doi:10.1152/jn.00714.2013 112:1169-1178, 2014. First published 28 May 2014;J NeurophysiolMichael C. Salling and Neil L. Harrisonare tonically activatedneurons in layers II/III of the mouse prefrontal cortex Strychnine-sensitive glycine receptors on pyramidal

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Strychnine-sensitive glycine receptors on pyramidal neurons in layers II/IIIof the mouse prefrontal cortex are tonically activated

Michael C. Salling1 and Neil L. Harrison1,2

1Department of Anesthesiology, Columbia University Medical Center, New York, New York; 2Department of Pharmacology,Columbia University, New York, New York

Submitted 4 October 2013; accepted in final form 27 May 2014

Salling MC, Harrison NL. Strychnine-sensitive glycine receptorson pyramidal neurons in layers II/III of the mouse prefrontal cortexare tonically activated. J Neurophysiol 112: 1169–1178, 2014. Firstpublished May 28, 2014; doi:10.1152/jn.00714.2013.—Processing ofsignals within the cerebral cortex requires integration of synapticinputs and a coordination between excitatory and inhibitory neu-rotransmission. In addition to the classic form of synaptic inhibition,another important mechanism that can regulate neuronal excitability istonic inhibition via sustained activation of receptors by ambient levelsof inhibitory neurotransmitter, usually GABA. The purpose of thisstudy was to determine whether this occurs in layer II/III pyramidalneurons (PNs) in the prelimbic region of the mouse medial prefrontalcortex (mPFC). We found that these neurons respond to exogenousGABA and to the �4�-containing GABAA receptor (GABAAR)-selective agonist gaboxadol, consistent with the presence of extrasyn-aptic GABAAR populations. Spontaneous and miniature synapticcurrents were blocked by the GABAAR antagonist gabazine and hadfast decay kinetics, consistent with typical synaptic GABAARs. Veryfew layer II/III neurons showed a baseline current shift in response togabazine, but almost all showed a current shift (15–25 pA) in responseto picrotoxin. In addition to being a noncompetitive antagonist atGABAARs, picrotoxin also blocks homomeric glycine receptors(GlyRs). Application of the GlyR antagonist strychnine caused amodest but consistent shift (�15 pA) in membrane current, withoutaffecting spontaneous synaptic events, consistent with the tonic acti-vation of GlyRs. Further investigation showed that these neuronsrespond in a concentration-dependent manner to glycine and taurine.Inhibition of glycine transporter 1 (GlyT1) with sarcosine resulted inan inward current and an increase of the strychnine-sensitive current.Our data demonstrate the existence of functional GlyRs in layer II/IIIof the mPFC and a role for these receptors in tonic inhibition that canhave an important influence on mPFC excitability and signal process-ing.

glycine; GABA; tonic current; prefrontal cortex

THE PREFRONTAL CORTEX (PFC) is a heavily interconnectedcortical subregion that plays a prominent role in the orga-nization of behavior. Abnormal PFC activity has been im-plicated in psychiatric illnesses such as schizophrenia anddepression (Elliott et al. 1997; Weinberger et al. 1986; Yoonet al. 2008), and environmental influences like stress anddrug addiction (e.g., alcohol, cocaine) can lead to a long-term depression of PFC activity (Abernathy et al. 2010;Goldstein and Volkow 2011). The rodent PFC has a con-siderably smaller volume relative to brain size than is thecase in humans, yet it remains a valuable model to study

PFC signaling as it retains many essential features of theprimate PFC, including the same cell types and circuitry,while mediating similar behavioral functions like workingmemory, behavioral flexibility, and attention (Chudasamaand Robbins 2006; Seamans et al. 2008; Uylings et al.2003). A better understanding of PFC connectivity andphysiology is essential in providing the context in whichprefrontal dysfunction can be understood.

The cortex has a distinct laminar organization that servesto integrate cortical and subcortical signals. In the rodentmedial (m)PFC, the conventional view is that the superficiallayers (I, II, III) are input layers receiving cortical andmediodorsal thalamic afferents while the deeper layers (V,VI) project to output regions like striatum and thalamus(Krettek and Price 1977; Ongur and Price 2000). Localcortical processing between layers is complex, with a myr-iad of interneuron subtypes forming distinct connectionswith dendritic, somatic, and axonal subcompartments ofpyramidal neurons (PNs) and other interneurons (DeFelipeet al. 2013; Fino et al. 2013; Rudy et al. 2011). Layer II/III(LII/III) PNs are known to be sparsely activated and receivea dense inhibitory synaptic input from a rich variety ofinterneurons including parvalbumin-containing fast-spiking(FS), somatostatin-containing, and neurogliaform interneu-rons, to provide tight control of neuronal excitability (Olahet al. 2009; Petersen and Crochet 2013).

