence of a GABAA receptor antagonist. All point histogram plots were constructed and the peak current value was selected as Ihold. The neurons were subjected to a family of rectangular voltage step pulses of 5 s duration from
80 to +50 mV with an increment of 10 mV at a holding potential of
70 mV to estimate the steady-state current at each potential. The picrotoxin-sensitive tonic current was calculated by subtracting the steady-state currents evoked by the voltage step pulses after picrotoxin application from the control. The tonic current responses of each cell were normalized to the maximum tonic current, which were then averaged to plot mean current–voltage (I–V) relationship. The tonic currents at different potentials from each cell were converted to conductance values using the equation Gtonic ¼ Itonic =ðV
Etonic Þ

ð1Þ

where Gtonic is conductance, Itonic is picrotoxin-sensitive current, V is the membrane potential step used to elicit the response and Etonic is the reversal potential (
79.02 mV) of the picrotoxin-sensitive tonic current. Conductance data were normalized using the maximum conductance of each cell individually and mean  SEM values were plotted against voltage (G–V plot) from all the pooled data. The G– V plot was fitted with a Boltzmann equation of the following form:

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 2241–2254

Tonic inhibition modulates subthreshold properties of subicular neurons 2243 ( S1 ¼ Gbasal þ

ð1
Gbasal Þ
 1 þ exp Vhalfk
V

) ð2Þ

where s∞ is the steady-state voltage activation, Gbasal is baseline conductance, Vhalf is the half-maximum of the steady-state voltage activation and k is the slope factor. Input resistance was determined from the slope of the linear regression fit through the V–I plot constructed by plotting the amplitude of the steady-state voltages (V) against the corresponding step current injections (I) from
50 to
200 pA of 500-ms duration. Voltage sag was measured as the percentage change between the maximum (Vmax) and steady-state (Vss) voltage change with respect to the baseline membrane voltage during hyperpolarizing current injection of
200 pA: Voltage sag ¼ ½ðVmax
Vss Þ=Vmax   100

Real time experimental interface-RTXI (dynamic clamp) The dynamic clamp recording configuration allows effective realtime application of the voltage- and time-dependent current associated with voltage-gated channels. Voltage-gated channels were modelled according to the conductance’s voltage and/or time-dependent properties (Hodgkin-Huxley equations) and were connected to the real cell, electronically. This was accomplished by interfacing a recording amplifier (Axopatch 200B) with a computer that calculated the current to be injected back into the real cell according to the modelled conductance in real time at each sampling interval (100 ls). We used a dynamic clamp configuration based on a system described earlier (Lin et al., 2010). Our dynamic clamp system consisted of an amplifier (Axopatch 200B), data acquisition device (NI-PCI 6221) and a computer running a real-time operating system (RTXI) which controlled the whole set-up. Plug-ins for appropriate functions were written in C++, which were compiled and implemented. Tonic inhibitory current model The tonic current was modelled as:

( S1 ¼ 0:237 þ

) 0:823
 1 þ exp 4:2
V 10:6

Mathematical model of h-current (Ih) We simulated the voltage gated ion-channel current through the HCN channel, Ih. The simulated conductance (Gh) was modelled as given below and dynamically linked to RTXI at run-time. All the parameters to simulate the Gh were taken from the model used by van Welie et al. (2006) to describe the experimentally recorded Ih using the following equations:

ð3Þ

The stimulus used for characterizing the impedance amplitude profile was a sinusoidal current of constant amplitude, with its frequency linearly spanning 0–15 Hz in 15 s (ZAP). The impedance amplitude profile was calculated from the magnitude of the ratio of the Fourier transform of the voltage response to the Fourier transform of the current stimulus. The frequency at which the impedance amplitude reached its maximum was the resonance frequency. The ratio of the impedance at the resonance frequency to the impedance at 0.5 Hz (Hutcheon et al., 1996) was used to quantify the strength of the resonance (resonance strength). The amplitude of the ZAP current was selected such that the initial peak-to-peak voltage response was ~12–17 mV.

Itonic ¼ Gtonic max  s1  ½V
Etonic 

sensitive tonic current (
79.02 mV). The dynamic clamp experiments were done at a holding potential of
65 mV. The simulated tonic conductance (Gtonic) was integrated with the real neuron through RTXI.

