Jay R. Gibson et al- Role for the Subthreshold Currents ILeak and IH in the Homeostatic Control of Excitability in Neocortical Somatostatin-Positive Inhibitory Neurons

8/3/2019 Jay R. Gibson et al- Role for the Subthreshold Currents ILeak and IH in the Homeostatic Control of Excitability in Neocortical Somatostatin-Positive Inhibitory N

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96:420-432, 2006. First published May 10, 2006; doi:10.1152/jn.01203.2005J NeurophysiolJay R. Gibson, Aundrea F. Bartley and Kimberly M. Huber

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Role for the Subthreshold Currents ILeak and IH in the Homeostatic Control

of Excitability in Neocortical Somatostatin-Positive Inhibitory Neurons

Jay R. Gibson, Aundrea F. Bartley, and Kimberly M. Huber

Center for Basic Neuroscience, Southwestern Medical Center, University of Texas, Dallas, Texas

Submitted 14 November 2005; accepted in final form 28 April 2006

Gibson, Jay R., Aundrea F. Bartley, and Kimberly M. Huber.Role for the subthreshold currents I

Leakand I

Hin the homeostatic

control of excitability in neocortical somatostatin-positive inhibitoryneurons. J Neurophysiol 96: 420432, 2006. First published May 10,2006; doi:10.1152/jn.01203.2005. Cortical circuitry reconfigures inresponse to chronic (13 days) changes in activity levels. To under-stand this process, we must know the role played by inhibitoryneurons because they crucially influence network properties by con-trolling action potential generation and synaptic integration. Usingpharmacological blockade of activity in neocortical organotypic slice

cultures, we examined the activity-dependent regulation of membraneexcitability in a specific inhibitory neuron subtype: the somatostatin-positive (SOM) neuron. Chronic action potential blockade (TTX,2.5 days) resulted in increased excitability in SOM neurons. Thisresult is consistent with a homeostatic process to maintain the averagefiring rate of SOM neurons at a particular level. Excitability changeswere not ascribed to changing cell size or alterations in voltage-dependent sodium current. Instead, the excitability increase waslargely the result of a decrease in the density of two subthresholdcurrents: a passive leak current (ILeak) and H-current (IH). Thedownregulation of these currents increased excitability mostlythrough a decrease in membrane input conductance. The coadaptationof ILeak and IH enabled a change in input conductance while helpingto preserve membrane potential. Evidence indicated that ILeak wasprobably mainly mediated by K. At earlier culture ages, this adap-

tation was superimposed on developmental changes, whereas at olderages, the same types of induced alterations occurred but with nodevelopmental component. Together with other studies, these dataindicate that both inhibitory and excitatory neurons increase mem-brane excitability with chronic reduction in activity, but throughdifferent mechanisms.

I N T R O D U C T I O N

Neocortical activity levels are chronically altered duringsensory map plasticity (Horton and Hubel 1981), neuronalcircuit maturation (Turrigiano and Nelson 2004), and certainpathological conditions, such as epilepsy or stroke. To under-stand how neural circuit properties are altered in these situa-tions, we must know the cellular alterations occurring indifferent cell types.

Across various species, most adaptations in neuronal prop-erties in response to chronic activity level changes (minutes todays) appear to be homeostatic (Davis and Goodman 1998;Marder and Prinz 2002; Turrigiano and Nelson 2000). Regu-lation of neocortical excitatory neurons is consistent with thehomeostatic maintenance of activity levels at a particular setpoint. In general, a reduction in circuit activity results in

changes at excitatory neurons that would promote more activ-ity in excitatory neurons. For instance, in response to long-termactivity suppression (days), excitatory synapses increase instrength (Desai et al. 2002; Lissin et al. 1998; Murthy et al.2001; Turrigiano et al. 1998), whereas inhibitory synaptic drivegenerally decreases (Hendry and Jones 1988; Kilman et al.2002; Marty et al. 2000; Micheva and Beaulieu 1995). Mem-brane excitability is enhanced mainly through upregulation ofvoltage-dependent sodium channels (VDSCs) and downregu-

lation of voltage-dependent potassium (K) channels, but notthrough subthreshold currents (Desai et al. 1999b; but see Liu2004). More acute forms of homeostatic plasticity (minutes)involve the subthreshold current I

H(Fan et al. 2005; van Welie

et al. 2004).Inhibitory neurons critically influence cortical circuit prop-

erties (Chagnac-Amitai and Connors 1989; Hensch 2004; Sil-lito 1974; Whittington and Traub 2003), but little is knownabout their activity-dependent regulation. Cortical inhibitoryneurons are divided into subtypes with distinct anatomical,biochemical, and electrophysiological properties (Gonchar andBurkhalter 1997; Gupta et al. 2000; Kawaguchi and Kubota1997). In dissociated culture, activity blockade for 2 days using

the VDSC blocker, tetrodotoxin (TTX), induces increasedexcitability in inhibitory neurons, but neither the inhibitoryneuron subtype nor the mechanism underlying this change wasknown (Desai et al. 1999a). We investigated activity-depen-dent regulation of membrane excitability in one inhibitorysubtype: the somatostatin-positive (SOM) neuron. This sub-type targets the distal dendrites of excitatory neurons, and thusmore likely controls synaptic input rather than spike timing inexcitatory neurons (Di Cristo et al. 2004; Miles et al. 1996;Somogyi et al. 1998). Accordingly, alterations in SOMneurons could alter synaptic integration and plasticity.

Intrinsic membrane excitability is affected by various cur-rent types and many of these have been described in SOMneurons: VDSCs, potassium currents, and H-current (I

H) (Lien

et al. 2002; Maccaferri and McBain 1996; Martina et al. 2000;Santoro et al. 2000). In other neuron types, each of thesecurrents has been demonstrated to be either acutely or chron-ically (long-term) modified by activity, and consequently toaffect neuron excitability (Aizenman et al. 2003; Brickley et al.2001; Chen et al. 2001; Coulter et al. 1989; Desai et al. 1999b;Fan et al. 2005; Nelson et al. 2003; Shah et al. 2004; Sourdetet al. 2003; Turrigiano et al. 1995; van Welie et al. 2006 2004).We demonstrate that excitability in neocortical SOM neuronsis homeostatically regulated in the long term (2.5 days), and

Address for reprint requests and other correspondence: J. R. Gibson,University of Texas, Southwestern Medical Center, Center for Basic Neuro-science, Box 9111, Dallas, TX 75390-9111 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J Neurophysiol 96: 420432, 2006.First published May 10, 2006; doi:10.1152/jn.01203.2005.

420 0022-3077/06 $8.00 Copyright 2006 The American Physiological Society www.jn.org


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this regulation appears to be mediated to a significant extent bysubthreshold currents, specifically an I

Leakand I

H.

M E T H O D S

Slice culture and pharmacological treatments

We performed all experiments using GIN (GFP-expressing inhib-itory neuron) mice, which express enhanced green fluorescent protein(EGFP) in only a subset of SOM neurons (Oliva et al. 2000)(Jackson Laboratories). The preparation of interface cultures wasbased on a previous study (Stoppini et al. 1991). Mice (P6) wereanesthetized with halothane in a manner consistent with the recom-mendations of the Panel on Euthanasia of the American VeterinaryMedical Association. The brain was removed and then dissected in aHEPES-based buffer containing kynurenic acid (1 mM) to obtain asquare sheet of somatosensory neocortex, 23 mm on each side,which was subsequently sliced into 400-m slices with a McIlwainchopper. Slices were transferred to 4C culture medium and thenplated onto semiporous membranes (Millicell, Millipore) in warmedculture medium. Slices were kept at 5% CO

2 /35C. Culture mediumwas exchanged the next day and every 2 days thereafter. The first two

exchanges involved adding a mitotic inhibitor to the culture medium(FUDR, 35 M; uridine, 80 M). Culture medium (based on Muslehet al. 1997) was 20% adult horse serum (Hyclone, defined, SH30074.02) and 80% MEM (GIBCO, 51200020) and contained thefollowing (in mM): 1 glutamine (Glutamax, Invitrogen), 0.7 ascorbicacid, 0.5 MgSO4, 12.9 dextrose, 5.3 NaCO3, 30 HEPES, and 1 g/mlbovine insulin (Sigma), pH 7.3, 310 mOsm. Chronic TTX (2 M)treatments were refreshed with new drug once per day. Indicating thatour results were not an artifact of drug application, effects wereidentical whether sucrose (2 M) was added to the control cultures.Usually, TTX was added to controls 10 min before removal from theinsert to control for any excitability phenomena occurring on trans-ferring the slice to the recording chamber. For any experiments thatinvolved sodium currents, all inserts were transferred into mediumcontaining 6,7-dinitroquinoxaline-2,3-dione (DNQX) and D-2-amino-

5-phosphopentanoic acid (AP5) for 10 min before recording towashout TTX applied during treatment. All experimental comparisonsrequired slices in the same plate obtained from the same animal. Sliceculture age was termed equivalent day (ED), which is the sum ofdays in vitro and the postnatal age of the dissected mouse.