Neuronal activity in the cortex may also be regulated bytonic inhibition. Tonic inhibition occurs when ambient levelsof GABA (originating from synaptic “spillover” or via releasefrom glia) cause sustained activation of a population of highlysensitive receptors located in the peri- or extrasynaptic space(Belelli et al. 2009; Farrant and Nusser 2005). This phenom-enon has been described in several brain regions, including thecerebellum (Brickley et al. 1996), hippocampus (Nusser andMody 2002), thalamus (Cope et al. 2005; Jia et al. 2005), andsomatosensory cortex (Salin et al. 1995; Yamada et al. 2007).The magnitude of these tonic currents can be increased byinhibiting GABA transporters (Semyanov et al. 2003; Vardyaet al. 2008), emphasizing the important role of transporters inregulating tonic currents through the local control of neu-rotransmitter availability.

GABAA receptors (GABAARs) that are located extrasynap-tically have been identified as essential to the generation oftonic inhibition. These receptors are composed primarily ofcombinations of �4/6-, �2/3-, and �-subunits (Belelli et al. 2009)or �5-, �2/3-, and �2-subunits (Belelli et al. 2009). Extrasyn-aptic receptors have a high affinity for GABA, with a slowdesensitization rate, permitting a sustained elevation in

Address for reprint requests and other correspondence: M. C. Salling, Depts.of Anesthesiology and Pharmacology, Columbia Univ. College of Physiciansand Surgeons, 630 West 168th St., Rm. 7-422, New York, NY 10032 (e-mail:ms4431@columbia.edu).

J Neurophysiol 112: 1169–1178, 2014.First published May 28, 2014; doi:10.1152/jn.00714.2013.

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chloride conductance even at very low concentrations ofGABA (Bai et al. 2001; Brickley et al. 1996; Saxena and Macdonald1994). In the ventrobasal thalamus, �4�2�-containing GABAARsmediate tonic current and control the excitability of relayneurons (Jia et al. 2005, 2008). In the superficial layers of therodent PFC these GABA �4- and �-subunits appear to behighly expressed (Peng et al. 2002; Pirker et al. 2000), yetGABA-mediated tonic inhibition has not been reported (Hoe-stgaard-Jensen et al. 2010). Here we test the hypothesis thattonic inhibition occurs in layer II/III of the prelimbic region ofthe mPFC, and we show that a tonic current mediated byglycine receptors (GlyRs), but not GABAARs, exists in themajority of these neurons.

METHODS

Experimental procedures were performed in male C57BL/6J mice(Jackson Laboratories) under the guidelines set forth in the NationalInstitutes of Health Guide for the Care and Use of LaboratoryAnimals and with the approval of the Institutional Animal Care andUse Committee of Columbia University.

Brain slice preparation. Mice (25–50 days old) were fully anes-thetized with sevoflurane and decapitated into ice-cold (4°C) artificialcerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 2.5 KCl, 26NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 2 MgSO4, and 10 glucose. Brainswere dissected and sectioned in cold ACSF with a vibrating mi-crotome (Leica VT1000S) into coronal slices (300 �m) that containedthe prelimbic region of the mPFC at between �2.8 and �1.78 mmfrom bregma, according to a mouse brain atlas (Franklin and Paxinos1997). Slices were then incubated at 32°C in oxygenated (bubbledwith 95% O2-5% CO2) ACSF for �30–45 min and then moved toroom temperature (22–25°C) for at least 45 min before recordingsbegan.

Slice electrophysiology. Slices were placed in a submersion cham-ber and continuously superfused with room-temperature oxygenatedACSF. mPFC neurons were visualized under an upright light micro-scope (Olympus BX51WI) using infrared and differential interferencecontrast. PFC cortical layers were identified under a �4 objective(layer II/III between �100 and 300 �m and layer V/VI between 350and 500 �m from the pial surface), and PNs were identified under a�40 objective by their characteristic size and shape. Pipettes (open tipresistance 2–5 M� for CsCl and 3–6 M� for K-gluconate solutions)were pulled from borosilicate glass (World Precision Instruments,Sarasota, FL) by a pipette puller (Sutter Instrument, Novato, CA) andused for electrophysiological recordings. Data were collected with aMulticlamp 700B amplifier (Axon Instruments, Union City, CA) andClampex 10.2 Software (Molecular Devices, Sunnyvale, CA) inidentified PNs after reaching a �1-G� seal and after minimization ofcapacitative currents. Data were collected at 10 kHz and low-passfiltered at 2 kHz. For whole cell recordings under current-clampconditions, a standard intracellular pipette solution was used (in mM:130 K�-gluconate, 5 NaCl, 2 MgCl2, 10 HEPES, 0.2 EGTA, 2ATP-K�, 0.3 GTP-Na�) and data collection was initiated �5 minafter achieving whole cell configuration. Passive membrane properties[input resistance (IR), membrane capacitance, time constant] weremeasured from the resting membrane potential (RMP) with 20-pAcommand increments (6 steps starting at �60 pA, 500 ms), and firingproperties (amplitude, frequency, accommodation, I/O relationship)were measured with 40-pA command increments (21 steps starting at�400 pA, 500 ms). To maximize chloride currents, recordings madeunder voltage-clamp conditions used a high-chloride intracellularsolution (in mM: 140 CsCl, 4 NaCl, 1 MgCl2, 10 HEPES, 0.05 EGTA,2 Mg-ATP, 0.3 GTP-Na�) adjusted to �295 mOsm by addingsucrose. To allow equilibration, 7 min of recording occurred prior todata acquisition and only cells demonstrating a stable baseline current