ð4Þ

Ih ðV; tÞ ¼ Gh ðV; tÞ  ðV þ 25Þ

ð6Þ

   V þ 76 Gh ðV; tÞ ¼ Gh max = 1 þ exp 8

ð7Þ

182  exp f0:033  ðV þ 75Þg 1 þ exp f0:083  ðV þ 75Þg

ð8Þ

sh ðV; tÞ ¼

where ionic reversal potential for Ih:
25 mV; conductance of HCN channel: Gh; maximal conductance of HCN channel: Gh max; steadystate kinetics of Ih: sh; and membrane potential: V. Statistical analysis Sample size (n) refers to the number of recordings from single neurons in different slices. Statistical tests were performed using SigmaPlot (Systat Software). Unless stated otherwise, differences between groups were examined using the appropriate paired or unpaired Student’s t test. Non-parametric tests (Mann–Whitney U test or Wilcoxon signed-rank test) were used when data distribution did not satisfy normality test (Shapiro–Wilk test). Differences were considered significant at P < 0.05. Chemicals (2S)-3-[[(1S)-1-(3,4-Dichlorophenyl)ethyl]amino-2-hydroxypropyl] (phenylmethyl) phosphinic acid hydrochloride (CGP 55845, 1 lM), picrotoxin (100 lM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 lM) and DL-2-amino-5-phosphonopentanoic acid (AP5, 50 lM), flumazenil (10 lM), 6-imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid (gabazine, 20 lM), L-655,708 (50 lM), allotetrahydrodeoxycorticosterone (THDOC, 20 nM), etomidate (3 lM) and 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol (THIP, 3 lM) were bath perfused wherever specified. N-(2,6-Dimethylphenyl-carbamoylmethyl) triethylammonium bromide (QX-314, 5 mM) and 4-Ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride (ZD-7288, 20 µM) were included in the pipette solution. Picrotoxin, CGP 55845, AP5, CNQX, flumazenil, gabazine, ZD-7288 and etomidate were purchased from Ascent Scientific (Bristol, UK) and all other chemicals and drugs were procured from Sigma– Aldrich (St Louis, MO, USA).

ð5Þ

where Itonic is the current through tonic conductance, Gtonic max is the maximum tonic conductance, s∞ is the steady-state voltage activation obtained from the fit of the G–V plot (shown in Fig. 5A and B), V is the membrane potential and Etonic is reversal potential of the picrotoxin-

Results Subicular pyramidal neurons exhibit phasic and tonic inhibition GABA is the primary inhibitory neurotransmitter in the hippocampus. Subicular pyramidal neurons receive significant GABAergic

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 2241–2254

2244 N. Sah and S. K. Sikdar Pharmacological profile of tonic current in subicular pyramidal neurons

inputs via presynaptic release of GABA at the synapses that cause phasic inhibition (Prida, 2003). Excessive inhibitory neuronal activity, however, can result in spillover of GABA that acts on the extrasynaptic GABAA receptors to cause tonic inhibition. We aimed to isolate the tonic component of inhibition from the synaptic component in subicular pyramidal neurons using GABAA receptor antagonists. To this effect, we used a non-competitive antagonist (picrotoxin) and competitive antagonist (gabazine) (Chandra et al., 2006; Curia et al., 2009; Panuccio et al., 2012). Application of picrotoxin (100 lM) to slices revealed an outward shift of the holding current along with blockade of phasic currents (n = 7, Fig. 1A). The outward current is due to the block of GABAA receptors responsible for the tonic inhibition. Gabazine (20 lM) could block the phasic currents but failed to reduce the amplitude of the tonic current substantially (n = 9, Fig. 1B). A higher affinity of extrasynaptic GABAA receptors to GABA could render gabazine (competitive antagonist) ineffective in blocking tonic inhibition (Stell & Mody, 2002). As gabazine application was unable to reveal the tonic component of GABAergic inhibition to a significant amount (Fig. 1C), we chose picrotoxin to probe the electrophysiological changes associated with tonic conductance.

Subunit composition of extrasynaptic GABAA receptor is cell typespecific and also varies according to the brain region. To investigate the subunit composition of the GABAA receptor contributing to the tonic current in subicular pyramidal neurons, we characterized the pharmacology of the tonic current. Several GABAA receptor subunits have been implicated in extrasynaptic GABAA receptor function – a1, a4, a5, a6 and d subunits (Brickley & Mody, 2012). Of these, a6 is the major subunit expressed in the cerebellum (Pirker et al., 2000). As a5 and d subunits are abundantly expressed and contribute to tonic inhibition in hippocampal neurons (Sperk et al., 1997; Pirker et al., 2000), we first examined the expression of the a5 and d subunits pharmacologically. L-655,708 is known for its selectivity for a5-containing GABAA receptors (> 50 fold) over other GABAA receptors (Quirk et al., 1996). We tested whether a5 subunit-containing receptors contribute to the tonic current using L-655,708 – the a5 subunit-selective inverse agonist. At 50 lM, L-655,708 produced a 53.95% decrease (control: 152.25  30.35 pA, L-655,708: 70.1  10.27 pA, n = 7, P = 0.02, paired t-test) in the tonic current amplitude (Fig. 2A).

A a

b

B a

b

C

Fig. 1. Presence of picrotoxin-sensitive tonic current in subicular pyramidal neurons. (A) Picrotoxin (100 lM) blocks inward inhibitory postsynaptic currents and reveals an outward shift in baseline holding current indicating the presence of a tonic current (a). (b) All-point histogram constructed from a control (black) and picrotoxin-treated (grey) region of the current trace shows the shift in the holding current. (B) Voltage-clamp recordings at
65 mV in the presence of gabazine (20 lM) abolished the spontaneous phasic currents without changing the holding current (a). (b) The all-point histogram illustrates the amplitude of the current in the absence and presence of gabazine. (C) Pooled data of change in holding current from control indicate significant block of the tonic current by picrotoxin (n = 7, *P = 0.023, one-sample t-test) but not gabazine (n = 9, P = 0.3, one-sample t-test). The picrotoxin and gabazine experiments were done on different slices. ns, not significant. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 2241–2254

