NEUROSCIENCE

Layer- and cell-type-specific tonic GABAergic inhibitionof pyramidal neurons in the rat visual cortex

Hyun-Jong Jang &Kwang-Hyun Cho &Myung-Jun Kim &

Shin Hee Yoon & Duck-Joo Rhie

Received: 25 April 2013 /Revised: 11 June 2013 /Accepted: 12 June 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Tonic inhibition mediated by persistent activationof +-aminobutyric acidA (GABAA) receptors by ambientGABA plays a crucial role in the regulation of networkexcitability and neuronal signal processing. Varying degreesin the strength of tonic inhibition were detected across differ-ent cell types throughout the brain. Since sensory informationflows through cortical layers in a specific order, the character-istics of tonic inhibition in different cortical layers are ofinterest. Therefore, we examined the properties of tonic inhi-bition in pyramidal neurons (PyNs) throughout the rat visualcortex. Layer 2/3 PyNs and burst-spiking PyNs in layers 5 and6 showed prominent tonic GABAA currents. Tonic GABAA

currents in layer 4 star PyNs and regular-spiking PyNs inlayers 5 and 6 were much weaker. The magnitude of toniccurrents correlated well with the inhibition of spike genera-tion. The amplitude of tonic GABAA currents measured withbicuculline and gabazine, the two different GABAA receptorblockers, did not differ. The differences in the expressionlevels of extrasynaptic GABAA receptors might be the majorcontributor to the differences in tonic GABAA currents amongcell types. Furthermore, α5 subunits might contribute signif-icantly to tonic currents in infragranular burst-spiking PyNs,especially in layer 5. These results suggest that ambientGABA might exert differential effects on the neuronal inte-gration in a layer- and cell-type-specific manner and thus

contribute to the processing of sensory properties by selec-tively tuning the signals flowing through the visual cortex.

Keywords Ambient GABA . Extrasynaptic GABAreceptor . GABA receptor subunit . Burst-spiking

Introduction

+-Aminobutyric acid (GABA), a major inhibitory neuro-transmitter, reduces excitability and controls the outputof postsynaptic neurons in the central nervous system.GABAergic neurotransmission exerts diverse roles in corti-cal functions, such as network excitability, oscillation, plastic-ity, and sensory development [38]. In addition to the phasicinhibition mediated by GABA released to the synaptic cleft,synaptically or nonsynaptically released ambient GABA alsoacts on extrasynaptic GABAA receptors, exerting tonic inhi-bition [9]. Tonic GABAA currents have been reported ingranule cells of the cerebellum [2, 17], and the dentate gyrus[33, 39], where the δ subunit associated with the α6 and α4subunits, respectively, is responsible for the tonic currents.The α4 and δ subunits also mediate tonic inhibition inthalamocortical neurons [5]. However, in some neuronal typesof the hippocampus and the somatosensory cortex, the α5subunit appears to make a major contribution to tonic GABAA

currents [3, 12, 46]. These regional differences in subunitcomposition might be important in modulation by endoge-nous modulators and exogenously applied drugs.

Excitatory pyramidal neurons (PyNs) of the neocortexreceive inputs from, and send outputs to, the correspondingbrain regions in a layer-specific manner. Moreover, PyNs inlayers 5 and 6 also exhibit cell-type-specific connectivity withdifferent brain regions. Layer-specific expression of receptorsand neuromodulators is one of the responsible factors in manycortical functions [20, 26, 43]. Tonic GABAA current densityin layer 5 neurons is higher than in layer 2/3 neurons of the rat

Electronic supplementary material The online version of this article(doi:10.1007/s00424-013-1313-1) contains supplementary material,which is available to authorized users.

H.<J. Jang :K.<H. Cho :M.<J. Kim : S. H. Yoon :D.<J. Rhie (*)Department of Physiology, College of Medicine,The Catholic University of Korea, 222 Banpo-daero,Seocho-gu, Seoul 137-701, Republic of Koreae-mail: djrhie@catholic.ac.kr

H.<J. Jang :K.<H. Cho : S. H. Yoon :D.<J. RhieCatholic Neuroscience Institute, The Catholic University of Korea,Seoul 137-701, Republic of Korea

Pflugers Arch - Eur J PhysiolDOI 10.1007/s00424-013-1313-1

somatosensory cortex [46]. There are also cellular differencesin tonic inhibition between PyNs and inhibitory interneuronsin the CA1 area of the hippocampus [36] and the neocortex[44]. Since tonic GABAA inhibition substantially affects theintegration of inputs and modulates the output of the neurons[16] and cortical functions [14, 28], systematic investigationof tonic GABAA currents in different layers of the neocortexshould provide information essential to the understanding ofcortical information processing.

Here, we investigated layer-specific tonic GABAA currentsin excitatory PyNs of the primary visual cortex of the rat.Tonic GABAA currents are relatively large in burst-spikingPyNs in layers 5 and 6 and are substantial in layer 2/3 PyNs,while they are small in layer 4 PyNs and in regular-spikingPyNs in layers 5 and 6. Furthermore, the major part of tonicGABAA currents was likely mediated byα5 subunits in burst-spiking PyNs in layer 5, but not in layer 2/3 PyNs. Themagnitude of tonic currents correlated well with the inhibitionof spike generation. Thus, ambient GABA might exert differ-ential effects on the cortical information processing in a layer-and cell-type-specific manner.

Materials and methods

Slice preparation

Coronal slices of visual cortexwere prepared from 5-week-oldSprague–Dawley rats of either sex (Orientbio Inc., Seoul,Korea), raised under standard conditions (23±1 °C, 12/12 hlight/dark cycle). Animal care and surgical procedures wereapproved by the Ethics Committee of the Catholic Universityof Korea and were consistent with the National Institutes ofHealth Guide for the Care and Use of Laboratory Animals.The animals were sedated with chloral hydrate (400 mg/kg,i.p.) and decapitated after the disappearance of the tail pinchreflex. The brains were quickly removed to ice-cold dissectionmedium, consisting of (in mM) 125 NaCl, 2.5 KCl, 1 CaCl2, 2MgSO4, 1.25 NaH2PO4, 25 NaHCO3, and 10 D-glucose,bubbled with 95 % O2/5 % CO2, and 300-μm-thick coronalslices of occipital cortex were prepared on a vibrotome(Campden Instruments, Leics, UK). Slices were kept in asubmerged-type chamber for 40 min at 37 °C in the dissec-tion solution for recovery from damage associated withslicing and then stored at room temperature in the samechamber and solution until use.