were used in the experiment. For phasic events, neurons were voltageclamped at �70 mV, 30-s epochs were collected for analysis ofspontaneous postsynaptic currents (sIPSCs), and 45-s epochs werecollected for analysis of miniature inhibitory postsynaptic currents(mIPSCs) before and after drug application, allowing time for stabi-lization of the baseline and drug application. Access resistance wasmonitored for all cells in voltage clamp via 10-mV hyperpolarizingtest pulses, and any cell with access resistance that changed �25%from the start of recording or reached �20 M� was omitted from thedata analysis.

Biocytin visualization. To identify neurons, recordings were per-formed with a modification of the current-clamp procedure and sliceswere cut at 150-�m thickness to enable better visualization of 0.5%biocytin (Invitrogen, Carlsbad, CA), which was added to the pipettesolution. After measurement of passive membrane and firing proper-ties, depolarizing current (�200 pA) was injected at 2 Hz for 15 min.After recording, slices were incubated in ACSF at room temperaturefor 45 min and then placed in 4% PFA for 1 h. Slices were thenprocessed with the Vectastain ABC kit (Vector Labs, Burlingame,CA) and reacted with the chromogen diaminobenzidine. Filled cellswere then examined under a low-power microscope, and images oflabeled cells were collected with imaging software (HC Image) on alight microscope (Olympus BX51WI).

Pharmacology. Drug solutions were applied to the slice by bathperfusion. Stock solutions were made by dissolving drugs in ACSF,adjusting pH to 7.4, and freezing. On the day of recording, drug stocksolutions were diluted to yield the appropriate concentration. Glycine,taurine, strychnine, picrotoxin, and sarcosine were purchased fromSigma (St. Louis, MO). Gabazine, gaboxadol {4,5,6,7-tetrahydroiso-thiazolo-[5,4-c]pyridine-3-ol hydrochloride (THIP)}, tetrodotoxin(TTX), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione (NBQX), and D-(�)-2-amino-5-phosphonopentanoic acid (D-AP5) were purchased from Tocris (Bristol, UK).

Data analysis. Off-line analyses of current-clamp and voltage-clamp data were performed with Clampfit 10.3.02 software. Forchanges in baseline current, 0.75- to 1.5-s epochs before and afterexperimental drug applications that contained minimal spontaneousevents were selected and all-points histograms were created. Thesedata were fitted with a single Gaussian curve. Responses to concen-trations of GABA and glycine were normalized to a common agonistconcentration in each cell (1 mM for GABA, 3 mM for glycine),analyzed with a nonlinear regression and fit to the Hill equation I �Imax � [agonist]nH/([agonist]nH � EC50

nH), where I is the peak current,Imax is the maximal whole cell current amplitude, [agonist] is theagonist concentration eliciting a half-maximal current response, andnH is the Hill coefficient. Mini Analysis software (Synaptosoft,Decatur, GA) was used to detect synaptic events �18 pA in amplitude4 times root mean square noise for all cells and 30 pA·ms in area.Amplitude, frequency, rise time (10–90%), and decay time weremeasured and compared before and after drug application. Graphingand statistical analyses were performed with GraphPad Prism soft-ware. Where appropriate, Student’s paired t-test and ANOVA wereused to test for statistical significance (P � 0.05). Data are expressedas means SE.

RESULTS

Characterization of pyramidal neurons in mouse prefrontalcortex. Many electrophysiological studies in the mouse PFChave focused on PNs in layer V/VI. In the present study, werecorded from a large number of neurons in layer II/III andmeasured their passive and active membrane properties usingpipettes filled with K-gluconate-based intracellular solutions.A subset of layer II/III cells identified by their large size andtriangular cell body shape were filled with biocytin (Fig. 1A)and confirmed to be LII/III PNs of the prelimbic region of the

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mPFC on the basis of morphological and electrophysiologicalproperties. PFC PNs have specific intrinsic and firing proper-ties that distinguish them from GABAergic interneurons, theother principal neuronal subtype within layer II/III (Amatrudoet al. 2012; Kawaguchi 1993).