Tonic inhibition modulates subthreshold properties of subicular neurons 2245 A number of specific steroids have been reported to modulate the activity of GABAA receptors. THDOC, an endogenous neurosteroid, is a potent allosteric modulator of GABAA receptors. At

physiological concentrations (10–30 nM), THDOC preferentially potentiates d subunit-containing GABAA receptor-mediated currents (Stell et al., 2003). Hence, we tested whether low concentrations of

A a

b

c

B a

b

c

C a

b

c

D a

b

c

Fig. 2. Pharmacological characterization of the GABAA receptor-mediated tonic current in subicular pyramidal neurons. Representative voltage clamp traces illustrate the effect of a GABAA receptor a5 subunit selective antagonist, L-655,708 (50 lM) (A–a), GABAA receptor d subunit selective modulator, allotetrahydrodeoxycorticosterone (THDOC; 20 nM) (B–a), a benzodiazepine antagonist, flumazenil (10 lM) (C–a) and GABAA receptor b subunit selective agonist, etomidate (3 lM) (D–a). Corresponding all-point histograms are shown in A–b, B–b, C–b and D–b, respectively. Averaged GABAA receptor-mediated tonic current plots before and after drug treatment are shown in A–c, B–c, C–c and D–c, respectively. The shift in the all-point histogram by L-655,708 (A–b) is consistent with a reduced tonic current as exemplified in the averaged data shown in A–c (n = 7, *P = 0.02). (B) Whole-cell voltage-clamp record shows an insignificant effect of 20 nM THDOC on the baseline current (B–a) as illustrated in the averaged data (B–c) (n = 5, P = 0.42). (C) Flumazenil caused a significant decrease in GABAA receptor-mediated tonic current (C–a, b, c) (n = 5, *P = 0.043). (D) Etomidate application increased the tonic current significantly, indicating the presence of the b subunit (D–c) (n = 6, **P = 0.007). Insignificant changes are indicated by ‘ns’ in the graphs. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 2241–2254

2246 N. Sah and S. K. Sikdar THDOC (20 nM) could modulate tonic inhibition in subicular pyramidal neurons. We found an insignificant effect of THDOC on tonic current amplitude (control: 121.67  48.56 pA, THDOC: 107.82  41.72 pA, n = 5, P = 0.42, paired t-test), which suggests that tonic inhibition is not contributed to by d subunit-containing GABAA receptors (Fig. 2B). Functional GABAA receptors are pentameric structures primarily composed of two a, two b and one c/d subunit in the hippocampus. As the d subunit is unlikely to be present in subicular pyramidal neurons (Fig. 2B), we checked for the presence of the c subunit. Previous work has shown that a and c subunits together compose the benzodiazepine binding site (Buhr & Sigel, 1997; Sigel, 2002). We sought to determine whether the GABAA receptors mediating tonic current have such a benzodiazepine binding site. To this effect, we used a benzodiazepine antagonist – flumazenil (Ro 15-1788) (Otis & Mody, 1992; Wafford et al., 1993) – and hypothesized that any decrease in tonic inhibition should indicate the presence of a benzodiazepine site composed of the c subunit. Flumazenil at 10 lM reduced the mean tonic current from 91.32  26.72 pA (baseline) to 28.41  10.54 pA during drug application (n = 5, P = 0.043, paired t-test; Fig. 2C). Next, we aimed to identify the presence of the b subunit in GABAA receptors that mediate tonic current. Etomidate (3 lM) is a positive allosteric modulator selective for the b subunit-containing GABAA receptor (Hill-Venning et al., 1997; Sanna et al., 1997). Etomidate application enhanced the tonic current amplitude to 193.68  39.32 from 120.37  28.96 pA of control (n = 6, P = 0.007, paired t-test; Fig. 2D). These observations suggest that the GABAA receptors mediating tonic current are primarily composed of a5bc subunits. THIP selectively increases GABAA-mediated tonic inhibition

(Minamoto et al., 1992) conditions in various brain regions and assessed the role of tonic inhibition in regulating membrane properties. Using THIP (3 lM) we studied how tonic inhibition modulates cellular subthreshold electrophysiological properties – input resistance, sag, resonance strength and resonance frequency (Fig. 4). These measurements are voltage sensitive; hence, the holding potential was kept constant before and after THIP (
65 mV) to study primarily the shunting effect of enhanced tonic inhibition. THIP decreased the input resistance by 10.3% (from 46.54  2.6 to 41.74  2.42 MΩ, n = 7, P = 0.006, paired t-test; Fig. 4A–c) and sag by 17.6% (from 36.74  1.12 to 30.26  2.37, n = 7, P = 0.016, Wilcoxon signed rank test; Fig. 4B–c). Resonance is a property that characterizes the frequency at which neurons respond maximally to an oscillating current injection. Consequently, if a neuron exhibits a resonant property then there is a specific frequency at which the neuron will have a higher impedance value (resonance frequency). We injected a sinusoidal current with constant amplitude but varying frequency from 0 to 15 Hz and recorded the voltage response. The impedance profile was calculated by taking the amplitude ratio of the fast Fourier transform (FFT) of the voltage response to FFT of the ZAP current (see Methods). Resonance strength, measured as the ratio of peak impedance to the impedance at 0.5 Hz, decreased significantly by 8.3% (from 1.44  0.04 to 1.32  0.05, n = 7, P = 0.016, Wilcoxon signed rank test; Fig. 4E– a). The resonance frequency remained unaltered (control: 2.88  0.2 Hz, THIP: 2.95  0.19 Hz, n = 7, P = 0.546, paired ttest; Fig. 4E–b). Voltage dependence of GABAA receptor-mediated tonic current in subicular pyramidal neurons