Electrophysiology

Whole cell recordings on layer 2/3 neurons were performed at 22and 32C (as indicated) in a submersion recording chamber (Gibson etal. 1999). Recordings were performed with IR-DIC visualization(Stuart et al. 1993) using a Nikon E600FN microscope and a CCDcamera (Hamamatsu), and most recordings lasted 15 min. Recordedcells were typically about 15 m below the slice surface. At 32C, weobserved a gradual depolarization of about 6 mV over the first 10 min.

Therefore any experiments that critically depended on resting poten-tial were performed within the first 5 min to assay properties in themore natural, initial state of the cell. Resting membrane potential andseries resistance were continuously measured to monitor recordingstability. Cell capacitance was always measured at recording onset toprovide a rough estimate of cell size and to normalize currents(filtered at 30 kHz, sampled at 50 kHz). Capacitance and inputresistance were measured in voltage clamp with a 400-ms, 10-mVstep from a 60-mV holding potential. The capacitance was obtainedby multiplying the series resistance by the fastest time constant of adouble-exponential decay calculated fitted to the first 20 ms of thecapacitive transient decay induced by a voltage step. Input resistance(or conductance) was based on the average voltage in the 80- to130-ms window after step onset. Data were not corrected for junctionpotential.

Excitability measurements

Current-clamp recordings were performed at 32C unless statedotherwise. Threshold current for firing a spike was determined usinga series of depolarizing current steps of 600-ms length at 25- or 50-pAintervals. Threshold voltage for firing a spike was defined as themembrane potential, in a 5-ms window preceding spike peak, at whichthe third derivative was a maximum (an inflection point). Firing

frequency versus injected current plots (FI plots) was made bymeasuring the initial firing frequency of a spike train evoked by aseries of incrementing current steps.

Voltage-ramp protocols and analysis examining ILeak

Voltage-clamp recordings were performed at 22C in BasicBlock artificial cerebrospinal fluid (ACSF) with 2.5 mM Cs andaccepted if series resistance was 15 M and compensation/predic-tion were 85%. Holding potential was 60 mV and a prestep wasapplied to 20 mV for 6 s to allow sufficient relaxation to asteady-state standing outward current. This was followed by avoltage ramp down to100 mV for 1.53 s (ramp durations indicatedin Figs. 4 and 5). Statistical difference between average traces (P

value, each cell was a sample) was the lower of treatment only andtreatmentvoltage interaction terms as obtained in a repeated-measures ANOVA. If these average traces were different, then theirdifference trace (subtraction of traces between the two groups) wastermed statistically significant. Thus any P values referring to adifference trace means the traces used in the subtraction were statis-tically different. Difference traces were always remapped into currentversus voltage (IV) plots, and statistical analysis on all ramp data wasapplied only to current obtained during the ramp. The SE for eachpoint in the difference current was the square root of the sum squaredof the SE values from the original two points (Zar 1999). If specificvoltages during the ramp were compared, a Fishers PLSD (protectedleast-significant difference) multicomparison test was applied.

When difference currents were obtained from two different externalsolutions in the same cell (all experiments in Fig. 5), the cell-to-cell

variability in the external solution conditions was correlated. Fordetermining a statistically significant effect on this type of differencecurrent between treatment groups (control vs. TTX-treated), rampcurrents from each cell were normalized to the first external solutioncondition (i.e., 1 mM [K] in Fig. 5, A and B). In effect, error bars existonly for current traces collected during the second external solutioncondition, and a repeated-measures ANOVA was again used tocompare traces between treatment groups.

To assess the ionic identity of currents in IVplots, passive currentsfor K and Cl were calculated according to GoldmanHodgkinKatz (GHK) current equations (Johnston and Wu 1995), adjusted for

junction potentials, and normalized to the peak current at the voltageramp apex (see Figs. 4, 5, and 9). A repeated-measures ANOVA wasused to determine whether the experimental data were different fromthe predicted passive currents (P 0.05 indicated that data matched

the predicted passive current).

H-current protocols and analysis

Voltage-clamp recordings were performed at 22C unless statedotherwise and accepted if series resistance was 15 M and com-pensation/prediction were 85%. When Basic Block ACSF wasused, 1 mM BaCl2 was added. IH was measured during a 2-s,hyperpolarizing voltage step. Even though steps were immediatelypreceded by a series of smaller,10-mV steps for the subtraction ofleak currents [akin to a p/4 protocol (Bezanilla and Armstrong 1977)],we found this was not effective in isolating a clear IH. This may havebeen a result of insufficient voltage clamp of dendritic current or someother nonlinear currents being modulated. Because repeated applica-tion of hyperpolarizing steps to 110 or below disrupted recording

421HOMEOSTASIS, INHIBITORY NEURON, SOMATOSTATIN, NEOCORTEX

J Neurophysiol VOL 96 JULY 2006 www.jn.org


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stability, we restricted our data collection to one to two traces percondition when such steps were required. All IH measurements werenormalized to total membrane capacitance. Dual-exponential fits forcurrent activation were performed with the generalized reduced gra-dient (GRG2) nonlinear optimization code (Excel software, Mi-crosoft).

Unless stated otherwise, we measured IH

with respect to the localmaximum during the step that represented the difference between themaximum current extent during the voltage step (usually within 200ms of step onset) and that immediately before step offset (see Fig. 7A,arrows). We preferred this method for its simplicity, and amplitudeswere nearly 79% less compared with IH isolated using pharmacolog-ical blockers (Cs, ZD7288; n 11, 10; see RESULTS).

Steady-state, voltage-dependent activation was inferred by firstinactivating IH with a prestep (2 s), and then determining how muchremained with a final test step to 110 mV (2 s) (Maccaferri andMcBain 1996). The percentage reduction in IH at this test stepindicated the percentage activated during the prestep. Holding poten-tial was 40 mV and bath solution was Basic Block ACSF.

Where indicated, IH

was also measured with a 40-mV step (2 s)from a potential of60 mV in normal ACSF (with DNQX, AP5, andpicrotoxin). This was an accurate measure of I

Hbecause ZD7288

blocked 96

2% of the slowly activating current (n

4, see Fig.10C). IH in normal ACSF was larger than that measured in BasicBlock external solution, making it easier to measure. Basic Blocksolution reduced IH by 61 7% (n 5). Much of this was mediatedby the 5 mM 4-AP which by itself reduced IH by 46 3% (n 5).The rest of the block may have arisen from Ba2 (van Welie et al.2005). We cannot explain this reduction by 4-AP, but one possiblereason is that a significant amount of IH occurred in the dendriteswhere voltage-clamp control was weak, and with addition of theseagents, the dendritic membrane was too depolarized for somatichyperpolarization to activate I

H. This explanation is supported by

abundant IH

in pyramidal neuron dendrites (Magee 1998), but nostudy has yet examined the localization of I

Hin SOM dendrites.

Na-current protocols

Recordings were performed at 22C and accepted if series resis-tance was 13 M and compensation/prediction were 90%. Acti-vation curves were based on the peak current evoked by incremental,10-ms depolarizing steps (Fig. 9A); inactivation curves were derivedfrom the maximum current evoked by a 10-ms step to 0 mV withvarying 100-ms, prestep potentials (Desai et al. 1999b). All stepprotocols were preceded by fifteen 10-ms depolarizing steps that wereone sixth the magnitude of the test step. All traces underwent leaksubtraction before measurements were made. Subsequent applica-tion of TTX revealed that the leak subtraction method was accurate(n 3).

Electrophysiology solutions

Normal ACSF contained (in mM): 126 NaCl, 3 KCl, 1.25NaH

2PO

4, 2 MgSO

4, 26 NaHCO

3, 10 dextrose, and 2 CaCl

2. Basic

Block ACSF was based on a previous study (in mM) (Maccaferri andMcBain 1996): 110 NaCl, 5 KCl, 1 MgCl

2, 24 NaHCO

3, 10 dextrose,

10 TEA-Cl, 5 4-AP, 0.2 CdCl2

, 0.1 NiCl2

, and 0.001 TTX. BasicBlock HEPES solution was (in mM): 110 NaCl, 5 KCl, 1 MgCl

2, 20

HEPES/Na-HEPES, 10 dextrose, 10 TEA-Cl, 5 4-AP, 0.2 CdCl2, and0.001 TTX. INa HEPES solution was as described (in mM) (Desaiet al. 1999b): 10 NaCl, 120 choline-Cl, 3 KCl, 20 HEPES/Na-HEPES,2 MgSO4, 0.4 CdCl2, 10 TEA-Cl, 5 4-AP, 14 dextrose, and 1.6 CaCl2.All solutions were pH 7.4 (7.8 and 6.2 for acidification experiments)and all included (in M): 20 DNQX, 100 picrotoxin, and 50 AP5.ACSF was saturated with 95% O2-5% CO2. The following were thepipette solutions (in mM): K-Meth 130 K-methanesulfonate, 6 KCl,3 NaCl, 10 HEPES, 0.2 EGTA, 4 ATP-Mg, 0.3 GTP-Tris, 14

phosphocreatine-Tris, 10 sucrose (pH 7.25, 290 mOsm); Cs-Meth 125 Cs-methanesulfonate, 16 CsCl, 10 NaCl, 10 HEPES, 2.5 BAPTA,4 ATP-Mg, 0.3 GTP-Tris, 14 phosphocreatine-Tris, 10 sucrose, 2QX-314-Cl, 10 TEA (pH 7.25, 290 mOsm); measured junction po-tentials were about 9 and 10 mV, respectively, in all the aboveexternal solutions (Neher 1992).