In total, identified LII/III PNs (n � 30) had a RMP of �67.9 1 mV, IR � 156 9 M�, membrane time constant (�m) � 28 2 ms, membrane capacitance (Cm) � 176 7 pF, rheobase �111 10 pA, and firing threshold � �38 1 mV (Table 1), allof which are consistent with previous reports for PNs in thisarea (Amatrudo et al. 2012; Kawaguchi 1993). When pro-longed depolarizing current injections were made at RMP, theLII/III neurons fired in a pattern characteristic of PNs, includ-ing pronounced accommodation of firing rate. Accommodationindex (AI) � 0.5 0.02 was expressed as the ratio of theinterspike frequency for the last two spikes versus the first twospikes (Fig. 1E, a and b) during a 500-ms, 240-pA command.A pronounced “sag” in the electrotonic potential (�3.0 0.5mV) was observed in response to larger hyperpolarizing cur-rents. This was expressed as the voltage difference between thepeak and the steady state during a 500-ms, �400-pA current(Fig. 1Ed). Afterdepolarization was defined as the peak voltagedifference between RMP and the overshoot following�400-pA current (Fig. 1Ec). Morphological analysis of LII/III

neurons successfully filled with biocytin and retrieved afterprocessing the slices (n � 8) revealed extensive apical den-drites that extended into layer I and cell bodies located in layerII/III of the prelimbic region of the PFC (Franklin and Paxinos2008). We conclude that we have been able to identify neuronsin layers II/III with the morphological and electrical signaturestypical of PNs (accommodation, voltage sag).

PNs were then recorded from layers V/VI to comparemembrane and firing properties between layers (Table 1).These neurons showed a slightly hyperpolarized RMP of�69.5 1. Student’s t-test revealed several significant differ-ences in PNs between layers, where layer V/VII PNs hadhigher IR (102 9 M�, P 0.001), �m (16 1 ms, P 0.001), and rheobase (155 14 pA, P 0.05) while Cm wassimilar (170 13 pF). Additionally, sag was significantlygreater in layer V/VI (sag � 4.0 0.5, P 0.05). It should benoted that when recorded at room temperature (Amatrudo et al.2012; Kawaguchi 1993), cortical PNs have higher IR and lowerrheobase than at more physiological temperatures withouteffects on RMP and Ih (Day et al. 2005; Thuault et al. 2013).For a detailed description of the effects of recording tempera-ture on cortical PNs, see Hedrick and Waters (2012).

Synaptic events. First, we recorded spontaneous synapticevents in layer II/III of the mPFC, using a CsCl-based intra-cellular solution. We found that gabazine (20 �M, n � 6)blocked 98% of all spontaneous transient events (Fig. 2A)recorded under voltage clamp at �70 mV (Fig. 2B), whichindicated that the majority of synaptic events were sIPSCsmediated by GABA acting at GABAARs. The GlyR antagoniststrychnine (1 �M) did not cause any significant changes infrequency (Fig. 2) or amplitude of the sIPSCs. Next, weisolated mIPSCs by blocking sodium channels with TTX andAMPA and NMDA receptor-mediated currents with NBQX(10 �M) and D-AP5 (50 �M). The properties of these mIPSCsare summarized in Table 2 and indicate that essentially all ofthese events are fast, with rise times �2 ms and decay timeconstant lasting �4–5 ms. After application of strychnine wefound that amplitude and frequency of mIPSCs were un-

12/3

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A B C

D

E

Fig. 1. A: schematic illustrating location of whole cell recordings in layer II/III region of prelimbic cortex (dashed line). B and C: pipettes were directed at layerII/III (�4, B), and pyramidal neurons (PNs) were identified by their triangular shape with infrared-differential interference contrast (IR-DIC) optics (�40, C).D: biocytin-filled neuron confirming PN morphology in recorded cell. E: current-clamp recording of firing pattern of filled neuron in D at resting membranepotential (�68 mV) showing single action potential after 80-pA stimulus (black spike) and multiple action potentials after 380-pA stimulus (gray spikes).Accommodation (index measured from a/b) that is characteristic of cortical PNs is present. Additionally, other properties of PNs including sag (d) and anafterdepolarization (c) are present in this neuron. Active and passive membrane properties of layer (L)II/III PNs are summarized in Table 1.

Table 1. Passive and active membrane properties of layer II/IIIand V/VI PNs located in the prelimbic region of the PFC

Layer II/III Layer V/VI P value

RMP, mV �67.9 1 �69.5 1 0.21Input resistance, M� 156 � 9 102 � 9 <0.001Membrane capacitance, pF 176 7 170 13 0.73�m, ms 28 � 2 16 � 1 <0.001Rheobase, pA 111 � 10 155 � 14 <0.05Sag, mV 2.7 � 0.4 4.0 � 0.5 <0.05ADP, mV 3.2 0.4 3.5 0.4 0.61

Values are means SE; n � 30 for each group. PN, pyramidal neuron; PFC,prefrontal cortex; RMP, resting membrane potential; �m, membrane timeconstant; ADP, afterdepolarization. P values, unpaired t-test; significant valuesare in boldface.

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changed, although we noted a small change in decay time ofmIPSCs (Table 2; P 0.05, t-test).