We next sought to determine whether Itonic alters the electrophysiological properties of the subicular pyramidal neurons. Studying the role of Itonic on sub-threshold electrophysiological properties entails the use of a pharmacological agent that is specific for tonic inhibition (Itonic) without altering phasic inhibition. THIP preferentially enhances tonic inhibition with an insignificant effect on phasic inhibition in neurons from various brain regions (Jia et al., 2005; Jo et al., 2011; Tang et al., 2011; Duguid et al., 2012; Herman et al., 2012). Hence, we tested the effect of bath-applied THIP (3 lM) on tonic and phasic inhibition in subicular pyramidal neurons (Fig. 3). We observed enhancement in tonic GABAA current amplitude without any effect on the phasic current amplitude or kinetics (Fig. 3). The tonic current increased from 144.97  29.83 to 226.11  30.7 pA (n = 7, P = 0.002, paired t-test; Fig. 3A–C) upon THIP perfusion. However, the characteristics of postsynaptic phasic currents did not change – rise time (control: 0.91  0.08 ms, THIP: 0.87  0.07 ms, n = 7, P = 0.591, paired t-test; Fig. 3C), current amplitude (control: 73.85  13.15 pA, THIP: 70.14  11.38 pA, n = 7, P = 0.572, paired t-test; Fig. 3D), frequency (control: 3.45  0.61 Hz, THIP: 3.22  0.48 Hz, n = 7, P = 0.563, paired t-test; Fig. 3E) and decay time constant (control: 19.75  1.19 ms, THIP: 18.91  0.92 ms, n = 7, P = 0.396, paired t-test; Fig. 3F).

We characterized the voltage dependence of the picrotoxin-sensitive tonic current with physiologically relevant chloride concentration inside the cell (7 mM, Cesium low chloride internal solution, see Methods). The I–V relationship was determined from the subtracted currents obtained by stepping the membrane potential from
80 to 40 mV before and after picrotoxin (Fig. 5A–a). The reversal potential was estimated to be
79.02 mV, which is close to the theoretical chloride reversal potential of
77.05 mV. The slope conductance was determined for each cell and was normalized with the maximal slope conductance. The conductance-voltage (G–V) plot shows two voltage ranges with distinct properties – a voltage-independent range close to resting membrane potential and voltagedependent range where the conductance rapidly increases near
30 mV and peaks around +40 mV (Fig. 5A–b). We fitted the G–V relationship with a Boltzmann equation (Eqn 2, for details see Methods) and used the fitted parameters for simulating tonic current in dynamic clamp experiments. As earlier studies have demonstrated outwardly rectifying tonic GABAA receptor current in CA1 pyramidal neurons (Pavlov et al., 2009), we further confirmed the rectifying behaviour using symmetrical chloride concentration to avoid Goldman–Hodgkin–Katz rectification. Analogous to CA1 pyramidal neurons, we observed outward rectification of GABAergic tonic current in subicular pyramidal neurons, where the current amplitudes were higher at positive potentials (Fig. 5B).

Enhanced tonic inhibition alters electrophysiological properties of subicular pyramidal neurons

Tonic inhibition modulates Ih-sensitive electrophysiological properties

Given that THIP specifically increased tonic inhibition, we used it to mimic the enhanced concentration of GABA that is observed in different physiological (Bianchi et al., 2003) and pathophysiological

The electrophysiological properties – input resistance, sag and resonance strength – showed a significant decrease with enhanced tonic inhibition in the presence of THIP (Fig. 4). These measurements are

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 2241–2254

Tonic inhibition modulates subthreshold properties of subicular neurons 2247 A a

b

c

B a

b

C

D

E

F

Fig. 3. THIP increases GABAA receptor-mediated tonic current without affecting spontaneous synaptic phasic current. (A–a) Continuous current recording (
65 mV) from a representative subicular pyramidal neuron shows the effect of THIP (3 lM), a GABAA receptor agonist. THIP produced a negative shift in the holding current that was reversed with picrotoxin. (A–b) The all-points histogram shows the relative amplitude of the THIP- (dark grey) and picrotoxin(light grey) induced current. (A–c) Pooled data illustrating a significant increase in mean tonic current by THIP (n = 7, **P = 0.002, paired t-test). (B–a, b) Sections of the voltage clamp raw trace in A–a (arrow marks) are expanded to illustrate that spontaneous phasic GABAA receptor-mediated synaptic currents are not affected by THIP. Averaged data from seven cells show an insignificant change in rise time (C), current amplitude (D), frequency (E) and decay time constant (F) of the spontaneous phasic GABAA receptor currents upon THIP application. ns, not significant.