DrugsFor all membrane current measurements, fast synaptic transmission

was blocked with the following: N-methyl-D-aspartate (NMDA) re-ceptor antagonist AP5 (Sigma), the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist DNQX(Sigma), and the -aminobutyric acid type A (GABAA)receptorantagonist picrotoxin (Sigma). Where stated, voltage-dependent so-dium currents were blocked with tetrodotoxin (TTX; 1 M, Sigma)and voltage-dependent potassium currents were blocked with tetra-ethylammonium chloride (TEA; 10 mM, Sigma) and 4-aminopyridine(4-AP; 5 mM, Sigma).

Anatomical labeling

Cells were filled with biocytin (Sigma) for 45 min using whole cellrecording and subsequently placed in cold fixative. Slices were notresectioned because the cultures, in layer 2/3, had thinned to about100120 m by the time of experimentation. After processing oftissue as described (Gibson et al. 1999), neurons were traced andprojected into a single plane using Metavue software and, from this,total dendritic length, branch point number, and Scholl crossings weremeasured (Jin et al. 2003; Scholl 1956). Fluorescent immunohisto-chemistry for somatostatin (#KO21, Santa Cruz Biotechnology, SantaCruz, CA) was performed as previously described (Gibson et al. 1999)on fixed, unresectioned slice cultures in neocortical layers 2/3. Theantibody did not penetrate 25 m into the slice, Therefore werestricted our analysis to all focal planes above the deepest focal planein which SOM could be clearly observed. GFP localization wasperformed with native EGFP signal.

Analysis

All statistics were performed using Statview (SAS Institute, 1998),and statistical significance was P 0.05. Unless otherwise stated, weused an unpaired t-test or a one-way ANOVA followed by a multi-comparisons test (Fishers PLSD). Sample number (n) was cellnumber and was always given in the following order: (Control,TTX-treated, Before) or similar order if one group was omitted (i.e.,Control, TTX-treated). All error bars are SE.

R E S U L T S

Cellular properties of SOM neurons are normalin slice culture

In slice culture, GFP was expressed almost exclusively insomatostatin-positive (SOM) neurons: 88% (21/24) of allGFP neurons co-localized with somatostatin at ED 16 and 91%(20/22) at ED 21 (ED is equivalent day; see METHODS). Thiswas consistent with immunohistochemistry in acutely preparedanatomical sections (Oliva et al. 2000). The electrophysiolog-ical profile of GFP neurons in culture was consistent with theSOM subtype previously described in acute slices. Restingpotential was similar and input conductance was in the upperrange (57.4 0.9 mV, 12.4 0.9 nS, n 25) (acute, 59mV and 3.4 to 12.2 nS from Bacci et al. 2003; Cauli et al. 2000;Deans et al. 2001). With simultaneous whole cell recordings ofneighboring GFP and non-GFP neurons, the GFP to non-GFP

422 J. R. GIBSON, A. F. BARTLEY, AND K. M. HUBER



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synaptic connection was always inhibitory as confirmed by thereversal potential of the postsynaptic potential (15/16 pairsconnected). All non-GFP to GFP synaptic connections wereexcitatory and displayed strong facilitation characteristic ofSOM neurons (10/16 pairs connected; Fig. 1A) (Gibson et al.1999; Reyes et al. 1998).

Activity blockade induces higher spontaneous activity

Activity blockade has commonly been used to assess the roleof activity on circuit and neuronal properties. Chronic activityblockade (15 days) is known to increase the spontaneousactivity of cultures when the blockade is removed (Corner andRamakers 1992; Gorba et al. 1999; Seil and Drake-Baumann1994; Turrigiano et al. 1998). To determine whether this wasthe case in our cultures, we applied TTX (2 M) for 2.5 daysstarting at late ED 14. At ED 17, slices were transferred to therecording chamber and bathed in modified ACSF (in mM: 1.8Mg2, 1.8 Ca2, 5 KCl; 32C). Spontaneous action potentialrate was indeed higher in TTX-treated slices (Fig. 1B) as

observed in both burst and individual spike rates in SOMneurons (0.1 0.1 vs. 10.6 2.0 burst/min, P 0.0008;0.6 0.4 vs. 17 2.5 spikes/min, P 0.0007; n 5, 5). Thisspontaneous activity was ascribed to intermittent, highly syn-chronous, excitatory input from other neurons because it wasblocked by the AMPA-receptor (AMPA-R) antagonist DNQX(20 M). The large excitatory drive was clearly observed involtage clamp.

The sparse activity in controls might be a concern becausethis would suggest that the effects of TTX are not specificallythe result of blocking activity. To address this issue, werepeated this experiment using 2.5-day treatment with DNQX(20 M) to chronically reduce activity. Recordings were per-formed in an ACSF in which [Mg2] more accurately reflectedthat in the culture medium (1.3 mM). Action potentials were

observed extracellularly with cell-attached patch recordings.Again, both burst and spike rates in SOM neurons wereincreased with activity reduction (1.4 0.4 vs. 3.7 0.6burst/min, P 0.01; 34 14 vs. 179 53 spikes/min, P 0.05; n 5, 7). Clear spontaneous activity was observed incontrol slices that, as observed with simultaneous recordings ofSOM/non-GFP pairs, was highly synchronized among neigh-boring neurons (n 3).

Increased excitability and decreased subthresholdconductance with activity blockade

Excitability was determined by plotting firing frequency as a

function of injected current (FI plot) and by measuring theminimum amount of current to evoke an action potential fromrest (threshold current; see METHODS). SOM neurons experi-encing chronic activity blockade were more excitable com-pared with same-age controls (n 25, 30) because they firedmore rapidly for a given amount of current injection (Fig. 2A).As observed in FI plots, this was true over the entire range ofinjected current intensities (Fig. 2B). Threshold currents de-creased by 49% with activity blockade (Fig. 2D). Thesechanges are consistent with a homeostatic regulation of excit-ability.

TTX-treated neurons also had a 24% reduction in subthresh-old input conductance (Fig. 2, C and D; 12.4 0.9 vs. 9.4 0.7 nS, P 0.001) and they had a more depolarized resting

potential (57.4 0.9 vs. 55.8 0.7 mV, P 0.03). Spikethreshold voltage may have been slightly hyperpolarized, butthis was not statistically significant (40.8 0.6 vs. 42.2 0.4 mV; P 0.053; see METHODS). To control for a possiblebias in our sampling, a subset of experiments were performedblind to the treatment history of the slices (n 7, 11), and thesame excitability and input conductance changes were ob-served (input conductance, P 0.02; FI curve, P 0.04).The same conductance decrease occurred when chronic activ-ity reduction was performed with the AMPA-R antagonistDNQX (P 0.001; n 7, 7). Assuming a simple ohmicprocess to achieve voltage threshold and using the numbersgiven above, the input conductance decrease would account for

most of the decrease in threshold current compared with thedepolarized resting potential (78 and 22%, respectively). Thestrong influence input conductance has on excitability is furthersupported by the positive correlation between input conduc-tance and threshold current in both Control and TTX-treatedcells (R 0.69, P 0.001; R 0.81, P 0.001; data set fromFig. 2B; Fishers r-to-z test, Statview). The same thresholdcurrent difference was observed when cells were held at 60mV (P 0.002, n 14, 13).

Is excitability decreased with activity-blockade or simplymaintained at an earlier developmental stage? A subset of theabove experiments included a before-treatment condition (n 18, 19, 18). Threshold current required to evoke a spike wassignificantly decreased in TTX-treated compared with before-

FIG. 1. Slice culture preparation is adequate studying homeostasis in so-matostatin-positive (SOM) neurons. A: while simultaneously recording froman excitatory and a SOM neuron, brief voltage steps were applied to theexcitatory neuron to induce a train of presynaptic action potentials (top).Resulting excitatory postsynaptic potentials (EPSPs) in the SOM neuron(bottom) displayed strong facilitation just like that observed in acute slices.Scale bars: 25 mV (top), 0.5 mV (bottom), 100 ms. B: spontaneously generatedaction potentials (APs) were examined in single SOM neurons. AP rate washigher in tetrodotoxin (TTX)-treated cells as a result of intermittent, strongexcitatory input. Scale (in A): 25 mV, 800 ms.




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treatment cells (P 0.02), suggesting an upregulation inexcitability induced by activity blockade (Fig. 2E). On theother hand, except for the lowest current, the FIplots were notdifferent (Fig. 2F). Furthermore, no difference was observedfor input conductance (P 0.25). These data suggest thatSOM neurons are still maturing in our experiments, and thatactivity blockade may partly involve a developmental arrest of

some electrophysiological properties.Although we focus on subthreshold currents, we briefly

examined action potentials for clues to altered suprathresholdcurrents. No differences were observed between all groups inmost action potential characteristics (i.e., spike width and risetime, spike height), but there was a significant 19% reductionin afterhyperpolarization (AHP) magnitude and a 39% increasein AHP duration in TTX-treated SOM neurons comparedwith same-age controls (P 0.0004 and P 0.05, respec-tively). No difference in AHP was observed between TTX-treated and before-treatment neurons. Therefore suprathresholdcurrents may have been altered, although the specific role thisAHP played was not examined.