Whole-cell patch-clamp recording

The slices were perfused (1.5–2 ml/min) with carbogenatedartificial cerebrospinal fluid (ACSF) in a recording chamberat 32–33 °C. ACSF consisted of (in mM) 125 NaCl, 2.5 KCl,2 CaCl2, 1 MgSO4, 1.25 NaH2PO4, 25 NaHCO3, and 10 D-

glucose. Cells were identified under infrared-DIC video-microscopy with an upright microscope (BX50-WI fittedwith a 40×/0.80 NA water immersion objective, Olympus,Tokyo, Japan). Layer discrimination was based on the ratbrain atlas [40] and pyramid-shaped cells with a prominentapical dendrite toward the pial surface were mainly targeted.The whole-cell recording was obtained with an EPC8 am-plifier (HEKA Elektronik, Lambrecht, Germany) andpClamp 9.0 software (Axon Instruments, Foster City, CA,USA). Data were low-pass filtered at 5 kHz and sampled ateither 10 or 20 kHz. Basic properties of neurons in differentlayers were studied using K-gluconate-based pipette solutioncontaining (in mM) 130 K-gluconate, 10 KCl, 4 Mg-ATP, 10Na2-phosphocreatine, 0.3 Na3-GTP, and 10 HEPES (pH 7.25with KOH). Typical pipette and access resistances were 3–5and 15–20 MΩ, respectively. Membrane potentials were notcorrected for an approximate 14 mV junction potential. Inputresistance (Rin) and membrane time constants were mea-sured by applying −50 pA of square current (200 ms).Hyperpolarizign sag ratio was calculated for cells in layers 5and 6 as the ratio between the peak deflection and the steady-state deflection by negative current injection (−50 pA,500 ms). Action potential (AP) discharge was evoked by agraded step-current injection (20 pA, 1 s). AP parameters weremeasured at the first AP, with minimal current injection. TheAP amplitude was measured from the AP threshold tothe peak. The width of AP was measured at its halfamplitude. Afterhyperpolarization (AHP) was estimatedfrom the AP threshold to the negative peak of the AHPand peak–trough (P-T) time was measured as the timebetween the AP peak and the trough nadir. AP adaptationwas calculated by dividing the fifth interspike interval bythe third one. In bursting cells, APs in the burst wereexcluded.

Measurement of tonic inhibition

Tonic inhibition was studied with a CsCl-based pipettesolution containing (in mM) 145 CsCl, 4 Mg-ATP, 10Na2-phosphocreatine, 0.3 Na3-GTP, 10 HEPES, and 3QX-314 (pH 7.25 with CsOH). The alpha-amino-3-hy-droxy-5-methyl-4-isoxazolepropionic acid receptor antag-onist 6,7-dinitroquinoxaline-2,3-dione (DNQX, 20 μM),the N-methyl-D-aspartic acid receptor antagonist D-(−)-2-amino-5-phosphonopentanoic acid (D-AP5, 50 μM), and theGABAB receptor antagonist CGP 52432 (1 μM) were addedto ACSF in order to isolate GABAA receptor-mediated cur-rent. The sodium channel blocker tetrodotoxin was not ap-plied. After whole-cell current stabilized at a holding potentialof −70 mV, the GABAA receptor antagonist bicuculline(10 μM) was applied to block tonic inhibition. The amplitudeof the tonic GABAA current was analyzed as the differencebetween the holding currents measured before and after the

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application of bicuculline. The holding current was calculatedfrom 100 ms epochs, containing no obvious spontaneoussynaptic events, taken every 4 s over an 80-s period. Mem-brane capacitance was estimated from the capacitance com-pensation function of the EPC8 amplifier. Tonic current den-sity was calculated by dividing the tonic current by the mem-brane capacitance. To enhance tonic inhibition, the endoge-nous neurosteroid tetrahydrodeoxycorticosterone (3α,21-dihydroxy-5α-pregnan-20-one; THDOC, 500 nM) or GABA(5 μM) was added to the bath solution. The effect of the bathapplication of the α5 subunit-specific inverse agonist L-655,708 (100 nM) was also investigated.

The effects of THDOC and bicuculline on spiking fre-quency were studied with a K-gluconate-based pipette solu-tion. First, spiking was measured with a graded step-currentinjection. Then, the effects of THDOC and bicuculline weremeasured at the end of 7-min applications.

Confocal reconstruction of recorded cells

In about 70 % of cells tested, biocytin (0.5 %) was includedin the pipette solution for the morphological reconstructionof recorded cells. After electrophysiological recording, brainslices were fixed with 4 % paraformaldehyde in 100 mMsodium phosphate buffer (pH 7.4) for 2 h and maintained in10 mM sodium phosphate-buffered saline (PBS) at 4 °Cbefore treatment with avidin. After overnight treatmentwith 0.5 % Triton X-100 in PBS to permeabilize cell mem-branes at 4 °C, the slices were reacted with 1 μg/mL ofstreptavidin (Alexa Fluor 488 conjugate, Invitrogen) atroom temperature for 2 h and then washed with PBS. Theslices were rinsed and mounted on glass slides with mount-ing medium (DakoCytomation, Carpinteria, CA, USA).The distribution of biocytin in the recorded cell was imagedwith confocal microscopy (FV-300, Olympus). Neuronalstructures were reconstructed from z-series stacks collectedat 0.5–1-μm increments.

Drugs

DNQX, D-AP5, bicuculline, gabazine (SR 95531), L-655,708,and CGP 52432 were purchased from Tocris (Bristol, UK).The other chemicals were purchased from Sigma (St. Louis,MO, USA).

Statistics

Data were expressed as the mean±SE. Statistical compari-sons were performed using a paired or unpaired two-tailedStudent’s t test, unless otherwise specified. One-wayANOVA followed by the Tukey’s post hoc test was also usedfor multigroup comparisons. The level of significance wasset at p<0.05.

Results

In our previous study, tonic GABAA receptor-medated cur-rents increased to a stable level at 5 weeks of age in layer 2/3PyNs [15]. Thus, in the present study, we investigated tonicGABAA receptor-mediated currents in 5-week-old rats.