sensitive to HCN-channels, as reported previously (Hutcheon et al., 1996; Wang et al., 2006). The potential interaction between membrane properties governed by Ih and tonic inhibition was investigated using dynamic clamp. We intended to block the native HCN channels and used controlled levels of modelled Gh and Gtonic through dynamic clamp. ZD-7288, a potent inhibitor of Ih, reduces presynaptic transmitter release (Chevaleyre & Castillo, 2002), if used in the bath solution. Hence, we decided to use ZD-7288 (20 lM) in the pipette solution (K+-internal solution) to limit potential undesirable effects on neurotransmitter release. K+-internal solutions with and without ZD-7288 were used to characterize the Ih-sensitive electrophysiological properties in the presence of picrotoxin (100 lM). ZD-7288 in the pipette solution increased the membrane input resistance (K+ internal: 44.51  4.27 MΩ, n = 9; ZD-7288 internal: 103.23  7.86 MΩ, n = 8; P ≤ 0.001, unpaired t-test) and decreased the voltage sag generated by hyperpolarizing current injection (K+ internal: 38.12  0.63, n = 9; ZD-7288 internal: 15.43  3.1, n = 8; P < 0.001, Mann–Whitney rank sum test),

indicating a significant block of HCN-channels. Similarly, the resonance strength (K+ internal: 1.49  0.03, n = 9; ZD-7288 internal: 1.08  0.02, n = 8; P < 0.001, unpaired t-test) and resonance frequency (K+ internal: 3.25  0.24, n = 9; ZD-7288 internal: 1.08  0.21, n = 8; P < 0.001, unpaired t-test) also showed a marked decrease. Next, we introduced 10 nS of h-conductance through dynamic clamp (10 nS Gh max) and checked the Ih-sensitive parameters – input resistance, sag and resonance (Fig. 6). Dynamic clamp-mediated addition of 10 nS of Gh decreased the input resistance significantly (control: 103.23  7.86 MΩ, 10 nS Gh max: 43.69  0.67 MΩ, n = 8, P < 0.001, paired t-test; Fig. 6A–c) while increasing the sag (control: 15.44  3.11, 10 nS Gh max: 40.73  1.06, n = 8, P < 0.001, paired t-test; Fig. 6B–c), resonance strength (control: 1.08  0.02, 10 nS Gh max: 1.52  0.04, n = 8, P < 0.001, paired t-test; Fig. 6E–a) and resonance frequency (control: 1.08  0.2 Hz, 10 nS Gh max: 3.77  0.07 Hz, n = 8, P < 0.001, paired t-test; Fig. 6E–b). Comparison of observed subthreshold

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 2241–2254

2248 N. Sah and S. K. Sikdar A a

b

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B a

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Fig. 4. Enhancement of the tonic conductance with THIP alters input resistance, sag and resonance strength. (A) Representative voltage traces in response to a series of hyperpolarizing and depolarizing current injections (
200 to +200 pA) during the control (a) and THIP treatment (b) in the same neuron. (A–c) Summary graph showing decreased input resistance after THIP application (n = 7, **P = 0.006). (B) Illustrative current clamp recording showing the sag before (a) and during THIP application (b). (c) Pooled data showing a significant drop in the sag (n = 7, *P = 0.016). Representative voltage traces in response to injection of the ZAP stimulus before (C–a) and during THIP application (D–a). The impedance amplitude profile shows a decrease in the maximum impedance during THIP application (D–b) with respect to the control (C–b). While average resonance frequency remained unchanged (E–b) (n = 7, P = 0.546), resonance strength showed a significant decrease (E–a) with THIP treatment (n = 7, *P = 0.016). An non-significant change is indicated by ‘ns’ in the graph.

properties with K+-internal solution and ZD-7288 internal solution after addition of 10 nS of Gh through dynamic clamp showed similar input resistance (K+ internal: 44.51  4.27 MΩ, n = 9; ZD-7288 internal +10 nS Gh max: 43.69  0.67 MΩ, n = 8; P = 0.34, Mann–Whitney rank sum test), resonance strength (K+ internal: 1.49  0.033, n = 9; ZD-7288 internal +10 nS Gh max: 1.52  0.04, n = 8; P = 0.61, unpaired t-test), resonance frequency

(K+ internal: 3.25  0.24 Hz, n = 9; ZD-7288 internal +10 nS Gh max: 3.76  0.07 Hz, n = 8; P = 0.06, Mann–Whitney rank sum test) and marginally higher sag (K+ internal: 38.12  0.63, n = 9; ZD-7288 internal + 10 nS Gh max: 40.73  1.06, n = 8; P = 0.046, unpaired t-test). Finally, we introduced increasing levels of tonic conductance, starting from 10 nS, using dynamic clamp (Gtonic max) while