Differences in subthreshold conductance do not arisefrom cell size

Our data suggested that subthreshold input conductancesplay a significant role in mediating increased membrane excit-ability induced by chronic activity blockade (see DISCUSSION).Alterations in this conductance may have arisen from changes

in either the conductance density of the membrane or theneuron size, or both. We expected alterations in conductancedensity to be more likely because no change in morphology ofneocortical inhibitory neurons is observed with 5-day TTXtreatment in slice cultures of similar age (Jin et al. 2003). Weexamined this issue with two experiments. First, we measuredinput conductance, total membrane capacitance, and soma sizeas seen under IR-DIC microscopy immediately before record-ing (n 22, 17). Although no change was observed inmembrane capacitance (12.6 0.6 vs. 14.1 0.6 pF, P 0.11) or soma size (235 11 vs. 250 14 m2, P 0.44),input conductance was less in TTX-treated SOM neurons(6.3 0.6 vs. 4.5 0.3 nS, P 0.005). This suggests thatcurrent density, not cell size, had changed.

Second, we measured the input conductance of neuronswhile filling them with biocytin for morphological analysis(n 14, 19, Fig. 3A). TTX was applied for 4 days toexaggerate any possible morphological effect. Even thoughinput conductance decreased with activity blockade (8.49 0.57 vs. 5.99 0.39 nS, P 0.0008), cell morphology wasunaltered for the same SOM neurons (Fig. 3, B and C). Wedid not observe any difference in total dendritic length(2,093 124 vs. 1,944 115 m, P 0.39) and branch pointnumber (10.4 0.9 vs. 12.3 0.8, P 0.13). As above, nochanges were observed in membrane capacitance or soma size.Finally, a Scholl analysis also failed to detect any difference indendritic ring crossings (Fig. 3C, P 0.52). Therefore the

alterations in input conductance appear to result from changesin membrane conductance density.

Decreased leak current (ILeak

) with activity blockade

We next examined the subthreshold currents that were al-tered in response to activity blockade. Initial voltage-rampexperiments indicated that both a Cs-sensitive and a Cs-insensitive component of subthreshold current were downregu-lated with activity blockade (data not shown). The Cs-sensi-tive current was reminiscent of H-current (I

H) because it was

an inward current activated at hyperpolarized potentials (Pape1996; Robinson and Siegelbaum 2003) and the Cs-insensitive

current appeared to be an outwardly rectifying passive current,which we tentatively called ILeak

because it resembled a pas-sive K or Cl leak current (based on the GHK equation).

We first characterized the Cs-insensitive component byapplying voltage ramps (20 to 100 mV, 3 s) to investigatecurrents as a function of voltage. Under these recording con-ditions, resting potential was clearly higher in TTX-treatedSOM neurons (55.5 1.3 vs. 47.8 1.1 mV). Activityblockade induced a clear change in current traces (Fig. 4A),which included a 43% decrease in the standing-outwardcurrent (measured at 20 mV, steady state; P 0.0003).From these traces, we derived the net current that accounted fordifferences induced by activity blockade, which we call theactivity-dependent current. The average TTX-treated trace

FIG. 2. Activity blockade (of 2.5 days) induces an increase in membraneexcitability in SOM neurons. A: for the same amplitude current step injected,a higher firing rate was evoked in TTX-treated neurons. B: firing frequency asa function of injected current (FI) plots based on data in A indicatingincreased excitability in TTX-treated neurons. All points are statisticallydifferent (P 0.02). C: examples of input conductance measurements forcontrol (left) and TTX-treated (right) neurons. Arrow indicates where conduc-tance was measured. Scale bars: 100 pA, 100 ms. D: TTX-treated neuronsrequire less current to evoke a spike (P 0.0002) and have lower totalconductance (P 0.001). E: a subset of experiments involved measurements

before treatment. A clear downregulation in threshold current is observed whencomparing before and TTX-treated neurons, but total input conductance wasnot statistically different. All control/TTX-treated comparisons were statisti-cally significant. F: only the first point in the FI plot was different betweenbefore and TTX-treated neurons (**P 0.05 for Control/TTX and Before/TTX comparisons). For D and E, *P 0.05.




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was subtracted from the average control trace to attain a differencetrace, and subsequently the difference trace was transformed intoa current versus voltage (IV) plot (Fig. 4B). This derived activity-dependent current represented a 25% decrease in input conduc-tance (based on slope at 60 mV), which indicated that I

Leak

played a large role in the activity-dependent conductance alter-ations described above (see DISCUSSION for details). Even thoughthis I

Leakwas not different from a predicted passive potassium

leak current as calculated from the GHK equation (see METHODS),it had a reversal potential (ER

) 20 mV depolarized to that pre-dicted for a pure K current (E

K 120 mV). The E

Rfor I

Leak

may explain the more depolarized resting potential of TTX-treated SOM neurons in normal ACSF.

To determine whether activity blockade induces a simple,isolated downregulation ofI

Leak, we also made comparisons with

cells before treatment. There was a clear developmental compo-nent in the experimental time window in which we observe theseactivity-dependent alterations (Fig. 4C). If the I

Leakchange was an

isolated downregulation, we would expect the before and controltraces to be identical and their difference trace to be a horizontalline at 0, but this was not the case. Instead, a difference trace(Control Before) revealed a developmentally regulated current

of unclear identity. In addition, there appears to be an activity-independent alteration of a DC, nonohmic current as indicated bythe Before TTX difference trace. In two other experiments, thisnonohmic component contributed an even higher proportion ofthe Before TTX difference trace (data not shown). The identityof this nonohmic current is unknown, but at the very least, thesedata indicate that activity-dependent alterations in I

Leakare super-

imposed on other developmental changes in intrinsic membranecurrents.

ILeak

at least partly mediated by potassium

In a subset of experiments in Fig. 4, we subsequently alteredextracellular [K] to 10 mM to see whether the activity-

dependent current would shift as predicted for a K-mediatedcurrent. The IV plot shifted in the depolarizing direction aspredicted (Fig. 5A; P 0.002, n 9, 8), but the shifted currentwas not identical to that predicted for K involvement (P 0.03). The E

Rshift was only 35% of the predicted shift. These

data suggest that ILeak

is partly mediated by K.We next examined a role for Cl. Experiments were iden-

tical except the pipette solution was altered by substituting 26

mM K-Meth with equimolar KCl for a total of 35 mM [Cl

].If Cl contributed to I

Leak, we would expect a distinct depo-

larizing shift in the reversal potential, although this was not thecase (Fig. 5B). The activity-dependent current was no differentfrom that predicted for a passive K current (P 0.82) butdifferent from a predicted passive Cl current (P 0.0001,n 8, 8). On washing in 10 mM K, the difference traceshifted to a similar extent as observed in Fig. 5A. The E

Rshift

was 57% of that predicted for the K-mediated current, ahigher contribution than observed above, which was probablya result of high cell-to-cell variability. Therefore Cl played aminimal role.

Even though the IV plot shifts with extracellular [K],

alterations indicate that K

is only partly involved; othercharacteristics of ILeak

suggest that K is the main ion: lack ofany contribution by Cl, reversal near E

K, and slight outward

rectification. Perhaps attempts to alter extracellular [K] areobstructed by buffering in our cultured slice preparation as isobserved for pH in acute slices (Chesler et al. 1994; Voipio andKaila 1993; see DISCUSSION). Some members of the two-pore(2P)domain family of potassium channels have been demon-strated to mediateI

Leakin various cell types (Goldstein et al. 2001;

Lesage 2003). We wanted to determine whether the activity-dependent I

Leakdisplayed characteristics of two members of this

family whose existence and function has been demonstrated inCNS neurons: TASK1 and TASK3 (Brickley et al. 2001; Meuthet al. 2003; Talley et al. 2000; Washburn et al. 2002).

FIG. 3. Size or morphology alterations do not account forinput conductance changes. A: examples of recorded cellsfilled with biocytin from which morphology was measured.

Left: control. Right: 4-day TTX-treated. B: input conductancewas reduced in these cells (left), whereas dendritic length wasunaffected (right). C: no difference in dendritic crossings wasobserved using a Scholl analysis (see METHODS).




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Acidification has been shown to block TASK channels(Brickley et al. 2001; Meuth et al. 2003; Talley et al. 2000).Acidification (pH 7.8 6.2) significantly changed ramp-in-duced currents obtained from both control and TTX-treatedSOM neurons (P 0.0001, P 0.03, n 15, 17). Acidi-

fication modulated more than one type of ionic current, and thiswas evident in our pH 7.8 pH 6.2 difference currents, whichhad E

Rvalues of60 and 38 mV for Control and TTX,

respectively. In spite of this apparent nonspecificity, acidifica-tion still clearly altered activity-dependent I

Leak(P 0.0001)

and reduced its standing outward current by 56% (Fig. 5C; P0.0002, n 15, 17).