Characterization of excitatory neurons

We initially examined the passive and active properties ofthe recorded neurons with K-gluconate-based pipette solu-tion in whole-cell current-clamp mode (Table 1). We alsoreconstructed the recorded neurons filled with biocytin(Fig. 1). Dendritic arbors were mainly reconstructed, whichmight be due to the short recording time in the presentexperimental condition. Layer 2/3 and 4 PyNs were lessvariable in the passive parameters and spiking patternsgenerated by somatic current injection. Layer 2/3 PyNsshowed a short apical trunk and widespread terminal arborin layer 1 with many fine basal dendrites [23]. PyNs in layer4 showed a short apical dendrite terminating in layer 2/3and many radiating short dendrites, largely in layer 4,characteristic dendritic structure of the star PyN [29]. Bycontrast, in layer 5, two distinct types of PyNs were iden-tified based on the shape and electrophysiological re-sponses [18]. One type of neurons showed a largepyramid-shaped cell body and a thick apical trunk on theinfrared-DIC monitor. These cells generated an initial burstfollowed by regular spiking at a low frequency with somat-ic current injection, indicating thick-tufted PyNs projectingtheir axons to the superior colliculus and the pons [30].Apical dendrites of these cells reached layer 1 with a wide-spread terminal arbor. The other type of cells had smaller,round soma with a less identifiable narrow apical trunk,indicating slender PyNs projecting their axons mainly tothe contralateral hemisphere. These cells showed regularspiking without burst firing with current injection, and hadhigher Rin than the thick-tufted PyNs. The cells in layer 6could also be divided into two main groups according totheir initial bursting or regular spiking with current injec-tion. The two cell types could be distinguished based on thedifferences in their dendritic morphologies. The burstingtype consisted of PyNs having apical dendrites reachingonly to layer 4 or the lower border of layer 2/3 with apaucity of apical tufts. The regular-spiking type showedshort apical dendrites that never exceeded layer 4 and manyperpendicular branches in an apical trunk confined to layer5 or 6. The most distinguishing difference in the dendriticmorphologies of these two cell types in layer 6 was thenumber of perpendicular branches in the proximal apicaltrunk. Regular-spiking PyNs had more than 8 main perpen-dicular branches, and burst-spiking PyNs had <8. Somecells showed intermediate characteristics with an initial

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short burst of only two spikes (data not shown). We did notinclude these cells in the present study. The electrophysio-logical properties of each cell type are summarized inTable 1. Cluster analysis based on the somatic locationand electrophysiological parameters showed well-suitedsegregation of each cell type with a few exceptions (Sup-plementary Figure 1).

Tonic GABAA currents in layer 2/3 and 4 PyNs

Tonic GABAA currents were measured by the application ofbicuculline (10 μM). Layer 2/3 PyNs showed a moderateamplitude of tonic GABAA currents (12.4±1.5 pA, n=10)and current density (0.16±0.02 pA/pF) (Fig. 2, a1 and a2).The experimental conditions using brain slices may wash outextracellular GABA. Thus, we tested the effect of the bathapplication of GABA (5 μM), which increased tonic GABAA

currents to 18.4±2.4 pA (n=8). To test the functional impli-cations of tonic GABAA currents, we applied a high concen-tration of THDOC (500 nM), which nonselectively potentiatesextrasynaptic GABAA currents, since 100 nM of THDOC didnot enhance tonic GABAA currents (14.88±1.67 pA, n=7,p=0.31 vs. control). THDOC in the bath increased the toniccurrents to ∼230 % of the control amplitude (29.0±2.6 pA,n=10, p<0.001). The effects of tonic inhibition on cell firingpatterns were investigated in current-clamp mode with a K-gluconate-based internal solution. Rin was decreased with theapplication of THDOC (control, 69.4±3.9 MΩ; THDOC,59.7±3.8 MΩ; n=8, p<0.01; Fig. 2, a4). The frequency ofspikes generated by step current injection was also decreased

with the application of THDOC (22.2 % at 500 pA stepcurrent; Fig. 2, a3 and a4). The subsequent application ofbicuculline reversed these decreases in Rin (75.6±4.0 MΩ)and spike frequency. Thus, an increase in tonic GABAA

currents significantly modulated the integrating properties oflayer 2/3 PyNs.

Layer 4 PyNs showed a significantly low amplitude for thetonic GABAA currents (5.5±0.6 pA, n=7, p<0.01) and cur-rent density (0.10±0.01 pA/pF, p<0.05) compared to layer2/3 PyNs (Fig. 2, b1 and b2). Exogenous GABA and THDOCincreased tonic currents (8.2±1.0 pA, n=8, p=0.054; 9.6±1.2pA, n=8, p<0.05; respectively), but the increases were muchsmaller than in layer 2/3 PyNs with the same treatment(p<0.01, p<0.001, respectively). In accordance with this,the change in Rin (control, 89.6±4.2 MΩ, n=7; THDOC,84.7±4.4 MΩ; p=0.08) and the slowing of spike frequencieswith the application of THDOC (7.8% at 500 pA step current)were slight (Fig. 2, b3 and b4). The small amplitude of tonicGABAA currents even in the presence of exogenous GABAsuggests that the contribution of tonic GABAA currents to theintegrating properties of layer 4 PyNs may be minimal.

Tonic GABAA currents in layer 5 PyNs

The two types of neurons in layer 5 were easily identifiedwith the morphological characteristics of the soma and theproximal apical trunk under the infrared-DIC microscopy,and the shapes were confirmed in the reconstruction afterrecording without exception (Fig. 1). Thick-tufted PyNsgenerated an initial burst discharge with current injection,

Table 1 Electrophysiological properties of pyramidal neurons (PyNs) in the visual cortex

L2/3 (n=11) L4 (n=12) L5 BS (n=10) L5 RS (n=13) L6 BS (n=7) L6 RS (n=10)