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 2241–2254

Tonic inhibition modulates subthreshold properties of subicular neurons 2249 A

a

injecting a fixed 10 nS of h-conductance (Gh max) (Fig. 7). The effect of increasing levels of Gtonic max on resonance properties of subicular pyramidal cells was studied. We found marked changes in subthreshold electrical properties where 10 nS of Gtonic max significantly decreased resonance strength (Gh max: 1.52  0.04, Gh max + 10 nS Gtonic max: 1.43  0.02, n = 8, P = 0.015, paired t-test) with a small change in the resonance frequency (Gh max: 3.77  0.07 Hz, Gh max + 10 nS Gtonic max: 4.07  0.09 Hz, n = 8, P = 0.027, paired t-test). Addition of progressively increasing Gtonic max decreased resonance strength linearly (Fig. 7C) with a minor change in resonance frequency until 40 nS of Gtonic max (Fig. 7D).

Discussion

b

In the present study, we used electrophysiological and pharmacological techniques to characterize tonic GABAA currents in the subicular pyramidal neuron and understand their role in modulating intrinsic cellular properties. Our data suggest that a5, b and c subunits together composed the functional GABAA receptors responsible for the tonic GABAergic currents observed in subicular pyramidal neurons. GABAergic tonic inhibition can modulate intrinsic electrophysiological properties by influencing input conductance of the subicular pyramidal neurons. Increased tonic conductance partially preserves the frequency selectivity of the subicular cells but significantly alters the gain at the resonance frequency due to HCN channels. Tonic inhibition and associated GABAA receptor subunits in subiculum

B

Fig. 5. Voltage dependence of the outwardly rectifying tonic current. (A–a) The normalized current–voltage (I–V) plot determined from subtracted steady-state currents before and after picrotoxin application (picrotoxin-sensitive tonic current) that were evoked by voltage steps between
80 mV and +40 mV in 10-mV increments from a holding potential of
70 mV (n = 7). The I–V relationship of picrotoxin-sensitive tonic current was obtained using physiological intracellular chloride concentration (7 mM, Cesium-low chloride internal solution, see Methods). (A–b) Plot of the normalized conductance– voltage (G–V) relationship for the tonic current. The grey line indicates the fit with Boltzmann’s equation (Eqn 2) with Vhalf = 4.2 and k = 10.6. (B) Outward rectification of the tonic current obtained from the I–V plot with symmetrical chloride concentration in the pipette and bath solution (Cesiumhigh chloride internal solution, see Methods).

The GABAergic system is crucial for physiological and pathophysiological responses of the brain (Brickley & Mody, 2012). Modulation of the GABAergic system at the cellular and network level endows neurons and networks with rhythmogenicity, plasticity and homeostasis (Mann & Paulsen, 2007; Schousboe & Waagepetersen, 2007; Wenner, 2011). Synaptic release of GABA can activate synaptic ionotropic GABAA receptors and cause ‘phasic’ inhibition. Additionally, GABA can activate a distinct pool of extrasynaptic GABAA receptors to mediate a persistent ‘tonic’ form of inhibition. Picrotoxin-sensitive tonic currents were recorded in subicular pyramidal neurons in acute rat hippocampal brain slices, consistent with previous reports of a tonic GABAA receptor-mediated conductance in subicular pyramidal neurons (Panuccio et al., 2012). The two major subunits involved in tonic GABAA currents in the hippocampus are a5 and d subunits. Immunocytochemical and mRNA studies have shown the marked presence of the d subunit in dentate gyrus and the a5 subunit in other parts of the hippocampus, including subiculum (Pirker et al., 2000). However, a recent study demonstrated equivalent abundance of a5 and d subunits in the subicular tissue at the protein and mRNA levels (Curia et al., 2009), although it is difficult to extrapolate these findings to individual pyramidal neurons in the subiculum. In the present study, we used electrophysiological and pharmacological profiling of the extrasynaptic GABAA receptors in subicular pyramidal neurons to get insight into their subunit composition (Figs 1 and 2). Our results showed the marked presence of an a5-subunit containing GABAA receptor that mediates tonic inhibition in the subicular pyramidal neurons and absence of the d subunit (Fig. 2). This contradicts earlier observations of d-subunit-containing GABAA receptors in subicular tissue (Curia et al., 2009), and suggests its presence in other cell types, such as inhibitory neurons that are dispersed in the subiculum.

