Synaptic organization and input-specific short-termplasticity in anterior cingulate cortical neuronswith intact thalamic inputs

Chia-Ming Lee,1,2 Wei-Chih Chang,2 Kung-Bo Chang2 and Bai-Chuang Shyu2

1Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan, ROC2Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan, ROC

Keywords: medial thalamus, mice, multi-channel electrode array, short-term plasticity, thalamocingulate pathway

Abstract

The absence of a slice preparation with intact thalamocortical pathways has held back elucidation of the cellular and synapticmechanisms by which thalamic signals are differentially transmitted to and processed in the anterior cingulate cortex (ACC). In thisreport we introduce an innovative mouse brain slice preparation in which it is possible to explore the electrophysiological properties ofACC neurons with intact long-distance inputs from medial thalamic (MT) nuclei by intracellular recordings; this MT–ACC neuronalpathway plays an integral role in information transmission. Biocytin-labeled fibers in a functional slice could be traced anterogradelyor retrogradely from the MT via the reticular thalamic nuclei, striatum and corpus callosum to the cingulate cortical areas. Eighty-seven cells downstream of the thalamic projections in 49 slices were recorded intracellularly. Intracellular recordings in the ACCshowed that thalamocingulate transmission involves both a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) ⁄ kainateand N-methyl-d-aspartate (NMDA) subtypes of glutamate receptors. Thalamus-evoked responses recorded extracellularly in theACC were activated and progressed along a deep–superficial–deep trajectory loop across the ACC layers. We observed enhancedpaired-pulse facilitation and tetanic potentiation of thalamocingulate synapses, suggestive of input-specific ACC plasticity andselective processing of information relayed by thalamocingulate pathways. Furthermore, we observed differential responses of ACCneurons to thalamic burst stimulation, which underscores the importance of MT afferents in relaying sensory information to the ACC.This new slice preparation enables the contribution of MT-evoked ACC synaptic transmission to short-term plasticity in the neuronalcircuitry underlying sensory information processing to be examined in detail.

Introduction

The cingulate cortex receives highly processed sensory input and is amajor organization network involved in visceral, cognitive, attention-al, emotional and motivational processes (Fuster, 2001; Miller &Cohen, 2001; Morgane et al., 2005). The anterior cingulate cortex(ACC) is reciprocally connected with the medial thalamus (MT) aswell as other limbic regions (Krettek & Price, 1977; Ongur & Price,2000; Vertes, 2002). A convergence of evidence suggests that thethalamocingulate division of the limbic system is critical in mediatingcognitive processes, such as mother–infant communication (Lorber-baum et al., 2002), pain perception (Vogt, 2005) and active avoidance(Smith et al., 2002).

The MT serves as a major relay in the medial pain system and in theconveyance of nociceptive information to the ACC (Albe-Fessardet al., 1985; Bushnell & Duncan, 1989; Sikes & Vogt, 1992; Vogtet al., 1993). Distinct activation patterns in different thalamocorticalpathways may underlie the functional specificity of different sensory-motor functions (Castro-Alamancos et al., 1995; Castro-Alamancos &Connors, 1996; Steriade, 1999). Synaptic plasticity studies of ACCneurons in vivo suggest that nociceptive information acquisition andstorage depends upon plasticity of synapses on cingulate neurons with

thalamic inputs (Wei & Zhuo, 2001; Kung & Shyu, 2002).Importantly, structural and functional specialization of these particularsynapses may have plasticity characteristics that differ fromneighboring synapses (Nicoll & Schmitz, 2005). Because thesesynapses could previously only be studied disconnected from theirthalamic inputs in slice preparations, the neuronal circuitries in theACC that are activated by specific thalamocingulate pathways arelargely unknown, and functional specification of the plastic propertiesof cingulate neurons receiving thalamic inputs relative to thosereceiving other inputs is unclear.Thus far, in vitroACC electrophysiological studies have been carried

out by stimulating locally or in the corpus collosum (CC; Vogt &Gorman, 1982; Sah & Nicoll, 1991; Hempel et al., 2000). Long-distance fibers are severed in conventional coronally cut medialprefrontal cortex (mPFC) slices, and the intermingling of corticipetaland corticofugal fibers with local neurites has precluded examinationof specific long-distance pathways involved in sensory informationprocessing (Orozco-Cabal et al., 2006). Given the integrative role ofthe ACC in the limbic frontal cortical system, an in vitro model systemin which the synaptic organization of the thalamocingulate pathwayand the synaptic plasticity and cellular mechanisms underlyingnociceptive signal processing can be examined is greatly needed.Thus, the purpose of the present study was first to develop a method

for functionally preserving the thalamocingulate pathway in aslice. We then sought to confirm functional preservation of the