One mM Ba2 blocks 80% of TASK1-mediated current(Han et al. 2002; Talley et al. 2000). Barium (1 mM) clearlyaltered ramp currents in both control and TTX-treated SOMneurons (P 0.0001, P 0.0001; n 19, 19). Unlikeacidification, no Ba2 Ba2 subtraction traces demon-strated a specific block of a potassium leak current. Upon Ba2

application, we observed a 60% reduction in the activity-

dependent standing outward current (P 0.0007, Fig. 5D). Insummary, both acidification and Ba2 block suggest a largecontribution of K to I

Leak.

Decreased H-current (IH) with activity blockade

Based on initial voltage-ramp experiments suggesting that a

Cs

-sensitive current, reminiscent of IH, was activity-depen-dent (data not shown), we examined regulation of I

H. Cortical

SOM neurons are known to display IH

(Lupica et al. 2001;Maccaferri and McBain 1996). We first established that I

H

existed in our cultured SOM neurons. Hyperpolarizing stepsdown to 60 mV elicited a clear, slowly activating inwardcurrent (Fig. 6A). The slowly activating current was blocked by

IH

blockers (93 3% reduction by 2.5 mM CsCl and 95 1%by 100 M ZD7288; n 11, 10; Fig. 6A) (Kilb and Luhmann2000; Maccaferri and McBain 1996). When traces collectedunder blocked conditions were subtracted from those undercontrol conditions, only the slowly activating inward current,characteristic of I

H, was isolated (Fig. 6B). Based on tail

currents, the IH

reversal was similar to that reported previously(28 6 mV; n 3).

In the same data set, we found that IH

was reduced with TTXtreatment. This reduction was similar using both the localmaximum measurement (64%; n 9, 12, P 0.02; Fig. 7A;see METHODS) and subtraction traces (60%, P 0.03; obtainedfrom blocker washins as described above). Because the localmaximum method appeared adequate for measuring I

Halter-

ations, it was thus always used.Time constants for I

Hactivation were no different when

obtained from local maximum and subtraction methods (Pvalues of 0.20 and 0.90; 311 21 and 2,774 124 ms, fromsubtraction traces), and these time constants were similar tothose observed in hippocampal SOM neurons in acute slices

FIG. 4. A putative leak current (ILeak) is downregulated in SOM neuronswith chronic activity blockade. A: average current as a function of time duringa voltage ramp is reduced with activity blockade (P 0.0001; 1 mM [K]Ext,20 to 100 mV, 3 s). B: control TTX difference traces mapped onto acurrentvoltage (IV) plot reveals an activity-dependent current that closelymatches a predicted leak current mediated by K (P 0.11, prediction is grayline). C: difference currents derived from cells before treatment indicate thatalterations in ILeak with activity blockade co-occur with developmentalchanges. Identity of these currents is not clear. n 15, 16, 15.

FIG. 5. Activity-dependent ILeak is at least partly mediated by a potassiumconductance. All figures are IVplots derived from voltage ramps of 3, 2, 1.5,and 2 s in AD, respectively. A: an incomplete shift ofILeak when external [K

]is increased indicates a partial contribution by K. Inset: traces are normalizedto eliminate correlated error in both [K] conditions. This scaling causeshighly variable behavior at midtrace because of variability of zero crossingsbetween [K]Ext conditions and between cells. B: with 35 mM [Cl

] in thepipette, no alterations in ILeak properties were observed. C and D: ILeak isconsiderably reduced with acidification (pH 6.2) or 1 mM Ba2. Insets, Cand

D: normalized plots as described in A. In all panels, gray lines are predictedIV plots for passive K and Cl conductances derived from the GoldmanHodgkinKatz equation. These prediction plots may be obscured by data. Cand D performed in 3 and 1 mM [K]Ext, respectively.




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(Santoro et al. 2000). However, it was not possible todetermine whether kinetics were altered in TTX-treated

SOM neurons because IH was too small for an accuratemeasurement.

We repeated measurements ofIH

under conditions that madeboth activation kinetics and amplitude more easily measured(n 13, 21; 40-mV step from 60-mV holding, normalACSF, 32C; see METHODS). We found a slight increase in thefast component (Fig. 7A) and no change in the slow componentof activation with activity blockade. Amplitude was againdecreased with activity blockade (45% drop), and this resultwas reproduced both with the experimenter blind to the groups(P 0.004, n 6, 11) and with 2.5-day treatment with theAMPA-R antagonist DNQX (P 0.003, n 7, 7). In sum-mary, I

Hkinetics were altered relatively little, whereas ampli-

tude was clearly decreased with activity blockade.Differences in voltage-dependent activation could explainthe decrease in I

H(Chen et al. 2001). An accurate activation

curve examining tail currents could not be obtained because wecould neither get a maximum tail current nor repeatedly hy-perpolarize cells to achieve this maximum (see METHODS). Theformer may possibly be explained by the poor voltage clamp ofdendrites. Therefore a rough estimate of steady-state, voltage-dependent activation was obtained by measuring the percent-age reduction in I

Hwith different prestep potentials (see

METHODS). This reduction represented the percentage of IH

activated during the prestep. No change in voltage-dependentactivation was observed even though I

Hamplitude was lower

with activity blockade (Fig. 7B).

To determine whether the IH

decrement was a result of eithera developmental arrest or an active downregulation, we com-pared I

Hamplitude between control, TTX-treated, and before-

treatment conditions (Fig. 7C). Although there was a clear IH

reduction in TTX-treated neurons compared with either controlor before groups, an age-dependent increase was also apparent.Thus there was a downregulation of I

Hsuperimposed on a

maturational increase of IH.

IH

modulation of resting potential and excitability is alteredwith activity blockade

If IH

were active at resting potential, it could play a criticalrole in controlling resting potential and excitability (Shah et al.2004). Although our activation data suggest a minimal contri-bution at resting potential (55 and 60 mV; see Fig. 7B),there may still be a significant I

Hcontribution at these poten-

FIG. 6. H-current (IH) exists in SOM neurons in the slice culture prepa-ration. A: IH was measured with a voltage step from 40 to120 mV and wasidentified by a slowly activating inward current that is blocked by both 2.5 mMCs and 100 M ZD7288. Scale bars: 450 pA, 500 ms. B: subtraction tracesobtained from no Cs and 2.5-mM Cs conditions show the voltagedependence ofIH in an untreated SOM neuron. Scale (in A): 100 pA, 500 ms.

FIG. 7. IH amplitude is reduced with chronic activity blockade. A: IHamplitude is decreased with activity blockade, whereas time constants foractivation change very little. B: a prestep protocol was used to measureactivation (see METHODS). Percentage reduction represented the percentageactivated during the prestep. No alteration in voltage-dependent activation withactivity blockade was observed even though IH magnitude was significantlydecreased (Inset: P 0.0002) (n 11, 11). C: IH amplitude is downregulatedwith activity blockade, but there also appears to be an age-dependent increasein IH [n 18, 15, 13; 40-mV step from 60-mV holding, Basic Blockartificial cerebrospinal fluid (ACSF), 22C]. *P 0.02.




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tials that was not detected. We examined the effect of ZD7288on resting potential and excitability in both control and TTX-treated SOM neurons. Recordings were performed with

high-resistance electrodes to better preserve the cytoplasmiccontents of cells (series resistance 21.6 1.1 M; 22C, 20M DNQX, 50 M AP5).

In control SOM neurons, ZD7288 induced a 3-mV hyper-polarization, indicating a significant contribution of I

Hat rest-

ing potential (Fig. 8, A and B), which is consistent withprevious studies (Lupica et al. 2001; Maccaferri and McBain1996). To our surprise, no detectable change occurred inTTX-treated cells. Based on our I

Hmeasurements above, we

expected a hyperpolarization nearly 40% of that observed forcontrols. Either I

Hwas too low to be detected using ZD7288 or,

contrary to our results above, there was a voltage-dependentactivation shift in I

H. Based on the average induced hyperpo-

larization, input conductance, and membrane capacitance, theIH

reduction at rest in TTX-treated SOM neurons was about2 pA/pF, which closely matched the reduction in ILeak, about 3pA/pF (Figs. 4 and 5). Because their reversal potentials areapproximately equidistant from rest (about 95 and28 mV),these data suggest I

Leakand I

Hchanges offset each other to

preserve resting potential. The greater reduction in ILeak

mayexplain the slightly higher resting potential in TTX-treatedcells. Using the reversal potential derived above, I

Hat rest

contributed 0.83 nS to total membrane conductance (10%).We found that decreases in I

Hincreased excitability in

SOM neurons, as previously reported for pyramidal neurons(Shah et al. 2004). In control SOM neurons, ZD7288 washincaused a 14% decrease in threshold current, whereas no de-

tectable change was observed with TTX-treated neurons (Fig.8C). When excitability from a slightly more hyperpolarizedpotential of70 mV was examined (Fig. 8D), the effects ofZD7288 were more dramatic, as expected for a hyperpolariza-tion-activated current. Both control and TTX-treated SOMneurons became more excitable, but the decrease in thresholdcurrent was proportionately bigger in control neurons (36 vs.

22%, P 0.01), consistent with their larger IH. The greaterincrease in excitability in control neurons is consistent with theassertion that a decrease in I

Hwith activity blockade promotes

an excitability increase.