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 2241–2254

2250 N. Sah and S. K. Sikdar A a

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Fig. 6. Addition of Ih through dynamic clamp in Ih -blocked neurons restores the sub-threshold Ih-sensitive properties. (A–a) Voltage traces from a subicular pyramidal neuron in response to step pulses (ranging from
200 to 100 pA) following block of Ih with pipette solution containing ZD-7288 (20 lM). The block of Ih is indicated by the absence of sag at hyperpolarized potentials. (A–b) Note changes in the voltage profiles to similar voltage steps following addition of the h-conductance (10 nS Gh max) through dynamic clamp (b) in the same neuron as demonstrated in (a). (c) Pooled data showing a significant decrease in the mean input resistance following addition of 10 nS Gh max to the neuron (A–b) from control (A–a) (n = 8, **P < 0.001). (Ba, b) Sample traces from A(a, b) upon
200 pA current injection to explicitly show the induction of sag following addition of 10 nS Gh max. (c) Pooled data from eight cells illustrating a significant increase in sag after addition of 10 nS of Gh max through dynamic clamp (n = 8, **P < 0.001). (C, D) Voltage responses to ZAP current injection from a subicular pyramidal neuron maintained at around
65 mV. The amplitude of membrane potential response to ZAP current injection increased within a narrow frequency window (resonance frequency) after addition of 10 nS of Gh max through dynamic clamp (D–a) but was absent when HCN channels were blocked with ZD-7288 in the internal solution (C–a). Impedance profiles calculated from the voltage responses after ZAP current stimulus are provided at the bottom of the respective raw traces (C–b, D–b). (E) Pooled data showing a significant increase in resonance strength (E–a) (n = 8, **P < 0.001) and resonance frequency (E–b) (n = 8, **P < 0.001) after addition of 10 nS of Gh max alone. All the experiments were conducted with 100 lM picrotoxin in the bath solution and 20 lM ZD-7288 in the pipette solution.

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 2241–2254

Tonic inhibition modulates subthreshold properties of subicular neurons 2251 A a

B

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Fig. 7. Tonic inhibitory conductance and HCN channels together modulate the resonance properties of subicular pyramidal neurons. (A) Sample voltage traces obtained from subicular pyramidal neurons during injection of a sinusoidal current waveform (ZAP). The voltage response to ZAP current with ZD-7288 in the pipette solution lacked a resonant hump (a), which became prominent following addition of artificial HCN channel conductance (Gh max =10 nS) using dynamic clamp (b). Further addition of tonic conductance (Gtonic max) from 10 to 40 nS decreased the resonant hump, as revealed in the raw traces (c–f). (B) Impedance as a function of ZAP frequency obtained using Fourier transform (see Methods) of the voltage traces shown in A. (C, D) Summary graphs illustrating a gradual decrease in resonance strength (C) with increasing tonic conductance with a relatively minor effect on resonance frequency (D) (n = 8). All the experiments were conducted with 100 lM picrotoxin in the bath and 20 lM ZD-7288 in the pipette solution.

Subicular excitability The subiculum is primarily a bursting structure and positioned between the hippocampus proper and entorhinal cortex. Its strategic location and connectivity allows it to modulate the hippocampal output to distinct cortical and sub-cortical regions (Witter et al., 1990). Cortical regions receiving efferents from the subiculum include the presubiculum, infralimbic, entorhinal, perirhinal, retrosplenial and postrhinal cortices. In addition, the subiculum also projects to subcortical regions such as the nucleus accumbens, the lateral septal region, the thalamus, the core of the nucleus gelatinosus, the medial

nucleus and the mammillary nuclei (Witter et al., 1990). Cellular excitability is a prime factor in processing and modulating incoming inputs to the downstream target neuronal population. The subiculum is endowed with unique cellular and network characteristics that regulate hippocampal output. The subicular pyramidal neurons express higher levels of HCN channels, which influence input resistance, resonance frequency, temporal synaptic integration and shape cellular excitability (Lorincz et al., 2002; Wang et al., 2006; van Welie et al., 2006). Additionally, a strong feedforward and feedback inhibitory network control provides an important regulatory mechanism

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 2241–2254

2252 N. Sah and S. K. Sikdar at the network level to fine-tune the information flow (Finch et al., 1988; Prida, 2003; Sah & Sikdar, 2013). An increase in ambient GABA has been reported for behaviours such as exploration (Bianchi et al., 2003) and pathologies such as epilepsy (Minamoto et al., 1992). Increases in local GABA concentrations can activate significant tonic conductance, which might act like a shunt, thus influencing electrical properties of the membrane. We have studied the effect of such shunting on subthreshold properties of subicular pyramidal neurons (Figs 4, 6 and 7). Although subthreshold oscillations cannot impact neuronal output directly in the form of altered rate or timing of suprathreshold action potentials, their presence may be critical for controlling the input–output relationship of neurons. Bursting in subicular neurons is not due to Ih (van Welie et al., 2006), but subthreshold membrane potential modulation through Ih (membrane resonance) can reinforce rhythmic theta field activity (Boehlen et al., 2009; Jackson et al., 2011; Yeung et al., 2013). The strength of such theta activity can be precisely tuned with activation of tonic inhibition with varying levels of ambient GABA. Interestingly, genetic deletion of the primary HCN channel type 1 (HCN1) induces compensatory changes by increasing the expression of a5-containing GABAA receptors in cortical pyramidal neurons (Chen et al., 2010). This homeostatically maintains the synaptic integration property of the pyramidal neurons (Chen et al., 2010). Moreover, mice lacking the a5 subunit gene (Gabra5
/
) express decreased levels of HCN1, bringing about the required homeostasis in the knockout mice (Bonin et al., 2013). These reports suggest that intricate mechanisms involving Itonic and Ih exist to regulate sub-threshold properties such as the resting membrane potential and physiological input–output function of the neuron. A higher density of HCN1 channels in the subiculum with respect to other pyramidal neurons in CA1/CA3 (Lorincz et al., 2002) along with our observations on tonic inhibition regulating Ih-mediated resonance strength reported here suggest that Ih and Itonic might together play a fundamental role in theta oscillations (Boehlen et al., 2009; Yeung et al., 2013). Future studies may reveal the precise role of these conductances in hippocampal oscillatory activity and behaviour. Tonic inhibition GABAA receptors contribute significantly to generate and maintain network synchrony (Mann & Paulsen, 2007). Synaptic and extrasynaptic GABAA receptors have different subunit composition and kinetic properties (Banks & Pearce, 2000). Simultaneous opening of a significant number of GABA gated Cl
channels alter the input conductance and membrane time constant, thus leading to shunting inhibition (Belelli et al., 2009; Pavlov et al., 2009; Song et al., 2011). This shunting effect lasts until the chloride channels are open irrespective of the direction of the chloride current. Extrasynaptic GABAA receptors have slow inactivation kinetics (Banks & Pearce, 2000) and hence induce a longer shunting inhibition, whereas in phasic inhibition it is transitory (Cossart et al., 2005). Phasic and tonic inhibition work together to influence neuronal excitability, as demonstrated in in vitro (Stell et al., 2003; Pavlov et al., 2009) and in vivo (Chadderton et al., 2004) experiments. In the present study, we found bimodal G–V characteristics of tonic current that can have distinct effects on subthreshold and suprathreshold signals (Fig. 5). We have demonstrated the influence of increasing tonic inhibition on subthreshold oscillatory properties of subicular pyramidal neurons (Figs 4E, 6E and 7). Finely tuned modulation of resonance strength with a minor change in the resonance frequency preserves the frequency selectivity of the neuron.