RMP (mV) −71.8±0.6bcdef −67.8±0.6a −65.6±0.6a −66.7±0.6a −67.0±0.6a −66.6±0.7a

Rin (MΩ) 63.8±3.5def 91.8±8.1 62.8±3.4def 109.3±9.7ac 108.7±8.0ac 90.0±4.3ac

tmemb (ms) 5.78±0.36d 5.54±0.39cd 7.71±0.36bf 8.69±0.75abf 6.66±0.92 4.86±0.17cd

Sag ratiog NA NA 1.11±0.01df 1.05±0.01c 1.06±0.02 1.05±0.01c

AP threshold (mV) −40.1±1.3 −40.6±1.0 −43.4±0.8 −43.1±0.7 −40.4±1.1 −38.8±1.8

AP amplitude (mV) 91.8±1.6 87.6±2.1 89.2±1.4 87.9±1.6 85.2±1.7 86.6±1.7

AP width (ms) 0.88±0.02bcdf 0.71±0.02a 0.69±0.02a 0.75±0.03a 0.82±0.02 0.73±0.01a

AHP (mV) 10.2±0.4 10.4±0.8 8.9±0.5 10.4±1.0 9.7±0.5 8.3±0.7

P-T time (ms) 36.1±1.1 31.6±2.6c 45.9±3.3b 44.5±4.6 43.7±4.2 33.0±4.7

Spike adaptation 1.16±0.03 1.30±0.06cdef 1.05±0.01b 1.06±0.02b 1.04±0.02b 1.06±0.03b

a ANOVAwith Tukey's post hoc test (p<0.05) vs. L2/3b ANOVAwith Tukey's post hoc test (p<0.05) vs. L4c ANOVAwith Tukey's post hoc test (p<0.05) vs. L5 BSdANOVAwith Tukey's post hoc test (p<0.05) vs. L5 RSeANOVAwith Tukey's post hoc test (p<0.05) vs. L6 BSf ANOVAwith Tukey's post hoc test (p<0.05) vs. L6 RSg Sag ratio was not calculated in L2/3 and L4 PyNs, since hyperpolarizing voltage sag was not noticeable.

BS burst-spiking, RS regular-spiking, NA not applicable

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which was followed by regular spiking, as shown in a previousstudy [18]. These bursting neurons showed sIPSCs with highfrequency (20.6±2.1 Hz, n=10) and amplitude (39.1±3.6 pA).They also showed a large amplitude of tonic GABAA currents(15.1±1.7 pA, n=10) and current density (0.13±0.01 pA/pF)(Fig. 3, a1 and a2). Exogenous GABA and THDOC signifi-cantly increased the tonic currents (25.7±1.6 pA, n=8,p<0.001 and 35.6±2.7 pA, n=8, p<0.001, respectively). Thefrequency of spikes was decreased by the application ofTHDOC (17.6 % at 500 pA step current, n=9), in accordancewith the decrease in Rin (control, 52.6±3.2 MΩ, n=7;THDOC, 45.6±3.4 MΩ; p<0.05; Fig. 3, a3 and a4). In somecells, burst spike numbers were also affected by THDOC (fourout of nine cells; Fig. 3, a3, insets). On the other hand, regular-spiking slender PyNs showed sIPSCs at a lower frequency(9.9±1.4 Hz, n=9, p<0.001) and amplitude (27.1±1.3 pA,p<0.01). These neurons showed a very low amplitude for thetonic GABAA currents (4.5±0.6 pA, n=9) and current density(0.06±0.01 pA/pF; Fig. 3, b1 and b2). Although exogenousGABA increased the tonic GABAA currents to ∼150 % of thecontrol, the current amplitude remained low (6.8±1.3 pA,n=9). The increase in the amplitude of tonic GABAA currentswith the application of THDOC was also not high enough

(9.9±1.1 pA, n=9) to change Rin (control, 108.65±6.1 MΩ,n=8; THDOC, 102.78±7.3 MΩ; p=0.09) or to decrease thespiking frequency (5.5 % at 500 pA step current) (Fig. 3, b3and b4).

Tonic GABAA currents in layer 6 PyNs

We initially targeted cells having pyramid-shaped soma andidentifiable apical dendrites in layer 6. Layer 6 PyNs were alsoeither burst-spiking or regular-spiking. Since morphologicdiscrimination of the two cell types was not feasible underinfrared-DIC microscopy and firing patterns could not beobtained with the CsCl-based internal solution, cell types weredetermined by reconstruction after the experiments. Cells witha strong sIPSC (43.3±3.1 pA, n=9), large tonic GABAA

currents (18.3±1.8 pA) and current density (0.23±0.02pA/pF)resembled burst-spiking PyNs that have relatively long apicaldendrites with few perpendicular branches and no terminaltufts. However, the cells with a weak sIPSC (25.2±1.0 pA,n=8), small tonic GABAA currents (5.9±1.4 pA) and currentdensity (0.07±0.02 pA/pF) showed apical dendrites extendingonly up to layer 4, with many perpendicular dendrites in layers5 and 6, which corresponded to regular-spiking PyNs (Fig. 1).

Fig. 1 Confocal reconstruction of pyramidal neurons (PyNs) in differentlayers of the rat visual cortex. B/W level of fluorescence intensity wasinverted. The position of each cell in the figure is based on the distancefrom the pial surface. Response to hyperpolarizing current injection and

AP firing pattern are also illustrated for each cell. a Layer 2/3. b Layer 4star. c Layer 5 burst-spiking, thick tufted. d Layer 5 regular-spikingslender. e Layer 6 burst-spiking. f Layer 6 regular-spiking PyNs. g Layer6 bipolar neuron. WM white matter

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Fig. 3 Tonic GABAA currents and their effects on cell firing in layer 5burst-spiking (L5 BS) and regular-spiking PyNs (L5 RS). a1, b1 Repre-sentative current traces with a control solution, with GABA and withTHDOC. a2, b2 Individual data (open circle) and averages (thick line) oftonic GABAA currents and the corresponding current density. a3, b3

Representative voltage responses to square current injection of 200 pAwith control solution, with the addition of THDOC and with the additionof bicuculline. Insets in a3 show the initial bursts with an extended timescale. a4, b4 Rin and the frequency of AP firing at each of the injectedcurrent amplitudes. *p<0.05, **p<0.01, and ***p<0.001 vs. control

Fig. 2 Tonic GABAA currents and their effects on cell firing in layer 2/3 (L2/3) and layer 4 PyNs (L4). Tonic GABAA currents were measuredin the ACSF solution (control) and in the presence of GABA (5 μM) orTHDOC (500 nM) with CsCl-based pipette solution. Changes of inputresistance (Rin) and firing frequency with the sequential application ofTHDOC and bicuculline (10 μM) were estimated by square currentinjection with K-gluconate-based internal solution. a1, b1 Representa-tive current traces with control ACSF solution, with GABA and with