No alteration in voltage-dependent sodium current

In excitatory neurons, upregulation of voltage-dependentNa current is involved in the activity-dependent regulation ofexcitability (Desai et al. 1999b). To determine whether this istrue for SOM neurons, we first measured Na current am-plitude with incremental voltage steps (Fig. 9A). These currentshad both the voltage- and time-dependent characteristics of

voltage-dependent Na

current (Desai et al. 1999b; Hamill etal. 1991; Huguenard et al. 1988) and were completely blockedby its specific blocker, TTX (1 M, n 3). We compared peakcurrent at steps up to 10 mV between TTX-treated and

FIG. 8. IH plays a role in maintaining resting potential and in regulatingexcitability. A and B: on washing in ZD7288, control SOM neurons hyper-polarized 3 mV, on average (P 0.0001; n 22; 2 min after washin). Nodetectable changes were observed in TTX-treated SOM neurons (n 17).Traces of individual experiments (A) are divided into 10-s epochs at 0, 1, and2 min after ZD7288 application. Average data (B) include SOM neuronswhere no ZD7288 was applied (n 8). C: ZD7288 decreased threshold currentin control (P 0.006) but not TTX-treated SOM cells when examined fromrest (n 21, 17). D: from a potential of 70 mV, threshold current wasdecreased by a greater fraction in control neurons (P 0.01, n 11, 11),consistent with more IH in control cells.

FIG. 9. No detectable alteration in voltage-dependent Na current withactivity blockade. A: average traces from a single experiment where 10-msdepolarizing steps were used to measure activation. Current traces underwentleak subtraction before peak current was measured. Scale bars: 250 pA, 5 ms.

B: using the protocol in A, no alteration in voltage-dependent activation wasobserved with activity blockade (P 0.9, n 13, 13). C: similarly, no changein voltage-dependent inactivation was observed (P 0.24, n 10, 11).




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The role of ILeak and IH in membraneconductance alterations

How much of the activity-dependent input conductancedecrease (24%) in our young SOM neurons is contributed by

ILeak

? This is difficult to measure because of the lack of aspecific antagonist for the current. Furthermore, experimental

conditions were very different when measuring overall inputconductance versus the voltage ramps measuring ILeak. Here,we make an approximate calculation. The average input con-ductance of control cells from all ramp experiments (Figs. 4and 5) was 6.6 nS, and in these same experiments, the averagedecrease in input conductance with activity blockade was 20%.By virtue of the IV plots (Figs. 4 and 5), this difference waspurely I

Leak. To scale for temperature differences between

experiments, a Q10

based on ion diffusion was applied (1.4;Hille 2001) and resulted in a modified 9.1-nS conductance incontrol cells (75% of the 12.4 nS reported in excitabilityexperiments; Fig. 2D). These data suggest that I

Leakaccounted

for nearly 63% [(0.2/0.24) 0.75] of the subthreshold con-ductance decrease induced by activity blockade.

How much of the activity-dependent input conductancedecrease was contributed by I

H? In our ZD7288 experiments,

we estimated that IH

contributed roughly 10% of the totalconductance at rest in control neurons and that this was totallyabsent with TTX treatment. This would suggest that I

Hac-

counts for 42% (0.10/0.24) of the conductance decrease. Theseapproximate calculations for I

Leakand I

Habove fully account

for the conductance decrement observed with activity block-ade.

The role of ILeak and IH in excitability alterations

We argue that the decrease in ILeak

and IH

with activity

blockade results in an increase in excitability. The best evi-dence for this would be to block both currents and eliminate thedifference in excitability. Unfortunately, we did not have aspecific blocker for I

Leak. We did demonstrate that excitability

differences were decreased with a specific blocker for IH

(Fig.8, C and D). Therefore we rely on this latter data and on thestrong relationship between input conductance and excitabilityto support our assertion.

Regarding IH

, our data are consistent with studies assertingmore I

Hdecreases excitability (Shah et al. 2004; van Welie et

al. 2004). We believe this is attributable to the proximity of theIH

reversal potential (28 6 mV, n 3; K-Meth pipettesolution) to threshold potential (41 1 mV, n 25). This

relationship means that IH contributes more shunting conduc-tance to prevent depolarization than actually providing directdepolarization when membrane potential approaches spikethreshold. This is substantiated by an analytical calculation ofpassive depolarizations in a membrane model using batteriesand conductances (data not shown) (Koester and Siegelbaum2000). We also believe there are circumstances where I

H

increases excitability (Chen et al. 2001) and the nature of the IH

influence on excitability is context dependent.The 49% decrease in threshold current compared with the

24% decrease in input conductance may appear inconsistentwith the assertion that the downregulation of I

Leakand I

H

mediate the excitability decrease (Fig. 2). If voltage thresholdfor spike generation and resting potential remained the same,

then according to Ohms law, the percentage change in thresh-old current and input conductance should be equal.

With this in mind, the input conductance change can accountfor nearly 50% of the change in threshold current, althoughthree additional factors suggest this percentage could behigher. First, we have already estimated that the conductancedecrement and the resting potential increase would account for

78 and 22% of the threshold current change, respectively. Thiscalculation accounts for 65% of the threshold decrement. Inaddition, we applied current steps in larger increments incontrol SOM neurons for faster data collection (100 vs. 50pA). Therefore because of sampling error, our control valuesmay be biased by an added 25 pA, which would mean thethreshold current decrease is actually roughly 40%. This wouldincrement the accounting to 80%. Third, a decreased mem-brane conductance decreases the amount of depolarizing cur-rent needed to evoke an action potential by virtue of a de-creased threshold voltage (Noble 1966), but although our datasuggested this to be true, it was not statistically significant (seeRESULTS).

In summary, we can account for most of the discrepancybetween threshold current and input conductance changes. Thissuggests that the major mechanism for excitability alterationsin SOM neurons is centered in subthreshold current modifi-cations. Alterations in voltage-dependent currents activatednear spike threshold may explain the remaining discrepancy.The same arguments hold for data from older slices.

Coregulation of ILeak and IH

Coregulation of IH

and ILeak

may provide a mechanism forregulating input conductance while preventing extreme mem-brane potential changes produced by regulation of either cur-rent alone. An activity-independent coregulation of IH and IA

occurs in lobster pyloric dilator neurons to maintain circuitoscillatory behavior (MacLean et al. 2003), although to ourknowledge, no activity-dependent coregulation of I

Hand a

potassium current has been reported in mammals. In this study,the activity-dependent change in I

Hat resting potential closely

matched the current provided by ILeak

, strongly suggestingthese offsetting currents were critical for maintaining constantresting potential. A homeostatic regulation of resting potentialmay be important for the proper baseline activation of voltage-dependent currents.

Ionic identity of ILeak

Extracellular alteration of [K] induced an IV plot shift in

the activity-dependent ILeak consistent with a nearly 40%contribution by K. However, the hyperpolarized reversalpotential ofI

Leak, its block by known K channel blockers, and

the absence of a Cl contribution suggest the K contributionmay be higher. Both Ca2 and Na are unlikely contributorsbecause Ca2-channel blockers were used and the 95-mVreversal potential in the IV plots is very distant from that ofCa2 and Na. We speculate the less-than-expected shift in

IV plots with [K]Ext

alterations may reflect buffering occur-ring within the slice like that observed for pH in acute slices(Chesler et al. 1994; Voipio and Kaila 1993). Such a bufferingeffect may be more pronounced in cultured slices because anastrocytic layer develops on the slice surface (Stoppini et al.1991).




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Implications for synaptic integration

Decreases in input conductance in response to activityblockade would increase the amplitude of postsynaptic poten-tials and their temporal summation (Johnston and Wu 1995),which in turn would homeostatically promote action potentialfiring. Interestingly, increased I

Hdecreases excitatory postsyn-

aptic potential size and temporal summation in hippocampalpyramidal neuron dendrites (Magee 1998). A similar processmay occur in SOM neurons, although it is not known howmuch I

Hexists in SOM neuron dendrites. In the same

long-term blockade paradigm used here, excitatory neuronhomeostasis has been reported not to involve subthresholdalterations, and thus subthreshold synaptic integration wouldremain unchanged (Desai et al. 1999b). This suggests thattransformation of synaptic input to action potential output maybe differentially regulated in these two cell types.

Homeostatic regulation of inhibitory circuitry

A previous study demonstrated that inhibitory neurons be-

come more excitable with activity blockade (Desai et al.1999a). We think it likely these neurons were parvalbumin-positive neurons based on the electrophysiological criteria usedand on the numbers of these neurons (roughly 50% of allinhibitory neurons) (Cauli et al. 2000; Kawaguchi and Kubota1997). If this were the case, both parvalbumin-positive andSOM neurons regulate their activity levels consistent with ahomeostatic process. From the excitatory neuron perspective,this regulation in inhibitory neurons would be nonhomeostaticbecause more inhibition is provided with activity blockade.Studies indicate that inhibitory synapses are homeostaticallyregulated (Kilman et al. 2002; Maffei et al. 2004) and maytherefore compensate for the increased excitability in inhibi-

tory neurons. Future studies examining the activity-dependentregulation of SOM inhibitory synapses targeting excitatoryneurons are needed to better understand how total inhibitorydrive originating from SOM neurons is regulated.

A C K N O W L E D G M E N T S

Thanks to Drs. Rolf Joho and Ege Kavalali for helpful discussions.