A significant population of subicular projection neurons are of burst firing type (Mattia et al., 1993; Taube, 1993). This and recurrent connections with nearby regions makes the subiculum a potential site for seizure generation and maintenance (Tamamaki & Nojyo, 1995; de la Prida et al., 2006). In temporal lobe epilepsy (TLE) patients and in animal models of TLE, neuronal death in the subiculum is lower than in other regions of the hippocampus (Fisher et al., 1998; Dawodu & Thom, 2005; Cross & Cavazos, 2007). Studies on the cellular mechanisms associated with these observations are scarce. Work from our laboratory suggests the up-regulation of Ca2+ transporter mechanisms (Srinivas & Sikdar, 2008) and increased inhibitory synaptic inputs (Sah & Sikdar, 2013) that may dampen hyperexcitability in these neurons. Quantitative pharmacological analysis performed on subicular GABAA receptors from chronic epilepsy models revealed a significant increase in GABAA receptor-mediated tonic inhibition (Biagini et al., 2010). Prolonged opening of high-affinity extrasynaptic GABAA receptors induces a greater shunting effect that can control the excitability of subicular pyramidal neurons and suppresses epileptiform activity in this region. In the present work, we show the significant presence of tonic inhibition in the subicular pyramidal neurons without any epileptogenic stimulus, suggesting the functional existence of high-affinity extrasynaptic GABAA receptors (Figs. 1 and 2). These active extrasynaptic GABAA receptors might predispose subicular pyramidal neurons to resist hyperexcitatory inputs through a shunting mechanism by modulating subthreshold electrophysiological properties.

Conclusion The subunit composition of GABAA receptors that underlie the tonic current in the subicular pyramidal neurons has not been determined using an electrophysiological and pharmacological approach. In the present study, we sought to study the presence of tonic inhibition, its pharmacological sensitivity to modulators selective for different GABAA receptor subunits, and its effect on subthreshold properties. Our data confirm a role for a5 GABAA receptors in the generation of tonic conductance besides b and c subunits, and demonstrate that tonic conductance can dynamically regulate resonance strength, sag and input resistance attributable to the additional presence of HCN channels. The influence of tonic conductance on various subthreshold properties can have implications on oscillations that are involved in learning and memory, spatial navigation and pathological conditions such as epilepsy.

Acknowledgements This study was partly funded by the Department of Science and Technology, Mathematical Biology programme at the Indian Institute of Science (SR/S4/ MS419/07) and the DBT-IISc Partnership programme. N.S. is a recipient of a senior research fellowship from the Council of Scientific and Industrial Research, Government of India. We thank J. C. Bettencourt, R. J. Lin and Tilman Kispersky for their help with RTXI. We thank Taro Ishikawa for excellent help with IGOR. Both N.S. and S.K.S. approved the final version of the manuscript for publication and neither of them has any conflict of interest to disclose.

Abbreviations ACSF, artificial cerebrospinal fluid; AP5, DL-2-amino-5-phosphonopentanoic acid; CGP 55845, (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid hydrochloride; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; FFT, fast Fourier transform; HCN, hyperpolarizationactivated cyclic nucleotide-gated; QX-314, N-(2,6-dimethylphenyl-carbamoylmethyl) triethylammonium bromide; THDOC, allotetrahydrodeoxycorticosterone; THIP, 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol; TLE, temporal lobe epilepsy.

© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 40, 2241–2254

Tonic inhibition modulates subthreshold properties of subicular neurons 2253

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