THDOC. Some sIPSCs with high amplitude were truncated to clearlyshow the tonic GABAA currents. a2, b2 Individual data (open circle)and averages (thick line) of tonic GABAA currents and the correspond-ing current density. a3, b3 Representative voltage responses to squarecurrent injection of 300 pA (a3) and 200 pA (b3) with control solution,with the addition of THDOC and with the addition of bicuculline. a4,b4 Rin and the frequency of AP firing at each of the injected currentamplitudes. *p<0.05, **p<0.01 and ***p<0.001 vs. control

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The amplitude of tonic GABAA currents was sufficiently in-creased by the application of exogenous GABA (26.6±2.0 pA,n=9, p<0.05) and THDOC (32.6±2.5 pA, n=9, p<0.001) inburst-spiking-like PyNs (Fig. 4, a1 and a2). The application ofTHDOC lowered Rin (control, 104.8±5.7 MΩ, n=8; THDOC,91.6±6.2 MΩ; p<0.01) and slowed spiking by the injectedcurrent (16.5 % at 500 pA step current) (Fig. 4, a3 and a4). Thenumber of spikes in the initial burst was decreased in five out ofnine cells (Fig. 4, a3, insets). By contrast, tonic GABAA

currents were minimally increased by the application of GABA(7.6±1.7 pA, n=7) and THDOC (9.3±1.0 pA, n=7) in regular-spiking-like PyNs (Fig. 4, b1 and b2). The application ofTHDOC did not exert a significant effect on the frequency ofspike generation (4.1 % at 500 pA step current) or Rin (control,92.6±5.1 MΩ, n=7; THDOC, 89.5±5.6 MΩ; p=0.12) in thistype of PyN (Fig. 4, b3 and b4).

Layer 6 consisted of diverse populations of excitatoryneurons, including PyNs [42, 47]. We further targeted neuronsthat did not show the shape of typical PyNs under infrared-DIC microscopy. Cells with bipolar soma unequivocallyexhibited an initial burst and were confirmed as bipolar cellsin the reconstruction of dendritic structures (n=5), which hadthe dendritic process extending into the white matter (Fig. 1).The bipolar cells exhibited a high amplitude of tonic GABAA

currents (17.5±1.9 pA, n=6), similar to those of burst-spik-ing-like PyNs (p=0.76). These neurons also showed sIPSCs

of a large amplitude (39.8±3.5 pA). Thus, our results suggestthat burst-firing cells in layer 6 exhibit stronger sIPSC andhigher tonic GABAA currents than regular-spiking PyNs,regardless of their diverse dendritic morphologies.

Tonic currents investigated with other GABAA receptorblockers

Although bicuculline could possibly block more compo-nents of tonic GABAA currents than gabazine [11, 31], onecaveat of using bicuculline might be that it may also inhibitSK channels [19]. Thus, we investigated the effect ofgabazine on tonic currents in neurons exhibiting prominenttonic currents: layer 2/3, layer 5 burst-spiking, and layer 6burst-spiking-like PyNs (Fig. 5a, c). Gabazine (10 μM) de-creased the holding currents in layer 2/3 (11.28±1.74 pA,n=9), in layer 5 burst-spiking (13.62±1.51 pA, n=9) and inlayer 6 burst-spiking-like PyNs (16.18±1.68 pA, n=7) to asimilar extents to that observed with bicuculline (p=0.631,p=0.543, and p=0.446, respectively). The effect of gabazineon firing frequencies in layer 2/3 and 5 burst-spiking PyNswere also comparable to that of bicuculline (see Supplemen-tary Figure 2). We further analyzed the changes in the prop-erties of AP and AHP with the application of bicuculline todetermine the possible involvement of the SK channel inhi-bition by bicuculline. In layer 2/3 PyNs, AP threshold, AP

Fig. 4 Tonic GABAA currents and their effects on cell firing in layer 6burst-spiking (L6 BS) and regular-spiking PyNs (L6 RS). a1, b1 Repre-sentative current traces with a control solution, with GABA and withTHDOC. a2, b2 Individual data (open circle) and averages (thick line) oftonic GABAA currents and the corresponding current density. a3, b3

Representative voltage responses to square current injection of 200 pAwith control solution, with the addition of THDOC and with the additionof bicuculline. Insets in a3 show the initial bursts with an extended timescale. a4, b4 Rin and the frequency of AP firing at each of the injectedcurrent amplitudes. *p<0.05, **p<0.01, and ***p<0.001 vs. control

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amplitude, AP width, AHP amplitude, and P-T time were notsignificantly changed by the application of bicuculline, withTHDOC in the bath (n=8, data not shown). In layer 5 burst-spiking PyNs, only AHP amplitude was slightly diminishedby bicuculline (9.8±0.2 mV to 8.9±0.5 mV, n=7, p=0.04).These results suggest that, if any, the SK channel inhibition bybicuculline did not significantly alter the assessment of theeffect of tonic GABAA currents in the regulation of cell firingproperties. Thus, at least in the rat visual cortex, bicucullineand gabazine at a concentration of 10 μM showed no signif-icant differences in the study of tonic inhibition.

Previous reports addressed the differences in the subunitcomposition of GABAA receptors mediating tonic currentsbetween the superficial and the deep layers of the neocortex,especially between layers 2/3 and 5 [7, 46]. We investigatedthe differences in layers 2/3, 5, and 6 with the α5 subunit-specific inverse agonist L-655,708 (Fig. 5b, d). In layer 2/3PyNs, the application of L-655,708 (100 nM) had no effect onholding currents (1.28±2.01 pA, n=6, p=0.552), sIPSC fre-quency (17.01±0.64 pA to 17.84±0.89 pA, n=6, p=0.107),and amplitude (37.07±3.51 pA to 36.90±2.89 pA, p=0.788).However, L-655,708 significantly decreased holding currentsin layer 5 burst-spiking PyNs (11.15±1.49 pA, n=10,p<0.001), which was about 74.2 % of the tonic currentsmeasured with bicuculline (Fig. 3, a2). In these cells, L-655,708 did not change the sIPSC frequency (19.11±1.63 Hzto 20.12±1.31 Hz, n=10, p=0.141), but it slightly decreasedthe sIPSC amplitude (35.39±2.06 pA to 34.03±1.87 pA,p<0.05). The decrease in amplitude of sIPSC by L-655,708

was minimal (3.9 %) compared to that in tonic currents(74.2 %). In layer 6 burst-spiking-like PyNs, L-655,708 alsosignificantly decreased the holding currents (6.33±1.73 pA,n=7, p<0.01). However, the tonic currents revealed by L-655,708were only about 28.9% of the tonic currents measuredwith bicuculline (Fig. 4, a2). These results suggest that theGABAA receptors containing the α5 subunit almost exclusive-ly participate in the tonic inhibition of PyNs in the deep layersof the visual cortex and the expression level of the subunitdiffers depending on the neuron types.