LII/III PN pharmacology: responses to GABA and THIP. Tofurther study the presence and sensitivity of GABAARs onPNs, exogenous GABA (1 �M–3 mM) was applied to PNsunder voltage-clamp conditions after stabilization of baselinerecording current. Responses to GABA were then normalizedto the response to 1 mM GABA (n � 7). A paired t-testrevealed a significant inward current following application of10 �M GABA (�40 13 pA, P 0.05) and above (Fig. 3A).A concentration-response curve was generated (Fig. 3B) andfitted to the data with the Hill equation, yielding a GABA EC50of 353 �M and Hill slope � 1.43.

We also investigated whether �-containing GABAARs werepresent with the selective agonist THIP. We observed a modestbut significant response (�14 4 pA, P 0.05, paired t-test)to a low concentration of THIP (0.3 �M, n � 5) that selectivelyactivates �-containing GABAARs. Application of 1.0 �MTHIP (n � 9) elicited a larger response (�56 7 pA, P 0.005, paired-t-test) (Fig. 4A). Responses to THIP were fullyblocked by the GABAAR antagonist gabazine (20 �M) (Fig.4). These results suggest that �-containing GABAARs arelikely present on LII/III PNs and that they can be fullyinhibited by gabazine.

Picrotoxin- and strychnine-sensitive tonic inhibitory current. Thepresence of a tonic inhibitory current is common in forebrainregions and can be routinely demonstrated by applying aGABAA receptor (GABAAR) antagonist and measuring anoutward (positive) shift in baseline current under conditionswhere internal chloride is high and tonic currents are inwardlydirected at �70 mV (e.g., Jia et al. 2009). To investigate thepresence of a GABAergic tonic current in mPFC LII/III PNs,we applied gabazine (20 �M) at a concentration that haspreviously been shown to block GABA-mediated tonic inhibi-

tion in thalamic relay neurons (Jia et al. 2009). We did notobserve a significant reproducible change in baseline current(�3.0 3 pA, n � 13, only 4 of 13 neurons with a shift � 6pA), despite a near-total block of synaptic events by gabazine(Fig. 5A), indicating the absence of a GABA-mediated toniccurrent in these neurons. Some labs report that higher concen-trations of gabazine are needed to demonstrate a tonic current(Bai et al. 2001). Increasing the concentration of gabazine (50�M) also did not alter the results in our hands (�3.9 6 pA,n � 7, 3 of 7 neurons with a shift � 6 pA). In our next seriesof experiments, we applied picrotoxin (PTX, 100 �M), acompound that blocks the chloride channel of open GABAARs.After application of PTX, we routinely observed a large pos-itive shift in baseline current (�21 4 pA, P 0.005, n � 9);eight of nine neurons had a shift � 6 pA (Fig. 5, B and D). Inaddition to GABAARs, PTX is known to inhibit other ligand-gated chloride channels, including homomeric GlyRs (Pribillaet al. 1992; Yoon et al. 2008). We subsequently found thatapplication of strychnine (1 �M), a selective GlyR antagonist,caused a reproducible (9 of 12 neurons � 6 pA) and significantshift (15 3 pA, P 0.005, n � 12) in baseline holdingcurrent (Fig. 5, C and D). From these results, we conclude thatLII/III neurons have a strychnine- and PTX-sensitive toniccurrent.

Glycine and taurine. The presence of a tonic strychnine-sensitive current in PFC LII/III neurons was surprising, asthere have been few reports on the existence of functionalGlyRs in the cortex using slice electrophysiology. In our nextexperiment, we set out to characterize LII/III PN sensitivity toendogenous ligands of GlyRs, beginning with glycine itself(0.01–10 mM). Bath-applied glycine elicited significant re-sponses at a concentration of 100 �M (P 0.05, n � 7) andEC50 of 447 �M, Hill slope � 2.56, and very large responses(�1.5 nA) at the highest concentrations tested (3 and 10 mM)

Table 2. Characteristics of synaptic events (sIPSCs and mIPSCs) summarized for neurons before and after application of 1 �Mstrychnine

Events (cells) Freq, Hz Amp, pA Rise, ms Decay, ms

sIPSCACSF 1,239 (6) 6.8 2 39.4 2 2.6 0.1 8.6 0.6STRY 1,175 (6) 6.7 2 38.8 3 2.7 0.1 7.8 0.6

mIPSCACSF 578 (5) 2.4 0.8 33 4 2.2 0.2 5.3 � 0.4STRY 439 (5) 1.8 0.7 29 3 2.2 0.2 4.3 � 0.3

Values are mean SE characteristics of synaptic events [spontaneous (sIPSC) and miniature (mIPSC) inhibitory postsynaptic currents] summarized forneurons before (ACSF) and after application of 1 �M strychnine (STRY). Significant changes in mIPSC decay time (P 0.05) are in boldface.

Fig. 2. A: representative recordings of spon-taneous synaptic events under voltage-clampconditions using high-internal chloride solu-tion under control (ACSF), 20 �M gabazine(GBZ, light gray), or 1 �M strychnine (STRY,dark gray) demonstrating near-completeblock of events by gabazine and no effect ofstrychnine. B: frequency (Hz) of synapticevents is plotted before (ACSF) and afterapplication of 20 �M gabazine or 1 �Mstrychnine. Properties of synaptic inhibitorycurrents are summarized in Table 2.