G R A N T S

This research was supported by a Klingenstein Foundation grant to J. R.Gibson, National Institute of Neurological Disorders and Stroke Grant NS-045711, and McKnight and FRAXA Research Foundations grant to K. M.Huber. K. M. Huber is a Southwestern Medical Foundation endowed scholarin biomedical research.

R E F E R E N C E S

Aizenman CD, Akerman CJ, Jensen KR, and Cline HT. Visually drivenregulation of intrinsic neuronal excitability improves stimulus detection invivo. Neuron 39: 831842, 2003.

Bacci A, Rudolph U, Huguenard JR, and Prince DA. Major differences ininhibitory synaptic transmission onto two neocortical interneuron sub-classes. J Neurosci 23: 96649674, 2003.

Bezanilla F and Armstrong CM. Inactivation of the sodium channel. I.Sodium current experiments. J Gen Physiol 70: 549 566, 1977.

Brewster A, Bender RA, Chen Y, Dube C, Eghbal-Ahmadi M, and BaramTZ. Developmental febrile seizures modulate hippocampal gene expressionof hyperpolarization-activated channels in an isoform- and cell-specificmanner. J Neurosci 22: 45914599, 2002.

Brickley SG, Revilla V, Cull-Candy SG, Wisden W, and Farrant M.

Adaptive regulation of neuronal excitability by a voltage-independent po-tassium conductance. Nature 409: 8892, 2001.

Cauli B, Porter JT, Tsuzuki K, Lambolez B, Rossier J, Quenet B, and

Audinat E. Classification of fusiform neocortical interneurons based onunsupervised clustering. Proc Natl Acad Sci USA 97: 61446149, 2000.

Chagnac-Amitai Y and Connors BW. Synchronized excitation and inhibi-tion driven by intrinsically bursting neurons in neocortex. J Neurophysiol62: 11491162, 1989.

Chen K, Aradi I, Thon N, Eghbal-Ahmadi M, Baram TZ, and Soltesz I.

Persistently modified h-channels after complex febrile seizures convert the

seizure-induced enhancement of inhibition to hyperexcitability. Nat Med 7:331337, 2001.

Chesler M, Chen JC, and Kraig RP. Determination of extracellular bicar-bonate and carbon dioxide concentrations in brain slices using carbonate andpH-selective microelectrodes. J Neurosci Methods 53: 129 136, 1994.

Corner MA and Ramakers GJ. Spontaneous firing as an epigenetic factor inbrain developmentphysiological consequences of chronic tetrodotoxinand picrotoxin exposure on cultured rat neocortex neurons. Brain Res Dev

Brain Res 65: 5764, 1992.Coulter DA, Lo Turco JJ, Kubota M, Disterhoft JF, Moore JW, and

Alkon DL. Classical conditioning reduces amplitude and duration of cal-cium-dependent afterhyperpolarization in rabbit hippocampal pyramidalcells. J Neurophysiol 61: 971981, 1989.

Davis GW and Goodman CS. Genetic analysis of synaptic development andplasticity: homeostatic regulation of synaptic efficacy. Curr Opin Neurobiol8: 149156, 1998.

Deans MR, Gibson JR, Sellitto C, Connors BW, and Paul DL. Synchronousactivity of inhibitory networks in neocortex requires electrical synapsescontaining connexin36. Neuron 31: 477485, 2001.

Desai NS, Cudmore RH, Nelson SB, and Turrigiano GG. Critical periodsfor experience-dependent synaptic scaling in visual cortex. Nat Neurosci 5:783789, 2002.

Desai NS, Rutherford LC, and Turrigiano GG. BDNF regulates the intrinsicexcitability of cortical neurons. Learn Mem 6: 284291, 1999a.

Desai NS, Rutherford LC, and Turrigiano GG. Plasticity in the intrinsicexcitability of cortical pyramidal neurons. Nat Neurosci 2: 515520, 1999b.

Di Cristo G, Wu C, Chattopadhyaya B, Ango F, Knott GW, Welker E,

Svoboda K, and Huang ZJ. Subcellular domain-restricted GABAergicinnervation in primary visual cortex in the absence of sensory and thalamicinputs. Nat Neurosci Advanced Online Publication, 2004.

Fan Y, Fricker D, Brager DH, Chen X, Lu HC, Chitwood RA, andJohnston D. Activity-dependent decrease of excitability in rat hippocampal

neurons through increases in I(h). Nat Neurosci 8: 15421551, 2005.Gibson JR, Beierlein M, and Connors BW. Two networks of electrically

coupled inhibitory neurons in neocortex. Nature 402: 7579, 1999.Goldstein SA, Bockenhauer D, OKelly I, and Zilberberg N. Potassium leak

channels and the KCNK family of two-P-domain subunits. Nat Rev Neurosci2: 175184, 2001.

Gonchar Y and Burkhalter A. Three distinct families of GABAergic neuronsin rat visual cortex. Cereb Cortex 7: 347358, 1997.

Gorba T, Klostermann O, and Wahle P. Development of neuronal activityand activity-dependent expression of brain-derived neurotrophic factormRNA in organotypic cultures of rat visual cortex. Cereb Cortex 9:864877, 1999.

Gupta A, Wang Y, and Markram H. Organizing principles for a diversity ofGABAergic interneurons and synapses in the neocortex. Science 287:273278, 2000.

Hamill OP, Huguenard JR, and Prince DA. Patch-clamp studies of voltage-

gated currents in identified neurons of the rat cerebral cortex. Cereb Cortex1: 48 61, 1991.Han J, Truell J, Gnatenco C, and Kim D. Characterization of four types of

background potassium channels in rat cerebellar granule neurons. J Physiol542: 431444, 2002.

Hendry SH and Jones EG. Activity-dependent regulation of GABA expres-sion in the visual cortex of adult monkeys. Neuron 1: 701712, 1988.

Hensch TK. Critical period regulation. Annu Rev Neurosci 27: 549 579,2004.

Hille B. Ion Channels of Excitable Membranes. Sunderland, MA: Sinauer,2001.

Horton JC and Hubel DH. Regular patchy distribution of cytochromeoxidase staining in primary visual cortex of macaque monkey. Nature 292:762764, 1981.

Huguenard JR, Hamill OP, and Prince DA. Developmental changes in Na

conductances in rat neocortical neurons: appearance of a slowly inactivatingcomponent. J Neurophysiol 59: 778 795, 1988.




14/15

Jin X, Hu H, Mathers PH, and Agmon A. Brain-derived neurotrophic factormediates activity-dependent dendritic growth in nonpyramidal neocortical in-terneurons in developing organotypic cultures.J Neurosci 23: 56625673, 2003.

Johnston D and Wu S. Foundations of Cellular Neurophysiology. Cam-bridge, MA: The MIT Press, 1995.

Kawaguchi Y and Kubota Y. GABAergic cell subtypes and their synapticconnections in rat frontal cortex. Cereb Cortex 7: 476486, 1997.

Kilb W and Luhmann HJ. Characterization of a hyperpolarization-activated

inward current in Cajal-Retzius cells in rat neonatal neocortex. J Neuro-physiol 84: 16811691, 2000.Kilman V, van Rossum MC, and Turrigiano GG. Activity deprivation

reduces miniature IPSC amplitude by decreasing the number of postsynapticGABA(A) receptors clustered at neocortical synapses. J Neurosci 22:13281337, 2002.

Koester J and Siegelbaum SA. Membrane potential. In: Principles of NeuralScience (4th ed.), edited by Kandel ER, Schwartz JH, and Jessel TM. NewYork: McGraw-Hill, 2000, p. 125139.

Lesage F. Pharmacology of neuronal background potassium channels. Neuro-pharmacology 44: 17, 2003.

Lien CC, Martina M, Schultz JH, Ehmke H, and Jonas P. Gating,modulation and subunit composition of voltage-gated K() channels indendritic inhibitory interneurones of rat hippocampus. J Physiol 538: 405419, 2002.

Lissin DV, Gomperts SN, Carroll RC, Christine CW, Kalman D, Kitamura

M, Hardy S, Nicoll RA, Malenka RC, and von Zastrow M. Activity

differentially regulates the surface expression of synaptic AMPA and NMDAglutamate receptors. Proc Natl Acad Sci USA 95: 70977102, 1998.

Liu G. Local structural balance and functional interaction of excitatory andinhibitory synapses in hippocampal dendrites. Nat Neurosci 7: 373379,2004.

Lupica CR, Bell JA, Hoffman AF, and Watson PL. Contribution of thehyperpolarization-activated current (I(h)) to membrane potential and GABArelease in hippocampal interneurons. J Neurophysiol 86: 261268, 2001.

Maccaferri G and McBain CJ. The hyperpolarization-activated current (Ih)and its contribution to pacemaker activity in rat CA1 hippocampal stratumoriens-alveus interneurones. J Physiol 497: 119 130, 1996.

MacLean JN, Zhang Y, Johnson BR, and Harris-Warrick RM. Activity-independent homeostasis in rhythmically active neurons. Neuron 37: 109120, 2003.

Maffei A, Nelson SB, and Turrigiano GG. Selective reconfiguration of layer 4visual cortical circuitry by visual deprivation. Nat Neurosci 7: 13531359,

2004.Magee JC. Dendritic hyperpolarization-activated currents modify the integra-tive properties of hippocampal CA1 pyramidal neurons. J Neurosci 18:76137624, 1998.