Correspondence: Dr B.-C. Shyu, as above.E-mail: bmbai@gate.sinica.edu.tw

Received 29 September 2006, revised 9 February 2007, accepted 14 February 2007

European Journal of Neuroscience, Vol. 25, pp. 2847–2861, 2007 doi:10.1111/j.1460-9568.2007.05485.x

ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

thalamocingulate pathway by assessing ACC responses to thalamicstimulation using intracellular neurophysiology and a multi-electrodearray (MEA) recording system. Additionally, we examined theglutamate receptor subtypes that mediate thalamocortical transmissionand tested whether short-term plasticity in this slice mimics thatobserved in vivo.

Materials and methods

Animals

Male C57BL ⁄ 6J mice (4–8 weeks old) were used in the present studyand purchased from the Center of Animals, Institute of BiomedicalScience, Academia Sinica. All experiments were carried out inaccordance with the guidelines established by the Academia SinicaInstitutional Animal Care and Utilization Committee. Every effort wasmade to minimize the number of animals used.

Reconstruction of the trajectory path

Brains were removed from anesthetized mice (4% halothane in pureoxygen) after transcardial perfusion of 100 mL of normal salinefollowed by 100 mL of 4% paraformaldehyde solution (PFA) in 0.1 m

phosphate-buffered saline (PBS, pH 7.4, Merck). Each brain wasbisected and the medial surface was examined under a dissectingmicroscope. The lipophilic, fluorescent carbocyanine dye DiI (1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate) was pre-mixed into a paste (NeuroTrace DiI tissue-labeling paste, MolecularProbes, Eugene, OR, USA). DiI paste was pressure injected into theMTvia glass micropipettes placed 500 lm below the medial surface. Thebrain was then submersed in PBS and kept warm (37 �C) for 4 weeks.DiI-labeled serial sections (100 lm thick) were examined under a

florescence microscope. The images were aligned and stacked in ImageJ (National Institutes of Health, Bethesda, MD, USA). Using there-slicing function, serial coronal slices were selected, and the ‘center ofmass’ ofDiI staining in each slicewas calculated. The 3D trajectory pathwas constructed by projecting the center of mass values onto X-, Y- andZ-planes together with their relative distance from the reference point(frontal cortical pole). The stacked images and constructed thalamo-cingulate trajectory path were viewed in 3D with Lightwave 3D�

(NewTek, San Antonia, TX, USA). The turning angles of the 3Dpathway were calculated from the slopes of the regression lines.

Preparation of acute slice for electrophysiological recording

Brains were quickly removed from anesthetized animals (4% haloth-ane in pure oxygen) and cooled for 3 min in chilled, oxygenatedartificial cerebrospinal fluid (aCSF; composition in mm: NaCl, 124;KCl, 4.4; NaH2PO3, 1; MgSO4, 2; CaCl2, 2; NaHCO3, 25; glucose,10; bubbled with 95% O2 and 5% CO2). The thalamocingulatepathway block was hand-cut with two sagittal cuts (lateral to thepathway) and two angled cuts that were ventral and parallel to thepathway. The brain block was attached to an angular plate withcyanoacrylate adhesive and a cut was made just above the turningpoint of the pathway. The stage was unfolded, flattened and glued ontothe chamber stage of a Vibratome (Series 1000, Vibratome, St. Louis,MO, USA); 500-lm sections were cut in ice-cold oxygenated aCSF.Slices were incubated in oxygenated aCSF at room temperature for1 h. A single slice was transferred to the recording chamber and keptat 29–30 �C under continuous perfusion (2 mL ⁄ min) of oxygenatedaCSF.