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(Fig. 6). Application of taurine (0.1–10 mM) also inducedmeasurable current at 100 �M (P 0.05, n � 7) but was lesspotent than glycine at several concentrations (100 �M–10mM). All responses to glycine and taurine were normalized tothe response to 3 mM glycine, established at the end of eachrecording after a washout period (�5 min) (Fig. 6B).

We then elicited responses (112 24 pA) to an �EC20concentration of glycine (200 �M) recorded in the absence andpresence of strychnine (1 �M) to determine whether theseglycine-activated currents are mediated by strychnine-sensitiveGlyRs. Strychnine applications blocked the response to glycine(Fig. 7A) and generated a positive shift in the baseline current(�22 9 pA) (Fig. 7B). Glycine is known to be an essentialcoagonist at the NMDA receptor (NMDAR). To examine thecontribution of NMDARs, we next measured the response to200 �M glycine before and after inhibition of NMDARs withD-AP5. We did not observe any significant difference of the200 �M glycine response in the presence of D-AP5 (Fig. 7C).

We next sought to examine the possible role of glycinetransporters (GlyTs) in regulating the availability of glycine inthe slice. We found that application of sarcosine, a potentinhibitor of GlyT1, caused a significant inward current(�63.5 13 pA) at 500 �M (P 0.001, t-test), which couldbe largely blocked (�75%) by strychnine (1 �M) (Fig. 8). Inaddition, GlyT1 inhibition increased the magnitude of the

strychnine-sensitive current by �30 pA, from �15 pA to 45pA (Fig. 8B).

DISCUSSION

Characterization of pyramidal neurons in mouse prefrontalcortex. Our investigation of passive and active membraneproperties in layers II/III and layers V/VI of mouse PFCidentified several layer-specific characteristics. PNs in layersII/II had higher IR, longer �m, and decreased rheobase, provid-ing evidence that they are more excitable than PNs located inlayer V/VI. Additionally, we found that both layers exhibit ameasurable sag and afterdepolarization in response to hyper-polarizing stimuli consistent with the presence of Ih. LayerV/VI PNs showed a larger sag on average, consistent withfindings that HCN1 channels predominate in layer V/VI PNs ofPFC, where they are important for establishing the RMP andfor the generation of persistent firing (Thuault et al. 2013).

Synaptic GABAAR signaling. Spontaneous synaptic GABA-mediated transmission was observed in LII/III PNs. Underconditions of high internal chloride, we observed frequent (�7Hz) spontaneous gabazine-sensitive currents averaging �40pA in amplitude. Measurements of mIPSCs show rise times of�2 ms and decay times of �5 ms. The relatively rapidtimescale of these events indicates that they were likely medi-

BAGABA

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.001 .003 .01 .03 0.1 0.3 1.0 3.0 10

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3 μM 30 μM 300 μM

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Fig. 3. A: representative traces recorded under voltage-clamp conditions using high-chloride internal solution demonstrating prefrontal cortex (PFC) LII/III PNresponse to 3, 30, and 300 �M GABA. Significant inward current was observed at concentrations � 10 �M, �40 13 pA, P 0.05. B: concentration-responsecurve of GABA as normalized to 1 mM GABA. EC50 � 353 �M; Hill slope � 1.43.

BA

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omB

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Fig. 4. A: representative trace demonstratinglarge inward current after application of theGABA agonist THIP (1 �M) that was com-pletely blocked by the GABA antagonistgabazine (20 �M). B: results including re-sponse to 0.3 and 1 �M THIP (*P 0.05,**P 0.005).

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ated by �1�2/3�2-GABAARs, as in cerebellar Purkinje neuronsand elsewhere in the brain (Vicini et al. 2001). Regionalexpression analysis supports the presence of �1�2/3�2-GABAARs in layer LII/III of the frontal cortex (Fritschy andMohler 1995; Pirker et al. 2000), and sIPSCs recorded incortical LII/III PNs of �1 knockout mice (Bosman et al. 2002)are significantly slower in time course.

Lack of tonic GABAAR current in LII/III PNs. Tonic inhibi-tion is widespread but by no means ubiquitous in the brain. Fortonic inhibition to occur, several requirements must be satis-fied. First, there needs to be sufficient expression of extrasyn-

aptic receptor subtypes with high ligand affinity and slowdesensitization rates in order to sustain chloride conductance.Second, an inhibitory neurotransmitter needs to be present atsufficiently high levels to activate these receptors. Expressionanalyses have demonstrated the presence of GABAAR subunits�4 and � in the outer layers of the PFC (Peng et al. 2002; Pirkeret al. 2000). GABAA �4�� receptors have been shown tohave a higher sensitivity to GABA (activation at 100 nM)compared with receptor subtypes associated with synapticreceptors (e.g., �1�2�2s GABAARs, activation at 3 �M) (Jiaet al. 2005).