Effect of enhanced ambient GABA

Despite layer- and cell-type-specific differences in the am-plitude of tonic GABAA currents among PyNs in the presentstudy, exogenous GABA (5 μM) enhanced tonic inhibitionto ∼150 % of the control amplitude in all the cell typesstudied. Since tonic GABAA currents are mediated by en-dogenously released ambient GABA, the removal of re-leased GABA from the extracellular space critically affectstonic GABAA currents. Thus, we investigated the effect ofthe GABA transporter 1 (GAT-1) inhibitor, NO-711, on tonicGABAA currents (Fig. 6). In layer 2/3 PyNs, tonic GABAA

currents were enhanced by NO-711 (5 μM) to 19.6±2.5 pA(n=7, p<0.05 compared to control). Tonic GABAA currentswere also enhanced by NO-711 in layer 5 burst-spiking PyNs(24.7±2.3 pA, n=7, p<0.01 compared to control). The am-plitudes of tonic currents in the presence of NO-711 weresimilar to those with 5 μMof exogenous GABA in both layer

Fig. 5 Tonic GABAA currents investigated with the GABAA receptorantagonist gabazine and the α5 subunit-specific inverse agonist L-655,708. a Representative current traces showing the effect of gabazine(10 μM) on the holding current for layer 2/3 (L2/3), layer 5 burst-spiking(L5 BS) and layer 6 burst-spiking PyNs (L6 BS). b Representative currenttraces showing the effect of L-655,708 (100 nM) on the holding current

for L2/3, L5 BS, and L6 BS. c Individual data (open circle) and averages(thick line) of tonic GABAA currents investigated with gabazine. dIndividual data and averages of tonic GABAA currents investigated withL-655,708. *p<0.05 between groups linked by line by ANOVA withTukey's post hoc test

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2/3 and 5 PyNs (p=0.74, p=0.71, respectively). These resultssuggest that the ambient GABA level might be criticallyregulated by GAT-1 activity, and thus, tonic GABAA currentscould change depending on the activity of GAT-1.

Characteristics of tonic GABAA currents in neuronsin different layers

Tonic GABAA currents, current density, amplitude of sIPSC,and the charges carried by tonic currents and sIPSC of all thecell types tested are summarized in Table 2. In layer 5 and 6PyNs, large tonic GABAA currents were accompanied bystrong sIPSCs. To clarify this relationship, we plotted theamplitude of tonic currents against that of sIPSC in each layer(Fig. 7). In the PyNs of layers 2/3 and 4, tonic currents had nocorrelation with the amplitude of sIPSC (r2=0.004, p=0.87 forlayer 2/3; r2=0.014, p=0.79 for layer 4; Fig. 7a, b). However,

tonic currents positively correlated to the amplitude of sIPSCin layer 5 and 6 neurons (r2=0.506, p<0.001 for layer 5;r2=0.638, p<0.001 for layer 6; Fig. 7c, d). The burstingneurons in both layers 5 and 6 showed a strong sIPSC andlarge tonic GABAA currents. To understand the relationshipbetween bursting properties and the strength of inhibitory in-nervations, we measured spiking patterns and sIPSC in layer 6PyNs with a K-gluconate-based internal solution. In this ex-perimental setting, sIPSC was measured at −20 mVof holdingpotential. It is of interest that the number of spikes in the burstshowed correlations with the frequency and amplitude ofsIPSC (Fig. 8). Since the amplitude of sIPSC correlated withthe strength of tonic currents (Fig. 7c, d), bursting propertiesmight also correlate with tonic GABAA currents in theseneurons. Another interesting finding was that the positivecorrelation between the amplitudes of tonic currents and sIPSCwas clearly revealed by the treatment with NO-711 in layer 2/3PyNs (r2=0.577, p<0.05; Fig. 7e) and in layer 5 PyNs(r2=0.593, p<0.05; Fig. 7f). This finding suggests that, inthe case of slowed GABA removal due to low GABA trans-porter activity, the positive relationship between the strength ofinhibitory synaptic transmission and the amplitude of tonicGABAA currents (which might reflect the level of ambientGABA) would be global.

Next, the overall relationship between electrical chargescarried by tonic inhibition and sIPSC was analyzed (Fig. 9a,Table 2). The ratios of charges carried by tonic inhibition andsIPSC (tonic/phasic ratio) were 5.73, 2.75, 2.69, 2.32, 4.32,and 4.69 for layer 2/3, layer 4, layer 5 burst-spiking, layer 5regular-spiking, layer 6 burst-spiking-like and layer 6 regular-spiking-like PyNs, respectively. Thus, tonic GABAA currentsin PyNs of layers 2/3 and 6 exerted a relatively higher inhib-itory influence than synaptic inhibitory currents, compared tothe neurons of layers 4 and 5. Despite these different charac-teristics of tonic inhibition between layers, the changes infiring frequency with the application of THDOC correlated

Fig. 6 Effect of the GABA transporter 1 antagonist NO-711 on the tonicGABAA currents. a Representative current traces showing the effect ofbicuculline on holding current in the presence of NO-711 for layer 2/3(L2/3) and layer 5 burst-spiking PyNs (L5 BS). b Individual data (opencircle) and averages (thick line) of tonic GABAA currents in the presenceof NO-711

Table 2 Summary of inhibitory parameters of PyNs in the visual cortex

L2/3 (n=10) L4 (n=7) L5 BS (n=10) L5 RS (n=9) L6 BS-like (n=9) L6 RS-like (n=8)

Tonic GABAA currents (pA) 12.4±1.5bd 5.5±0.6ace 15.1±1.7bdf 4.5±0.6ace 18.3±1.8bdf 5.9±1.4ce