Marder E and Prinz AA. Modeling stability in neuron and network function:the role of activity in homeostasis. Bioessays 24: 11451154, 2002.

Martina M, Vida I, and Jonas P. Distal initiation and active propagation ofaction potentials in interneuron dendrites. Science 287: 295300, 2000.

Marty S, Wehrle R, and Sotelo C. Neuronal activity and brain-derivedneurotrophic factor regulate the density of inhibitory synapses in organo-typic slice cultures of postnatal hippocampus. J Neurosci 20: 80878095,2000.

Meuth SG, Budde T, Kanyshkova T, Broicher T, Munsch T, and Pape

HC. Contribution of TWIK-related acid-sensitive K channel 1 (TASK1)and TASK3 channels to the control of activity modes in thalamocorticalneurons. J Neurosci 23: 64606469, 2003.

Micheva KD and Beaulieu C. An anatomical substrate for experience-

dependent plasticity of the rat barrel field cortex. Proc Natl Acad Sci USA92: 1183411838, 1995.

Miles R, Toth K, Gulyas AI, Hajos N, and Freund TF. Differences betweensomatic and dendritic inhibition in the hippocampus. Neuron 16: 815823,1996.

Murthy VN, Schikorski T, Stevens CF, and Zhu Y. Inactivity producesincreases in neurotransmitter release and synapse size. Neuron 32: 673682,2001.

Musleh W, Bi X, Tocco G, Yaghoubi S, and Baudry M. Glycine-inducedlong-term potentiation is associated with structural and functional modifi-cations of alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid re-ceptors. Proc Natl Acad Sci USA 94: 94519456, 1997.

Neher E. Correction for liquid junction potentials in patch clamp experiments.Methods Enzymol 207: 123131, 1992.

Nelson AB, Krispel CM, Sekirnjak C, and du Lac S. Long-lasting increasesin intrinsic excitability triggered by inhibition. Neuron 40: 609620, 2003.

Noble D. Applications of HodgkinHuxley equations to excitable tissues.Physiol Rev 46: 150, 1966.

Oliva AA Jr, Jiang M, Lam T, Smith KL, and Swann JW. Novelhippocampal interneuronal subtypes identified using transgenic mice thatexpress green fluorescent protein in GABAergic interneurons. J Neurosci20: 33543368, 2000.

Pape HC. Queer current and pacemaker: the hyperpolarization-activatedcation current in neurons. Annu Rev Physiol 58: 299 327, 1996.

Reyes A, Lujan R, Rozov A, Burnashev N, Somogyi P, and Sakmann B.Target-cell-specific facilitation and depression in neocortical circuits. Nat

Neurosci 1: 279285, 1998.Robinson RB and Siegelbaum SA. Hyperpolarization-activated cation cur-

rents: from molecules to physiological function. Annu Rev Physiol 65:453480, 2003.

Santoro B, Chen S, Luthi A, Pavlidis P, Shumyatsky GP, Tibbs GR, and

Siegelbaum SA. Molecular and functional heterogeneity of hyperpolariza-

tion-activated pacemaker channels in the mouse CNS. J Neurosci 20:52645275, 2000.

Scholl DA. The Organization of Cerebral Cortex. London: Methuen, 1956.Seil FJ and Drake-Baumann R. Reduced cortical inhibitory synaptogenesis

in organotypic cerebellar cultures developing in the absence of neuronalactivity. J Comp Neurol 342: 366 377, 1994.

Shah MM, Anderson AE, Leung V, Lin X, and Johnston D. Seizure-induced plasticity of h channels in entorhinal cortical layer III pyramidal

neurons. Neuron 44: 495508, 2004.Sillito AM. Proceedings: modification of the receptive field properties of

neurones in the visual cortex by bicuculline, a GABA antagonist. J Physiol239: 36P37P, 1974.

Somogyi P, Tamas G, Lujan R, and Buhl EH. Salient features of synapticorganisation in the cerebral cortex. Brain Res Brain Res Rev 26: 113135, 1998.

Sourdet V, Russier M, Daoudal G, Ankri N, and Debanne D. Long-termenhancement of neuronal excitability and temporal fidelity mediated by metabo-tropic glutamate receptor subtype 5. J Neurosci 23: 1023810248, 2003.

Stoppini L, Buchs PA, and Muller D. A simple method for organotypiccultures of nervous tissue. J Neurosci Methods 37: 173182, 1991.

Stuart GJ, Dodt HU, and Sakmann B. Patch-clamp recordings from thesoma and dendrites of neurons in brain slices using infrared video micros-copy. Pfluegers Arch 423: 511518, 1993.

Talley EM, Lei Q, Sirois JE, and Bayliss DA. TASK-1, a two-pore domainK channel, is modulated by multiple neurotransmitters in motoneurons.

Neuron 25: 399410, 2000.Thoby-Brisson M and Simmers J. Long-term neuromodulatory regulation ofa motor pattern-generating network: maintenance of synaptic efficacy andoscillatory properties. J Neurophysiol 88: 29422953, 2002.

Turrigiano G, LeMasson G, and Marder E. Selective regulation of currentdensities underlies spontaneous changes in the activity of cultured neurons.

J Neurosci 15: 36403652, 1995.Turrigiano GG, Leslie KR, Desai NS, Rutherford LC, and Nelson SB.

Activity-dependent scaling of quantal amplitude in neocortical neurons.Nature 391: 892896, 1998.

Turrigiano GG and Nelson SB. Hebb and homeostasis in neuronal plasticity.Curr Opin Neurobiol 10: 358 364, 2000.

Turrigiano GG and Nelson SB. Homeostatic plasticity in the developingnervous system. Nat Rev Neurosci 5: 97107, 2004.

van Welie I, van Hooft JA, and Wadman WJ. Homeostatic scaling ofneuronal excitability by synaptic modulation of somatic hyperpolarization-activated Ih channels. Proc Natl Acad Sci USA 101: 51235128, 2004.

van Welie I, van Hooft JA, and Wadman WJ. Background activity regulatesexcitability of rat hippocampal CA1 pyramidal neurons by adaptation of aK conductance. J Neurophysiol 95: 20072012, 2006.

van Welie I, Wadman WJ, and van Hooft JA. Low affinity block of nativeand cloned hyperpolarization-activated Ih channels by Ba2 ions. Eur

J Pharmacol 507: 1520, 2005.Voipio J and Kaila K. Interstitial PCO2 and pH in rat hippocampal slices

measured by means of a novel fast CO2/H()-sensitive microelectrodebased on a PVC-gelled membrane. Pfluegers Arch 423: 193201, 1993.

Washburn CP, Sirois JE, Talley EM, Guyenet PG, and Bayliss DA.

Serotonergic raphe neurons express TASK channel transcripts and a TASK-like pH- and halothane-sensitive K conductance. J Neurosci 22: 12561265, 2002.

Whittington MA and Traub RD. Interneuron diversity series: inhibitory inter-neurons and network oscillations in vitro. Trends Neurosci 26: 676 682, 2003.

Zar JH. Biostatistical Analysis. Upper Saddle River, NJ: Prentice Hall, 1999.




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Corrigenda

Volume 96, July 2006

Pages 420 432: Gibson JR, Bartley AF, and Huber KM. Role for the Subthreshold Currents ILeakand IH in the Homeostatic Control of Excitability in Neocortical Somatostatin-Positive InhibitoryNeurons (doi:10.1152/jn.01203.2005; http://jn.physiology.org/cgi/content/full/96/1/420). The fol-lowing sentence in the 2nd paragraph in METHODS (9th sentence under heading Electrophysiology)is in error:

The capacitance was obtained by multiplying the series resistance by the fastest time constant ofa double-exponential decay calculated fitted to the first 20 ms of the capacitive transient decayinduced by a voltage step.

The sentence should read:The capacitance was calculated by first obtaining the decay time constant of a current transient

induced by a voltage step (the faster time constant of a double-exponential decay fitted to the first20 ms) and then dividing this by the series resistance.

Volume 96, December 2006

Pages 28892904: Orbn G, Kiss T, and rdi P. Intrinsic and Synaptic Mechanisms Determiningthe Timing of Neuron Population Activity During Hippocampal Theta Oscillation (doi:10.1152/

jn.01233.2005; http://jn.physiology.org/cgi/content/full/96/6/2889). In the methods section, underthe heading Neuron types, in the first sentence, Warman et al. (1994) is erroneously cited; thecorrect reference is Varona et al. (2000), which was not included in the reference list. Therefore,the sentence should read: The multicompartmental pyramidal cell model was an extended versionof the 256 compartmental model of Varona et al. (2000). The full citation information is asfollows: Varona P, Ibarz JM, Lopez-Aguado L, Herreras O. Macroscopic and subcellular factorsshaping population spikes. J Neurophysiol 83: 2192-2208, 2000.

J Neurophysiol 97: 1869, 2007;doi:10.1152/jn.z9k-8066-corr.2007.

Documents

Jay R. Gibson et al- Role for the Subthreshold Currents ILeak and IH in the Homeostatic Control of Excitability in Neocortical Somatostatin-Positive Inhibitory Neurons