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Fig. 5. A and B: representative recordingsunder voltage-clamp conditions showing thatapplication of 20 �M gabazine (GBZ) didnot detect a tonic GABA current; however,application of 100 �M picrotoxin (PTX) did,causing a significant positive shift from base-line current. C: a similar positive shift wasrevealed after application of 1 �M strych-nine. Results are summarized in D (**P 0.005, ***P 0.001).

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Fig. 6. A, top: representative recordings under voltage-clamp conditions showing the response to exogenously applied glycine (30 �M–3 mM). Bottom: taurineapplication (100 �M–10 mM) causes a similar inward current at higher concentrations. B: glycine and taurine applications are shown on concentration-responsecurve normalized to 3 mM glycine for each neuron, where glycine EC50 � 447 �M and Hill slope � 2.56.

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Determining neurotransmitter concentrations in brain slicesis obviously difficult, but we can infer that ambient GABAconcentrations in slices of the mouse PFC must be very low,since although THIP (a selective, highly efficacious agonist at�4,�-containing GABAARs) can elicit a significant current at0.3 and 1.0 �M, there is little to no gabazine-sensitive toniccurrent, perhaps 100 nM. The lack of a tonic GABA currenthere is consistent with previous reports in LII/III PFC neuronsin rodents (Drasbek and Jensen 2006; Hoestgaard-Jensen et al.2010). Variations in brain levels of GABA are presumed to bea result of regional differences in the subtypes and expressionlevels of the GABA transporters. Tonic inhibition revealed bygabazine has been identified in cortical LII/III PNs, but fol-lowing extensive blockade of GABA transporters with NO-711(Vardya et al. 2008). In reports investigating layer V PFCneurons GABA-mediated tonic inhibition was absent (Weitlaufand Woodward 2008), but it has been demonstrated in thesomatosensory cortex (Salin et al. 1995), suggesting that inanterior cortical subregions such as PFC ambient GABA maybe more tightly regulated.

Presence of tonic glycine currents in LII/III PNs. An alter-native source of cortical inhibition can arise from the presenceof glycine, the other major inhibitory neurotransmitter in thecentral nervous system. Like GABAARs, GlyRs are pentamericligand-gated ion channels that are permeable to chloride(Kirsch 2006). In the spinal cord, synaptic GlyRs are formed asa heteropentamer of �1- and �-subunits; the presence of a�-subunit confers synaptic localization through its binding to

gephyrin, an anchoring protein that is enriched at synaptic sites(Kirsch 2006). In structures of the postnatal forebrain, there isvery low expression of GlyR�1 mRNA compared with thespinal cord, but the expression of GlyR�2 and GlyR�3 has beenreported in several brain regions, including the PFC (Jonsson etal. 2009; Malosio et al. 1991).

In the hippocampus functional, strychnine-sensitive GlyRshave been identified and are believed to be homomeric assem-blies of �-subunits because of their sensitivity to PTX (Chat-tipakorn and McMahon 2002, 2003; Zhang et al. 2008b) aswell as cyclothiazide, a proposed �2-GlyR antagonist (Zhanget al. 2008b). In hippocampal slices, a tonic GlyR current wasidentified in the CA1 region after strychnine application(Zhang et al. 2008a). These data support the idea that �homomeric GlyRs mediate tonic inhibition, analogous to therole that �-containing GABAARs play in tonic GABAinhibition.

In layer II/III of the PFC, using PTX (100 �M), we foundthat we could elicit a positive shift in baseline current. Indissociated PFC neurons, PTX has been shown to block gly-cine currents (Lu and Ye 2011). We subsequently demon-strated the presence of tonic GlyRs by applying strychnine,which elicited a significant shift (�15 pA) in baseline currentin the absence of an effect on the frequency of GABA-mediated sIPSCs or isolated mIPSCs. Applications of exoge-nous glycine further showed that a large population of GlyRsis present in PFC LII/III PNs and can generate large inwardcurrents in response to exogenous glycine, which could be

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Fig. 7. A: representative recording showing that the shift in current by glycine (200 �M) is completely blocked by coapplication of strychnine (1 �M)(summarized in B; **P 0.01). C: graph demonstrating that the NMDA antagonist D-(�)-2-amino-5-phosphonopentanoic acid (AP5) does not affect the shiftin current by glycine (200 �M).

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Fig. 8. A: representative recording showingthat sarcosine (SARC; 500 �M) causes anegative shift in baseline current that can beprimarily blocked by coapplication of strych-nine (1 �M). B: graph showing concentra-tion-dependent action of sarcosine on hold-ing current and blockade by strychnine(***P 0.001).