Tonic current density (pA/pF) 0.16±0.02d 0.10±0.01 0.13±0.01 0.06±0.01ae 0.23±0.02df 0.07±0.02e

sIPSC (pA) 33.1±1.4 38.1±4.7df 39.1±3.6f 27.1±1.3be 43.3±3.1df 25.2±1.0bce

Charge carried by tonic currents (pC/s) 12.4±1.5bd 5.5±0.6ace 15.1±1.7bdf 4.5±0.6ace 18.3±1.8bdf 5.9±1.4ce

Charge carried by sIPSC (pC/s) 2.16±0.25cef 2.02±0.24ce 5.58±0.55abdf 1.94±0.30c 4.24±0.49ab 1.26±0.08ac

a ANOVAwith Tukey's post hoc test (p<0.05) vs. L2/3b ANOVAwith Tukey's post hoc test (p<0.05) vs. L4c ANOVAwith Tukey's post hoc test (p<0.05) vs. L5 BSdANOVAwith Tukey's post hoc test (p<0.05) vs. L5 RSeANOVAwith Tukey's post hoc test (p<0.05) vs. L6 BS-likef ANOVAwith Tukey's post hoc test (p<0.05) vs. L6 RS-like

BS burst-spiking, RS regular-spiking

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well with the tonic current density of each neuron type(r2=0.843, p<0.01; Fig. 9b). Thus, the regulation of firingby tonic inhibition would mainly depend on the level of tonicGABAA currents on the unit surface of the cell membrane.

Discussion

The levels of tonic GABAA currents were layer- and cell-type dependent in the rat visual cortex. The magnitude oftonic GABAA currents correlated with decrease in cell firingrates. Contribution of α5 subunits of GABAA receptor to

tonic currents was also layer specific. Thus, information flowin the visual cortex might be regulated by ambient GABAand by the subunit specific modulators of GABAA receptorsin layer- and cell-type-specific manners.

Layer-specific tonic GABAA currents

In the present study, the cells in each layer showed differentamplitudes of tonic GABAA currents. This variability mightbe the result of differences in the receptor expression levels,the subtypes of extrasynaptic GABAA receptors or the ambi-ent GABA levels. The application of exogenous GABA(5 μM) enhanced tonic GABAA currents to ∼150 % of thecontrol in all cell types in the present study. Since the level ofambient GABA in the visual cortex has been estimated lowerthan 1 μM [8], 5 μM of exogenous GABA could largelycompensate for the differences in the levels of ambient

Fig. 7 Correlation of tonic GABAA currents with sIPSC. The amplitudeof tonic GABAA currents are plotted against the amplitude of sIPSCobtained before the application of bicuculline in layer 2/3 (a), layer 4(b), layer 5 (c), and layer 6 PyNs (d). The same plots in the presence ofNO-711 in layer 2/3 (e) and layer 5 PyNs (f) are shown.Open circles andtriangles represent regular-spiking slender and burst-spiking thick-tuftedPyNs, respectively, in c. Circles and triangles represent regular-spikingand burst-spiking neurons, respectively, in d, where closed symbolsrepresent cells confirmed by reconstruction. Dotted lines are virtualborders that might help to distinguish the two cell types. Lines representline-fitting results in all figures

Fig. 8 Correlation of sIPSC with burst-firing patterns in layer 6 PyNs.Firing was evoked by square current injection in current-clamp modeand sIPSCs were recorded at −20 mV of holding potential in voltage-clamp mode with K-gluconate-based internal solution in a cell. a Theleft traces show the firing patterns of four different layer 6 PyNs. Theinsets show the initial bursts on an extended time scale. The right tracesare the recorded sIPSC in the cells corresponding to the left traces. bIndividual data (open circle) and averages (thick line) of sIPSC fre-quency and amplitude are plotted against spike number of the burst.Lines are the results of a line fitting

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GABA. However, the differences in tonic GABAA currentsbetween cell types were not diminished by the application of5 μM of GABA. Thus, differences in the level of ambientGABA might not be the cause of different tonic GABAA

currents in different cell types in our slice preparation.A majority of extrasynaptic receptors are assumed to con-

tain the δ subunit [48]. In the visual cortex, the δ subunit alsoexists and thus would participate in the tonic GABAA currents[13]. The application of THDOC (100 nM), a δ subunit-specific agonist at a low concentration, did not enhance tonicinhibition in the somatosensory cortex despite the fact that∼50 % of the cells were positive for messenger RNA of the δsubunit [46]. We also observed no changes in tonic currentswith THDOC at the same concentration (100 nM) in thepresent experiment (14.88±1.67 pA in layer 2/3 PyNs;17.37±2.67 pA in layer 5 burst-spiking PyNs; p=0.31,p=0.45 vs. control, respectively), suggesting minimal involve-ment of δ subunit-containing GABAA receptors in tonicGABAA currents of the visual cortex. In contrast, even10 nM of THDOC enhanced the tonic currents in the dentategyrus granule cells [39]. Although 500 nM of THDOC signif-icantly increased tonic GABAA currents in the present study,the amplitude and decay time constant of evoked IPSC in bothlayer 2/3 and 5 PyNswere also significantly increased (data notshown), indicating the nonspecific effect of THDOC at

500 nM. Thus, we conclude that δ subunit-containing GABAA

receptors might not be an important contributor to the tonicGABAA currents in the rat visual cortex.

In the somatosensory cortex, the α5 subunit-specific in-verse agonist L-655,708 revealed tonic currents in layer 5 butnot in layer 2/3 [46]. In our experiments, the effects of L-655,708 on holding currents were most strong in layer 5 andalmost negligible in layer 2/3. Thus, the composition ofextrasynaptic GABAA receptors in the visual cortex mightdiffer in different layers as in the somatosensory cortex. Thebath application of GABA (5 μM), NO-711 (5 μM), andTHDOC (500 nM) enhanced tonic GABAA currents in sim-ilar proportions, to ∼150, ∼150, and ∼200 % of the controls,respectively, regardless of cell types. These treatments mightnon-specifically stimulate GABAA receptors on the cell sur-face. Thus, besides differences in the subunit composition, thedifferences in the expression levels of extrasynaptic GABAA

receptors might be a major contributor to the differences intonic GABAA currents among cell types.