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fully blocked by strychnine. Previous reports have shownslightly higher sensitivity of exogenous glycine on hippocam-pal PNs from 3- to 4-wk-old rats (EC50 � 270 �M) and onPFC PNs from periadolescent rats (EC50 � 117 �M) and thatthese currents could be blocked by strychnine and PTX (Chat-tipakorn and McMahon 2002; Lu and Ye 2011). The measure-ments made in PFC PNs were from freshly dissociated neu-rons, and differences in glycine sensitivity are likely due toGlyTs actively taking up glycine. Importantly, application ofsarcosine (500 �M), a GlyT1 antagonist, elicited a 64-pAinward current that could be blocked by strychnine. GlyT1s areprimarily located on astrocytes in the cortex (Kunz et al. 2012),and we conclude that astrocytic GlyT1 plays an important rolein regulating the magnitude of tonic GlyR inhibition in themouse PFC.

A recent report in the mouse lateral orbital frontal cortex(LOFC) is consistent with our findings and has implicatedextrasynaptic GlyRs as targets of ethanol modulation (Badan-ich et al. 2013). Specifically, these authors found that highconcentrations of ethanol (66 mM) shift the holding current ofdeep-layer PNs of the LOFC and that this could be blocked byPTX and strychnine (1 �M) but not by the GABAAR antago-nist bicuculline. In addition, ethanol, a known positive alloste-ric modulator of GlyRs, decreased neuronal excitability, aneffect that was blocked by the application of strychnine but notbicuculline. They conclude that strychnine-sensitive GlyRsmodulate ethanol’s effects on deep-layer LOFC neurons. Un-like our findings in LII/III PFC, they did not detect thepresence of a strychnine-sensitive tonic current in LOFC. Thismay be due to a reduced availability of ambient glycine orother GlyR ligands in this subregion.

Endogenous ligand responsible for tonic GlyR activation.Our evidence supports the expression of functional, extrasyn-aptic GlyRs, but the source of the endogenous ligand for GlyRsin the PFC remains unclear. In vivo microdialysis has shownthat 10 �M of extracellular glycine exists in the extracellularspace of the hippocampus (Horio et al. 2011). Additionally,glutamatergic synapses (where concentrations of glycine havebeen estimated to reach 1 mM) may be a second source ofglycine due to synaptic spillover (Vandenberg and Aubrey2001), although it is believed that GlyTs limit glycine spillover100- to 1,000-fold (Harsing and Matyus 2013). In addition toglycine, the GlyR partial agonists taurine and �-alanine areknown to be abundant in the brain (Dahchour et al. 2000;Murakami and Furuse 2010), and we show here that exogenoustaurine can cause an inward current, supporting its role as aGlyR agonist on PFC PNs. In early postnatal cortical PNs,taurine has been shown to mediate tonic nonsynaptic GlyRactivation (Flint et al. 1998). In hippocampal PNs, the inhibi-tion of taurine and �-alanine transporters by guanidinoethane-sulfonic acid can generate a GABA-independent, strych-nine-sensitive current of 15 pA (Mori et al. 2002). Ourfinding that sarcosine, which selectively inhibits GlyT1,increased the measurable strychnine-sensitive tonic currentby �30 pA indicates that glycine is a major mediator of thestrychnine-sensitive current; however, it seems reasonableto propose that sufficient levels of multiple endogenousGlyR ligands can arise to tonically activate GlyRs in theforebrain.

Physiological and functional significance. In the PFC, LII/III PNs are uniquely positioned to integrate several inputs from

cortical and subcortical structures. We have shown that inhi-bition of these neurons is more diverse than previously real-ized, including a novel role for tonic GlyR activation. Tonicinhibitory conductances of this scale have previously beenshown to inhibit neuronal activity (Jia et al. 2008). This novelmechanism may contribute to the sparse action potentialsobserved in LII/III neurons around resting potential and permitincreased fidelity of neuronal output, a proposed tuning mech-anism involved in attention and working memory in the PFC(Rao et al. 2000). As GlyR activation increases, we wouldexpect increased fidelity at lower levels of activation andsubsequent silencing of PFC PNs at high concentrations ofGlyR ligands or positive GlyR modulation.

Summary. We investigated excitability and inhibitory cur-rents in neurons of layer II/III of the prelimbic region of themouse PFC and found distinct roles for GABA and glycine.Our results indicate that GABAARs mediate primarily synapticinhibitory currents and strychnine-sensitive GlyRs mediateprimarily tonic currents on LII/II PNs. These neurons exhibitlarge currents after the application of glycine and taurine,suggesting the existence of functional GlyRs. These findingsreveal a novel form of inhibition in the PFC that may regulateneuronal excitability and contribute to the balance of excitationand inhibition that is critical to maintaining proper corticalfunction.

GRANTS

This work was supported by National Institute on Alcohol Abuse andAlcoholism (NIAAA) Grant R01 AA-19801 to N. L. Harrison. M. C. Sallingwas supported by Postdoctoral Training Grant F32 AA-022028 from NIAAA.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: M.C.S. and N.H. conception and design of research;M.C.S. performed experiments; M.C.S. analyzed data; M.C.S. and N.H.interpreted results of experiments; M.C.S. prepared figures; M.C.S. draftedmanuscript; M.C.S. and N.H. edited and revised manuscript; M.C.S. and N.H.approved final version of manuscript.

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