In layers 5 and 6, the tonic GABAA currents were wellcorrelated with the amplitude of sIPSCs (Fig. 7c, d). Thus,the expression level of extrasynaptic receptors in these layersmight also be correlated with the level of inhibitory synaptictransmission. Application of the GAT-1 inhibitor, NO-711,clearly revealed the relationship between sIPSCs and tonicGABAA currents (Fig. 7e, f). This observation indicates thatthe major source of ambient GABA might be GABA synap-tically released onto the surface of the respective cells in thevisual cortex, as shown in the auditory cortex [41]. However,the involvement of nonsynaptic origins such as release fromglia [24] could not be excluded, at least in layers 2/3 and 4,since sIPSCs and tonic GABAA currents did not correlate inthese layers under the control conditions.

Cell type-specific tonic GABAA currents in layers 5 and 6

Previous studies reporting tonic GABAA currents in layer 5neurons of the somatosensory cortex did not distinguishregular-spiking and burst-spiking neurons [35, 46]. In thepresent study, we confirmed their different morphologicaland electrophysiological characteristics and found differentialcontribution of tonic GABAA currents. The burst-spiking,thick-tufted PyNs showed large tonic currents, and the spikingwas substantially regulated by the changes in tonic inhibition.However, regular-spiking slender PyNs exhibited smaller tonicGABAA currents, and the spiking was minimally changed bythe application of THDOC. The significant difference in theamplitude of tonic GABAA currents between the neighboringneuronal subtypes in the same layer also suggests differentlevels of expression of extrasynaptic GABAA receptors be-cause they might share the same pool of ambient GABA.

Layer 6 PyNs are also either burst-spiking or regular-spiking. The dendritic morphologies and the firing patterns

Fig. 9 Charge carried by tonic GABAA currents and their effect onfiring frequencies in neurons of different layers. a Charges carried bysIPSC (phasic charge) and tonic GABAA currents (tonic charge) arecompared in PyNs of different layers (scaled in the left vertical axis).The ratios of tonic charges and phasic charges were also compared(scaled in the right vertical axis). L2/3 layer 2/3, L4 layer 4, L5BS layer5 burst-spiking, L5RS layer 5 regular-spiking, L6BS layer 6 burst-spiking-like, and L6RS layer 6 regular-spiking-like PyNs. b THDOC-induced frequency change in firing at square current injection of 500 pAis plotted against the tonic density of each type of PyNs

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suggest that burst-spiking and regular-spiking neurons inlayer 6 might correspond to cortico-cortical and cortico-thalamic neurons, respectively, despite the lack of the recon-struction of axonal ramification in the present study [22, 32,47]. In layer 6, in contrast to layer 5, regular-spiking neuronsare subcortical projecting neurons and burst-spiking neuronsare cortical projecting neurons. Despite this disparity in con-nectivity between layers 5 and 6, tonic GABAA currents arestronger in bursting neurons in both layers. A burst is thoughtto increase the reliability of neural coding [25]. Stronger tonicinhibition in bursting neurons might exert an important influ-ence on the flow of visual information to other brain regions.Since the two different types of cells in layers 5 and 6 projecttheir axons to different targets, it could be assumed that tonicinhibition might regulate outputs from the infragranular layersof the visual cortex in a pathway-specific manner.

The bursting properties of neurons are regulated byhyperpolarization-activated cationic current (Ih), which ismediated by hyperpolarization-activated cyclic-nucleotidegated (HCN1) channels. Loss of HCN1 increased the burstfiring in layer 5 PyNs [21]. In HCN1 knockout mice, com-pensatory upregulation of tonic GABAA currents restoredthe normal synaptic summation [4]. Those results suggest thecomplementarity between HCN1 and tonic inhibition. Basedon our experiments, the number of spikes in a burst mightcorrelate with the strength of tonic GABAA currents. Thus, theinverse relationship between the expression levels of HCN1and extrasynaptic GABAA receptors might be general in thebursting neurons. This may explain the seemingly paradoxicalconclusion that the level of tonic GABAA currents, whichcould suppress the bursting (Figs. 3 and 4, a3 insets), correlatepositively with the number of spikes in a burst (Fig. 8b). Otherfactors such as the level of HCN1 expression might be moreimportant regulator of the bursting properties than the tonicGABAA currents.

Physiological implications

Visual cortical neurons exhibit characteristic properties suchas orientation tuning and directional selectivity, which couldnot be observed in thalamic relay neurons. Thus, these prop-erties are thought to emerge by complex processing throughcortical networks [10, 27]. Fine tuning of the gain and offsethas an important role in the contrast invariance of orientationtuning or directional selectivity [1, 27]. Tonic inhibition couldregulate the offset or gain of neuronal responses [34, 37].Thus, tonic inhibition might participate in the processing ofvisual signals and the emergence of many characteristic prop-erties of visual cortical neurons. Previous efforts to elucidatethe roles of inhibition in the processing of visual signalsthrough cortical layers have focused mainly on phasic synap-tic inhibition. In the present study, we demonstrated that tonicinhibition is generally correlated with the amplitude of sIPSC

and has a much higher charge carrying capacity than phasicinhibition. Thus, the differences in the amplitude of tonicinhibition and its effect on cell firing across layers and cellsin the present study suggest that studies on the roles of tonicinhibition are also necessary to fully understand the mecha-nism of information processing in the visual cortex. A recentfinding that tonic inhibition sharpens auditory coincidencedetection in the auditory cortex [41] suggests the generalimportance of tonic inhibition in sensory processing.

Another interesting finding was that burst-spiking neu-rons consistently showed stronger tonic inhibition thanregular-spiking neurons in layers 5 and 6. Bursting neuronsare important in gamma oscillation in the cortex [6]. Theroles of shunting inhibition in the maintenance of gammaoscillation [45] and of tonic inhibition in the control ofgamma oscillation [28] have been well established in thehippocampal interneuron networks. Therefore, the strongtonic inhibition in bursting neurons might also regulate thenetwork oscillation in the visual cortex and thus contribute tothe processing of visual signals. Further study on the role ofburst-spiking PyNs in the network oscillation and the effectof tonic inhibition in the regulation of the oscillation mightenhance our understanding of the visual signal processing.

Acknowledgments This study was supported by the Basic ScienceResearch Program (2012-046621) and the Science Research CenterProgram (2012-0009133) through the National Research Foundationof Korea, which is funded by the Korea Ministry of Education, Scienceand Technology.

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