Intracellular recording

Intracellular electrodes were filled with 3 m potassium acetate with orwithout 5% biocytin (impedance 120–200 MW). Recordings weremade using an Axoclamp 2A (Molecular Devices, Sunnyvale, CA,USA) microelectrode amplifier with 10 · amplification. The amplifiedsignals were digitized by an A ⁄ D converter card in an IBM-compatible computer, recorded by a windows-based data acquisitionprogram and analysed with a homemade MATLAB program (Math-Works, Natick, MA, USA). Input resistance was calculated bymeasuring Vm deflections produced in response to constant hyperpo-larizing current pulses (0.1–0.6 nA, 500 ms). Only cells with a stableresting Vm ¼ )60 mV were used. Stimulation was applied with anisolated pulse stimulator (Model 2100, A-M Systems, Carlsborg, WA,USA) through a bipolar tungsten electrode. Stimulus parameters(biphasic, duration 200 ls; intensity 50–600 lA) were adjusted toyield consistent synaptic responses at a frequency of 0.033 Hz.

Extracellular recording

An array of 60 pyramidal-shaped electrodes with the followingproperties was used: electrode diameter, 40 lm; electrode spacing,200 lm; tip height, 50 lm; impedance, 1000 kW at 200 Hz (AyandaBiosystem, Lausanne, Switzerland). Slices were mounted so that theACC was orientated above the electrodes. The MEA chamber waskept at 29–30 �C by a Temperature Controller TC02, PH01 (MultiChannel Systems, Reutlingen, Germany) and continuously perfusedwith warm oxygenated aCSF (8 mL ⁄ min). Local field potentials(LFPs) at each electrode were recorded relative to the bath electrode.A 60-channel amplifier was used with a band-pass filter set between0.1 Hz and 3 kHz, 1200 · amplification (MEA-1060-BC, MultiChannel Systems), and data were acquired by PC-based data softwareMC Rack (Multi Channel Systems) at a 10 kHz sampling rate. Toimprove the signal-to-noise ratio, 10–20 traces were averaged. Datawere analysed with MEA-Tools and a homemade MATLAB program.Extracellular current pulses were generated by a programmablestimulus generator (model STG 1002, Multi Channel Systems) anddelivered via bipolar tungsten electrodes. Rectangular biphasic(negative pulse followed by a positive pulse) current pulses weredelivered. In preliminary intensity tests, ± 350 lA (200 lS per phase)elicited 80% maximal-induced activity in the ACC.

Drug application

The N-methyl-d-aspartate (NMDA) receptor antagonist d,l-2-amino-5-phosphonopentanoic acid (APV), the a-amino-3-hydroxy-5-methy-lisoxazole-4-propionic acid (AMPA) ⁄ kainate receptor antagonist6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX) and thec-aminobutyric acid (GABA)A receptor antagonist picrotoxin werepurchased from Tocris Cooks (Ellisville, MO, USA). Stock solutions(APVand CNQX in double-distilled water, picrotoxin in ethanol) wereprepared, divided into small aliquots and stored at )80 �C. Thealiquots were thawed on experimental days and added to aCSF toachieve final concentrations of 30 lm APV, 15 lm CNQX and 30 lm

picrotoxin. All drugs were applied to the bath solution.

Data analysis

Responses to minimal stimulation

A minimal stimulation protocol was performed to distinguish thesynaptic characteristics of cingulate neurons stimulated from different

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ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 25, 2847–2861

stimulation sites (Stratford et al., 1996; Gil et al., 1999). aCSFcontaining 2.5 mm [Ca], 1 mm [Mg] and 25 lm APV was used in thisexperiment. The stimulus intensity was adjusted to produce all-or-none synaptic events for each stimulation site. Negligible variation inexcitatory postsynaptic potential (EPSP) latency, amplitude and shapeof synaptic events was observed. The EPSP rising time was defined asthe time during which the amplitude increased from 10% to 90% ofthe peak amplitude. The initial slope was calculated by fitting astraight line of the EPSP during the rising time. The time period duringwhich an exponential curve fit the falling phase of the EPSP from 90%to 10% of its peak amplitude was taken as the decay time. The half-width was defined as the duration of the period from 50% peakamplitude of rising phase to 50% peak amplitude of falling phase ofthe EPSP.

Trajectory path

DiI center of mass values in the serial coronal sections were calculatedin Image J. The center of signal source of 60 channels’ LFPs recordedat specific time points t (Xcm, Ycm) were calculated according to thefollowing formulae:

Xcm ¼

P

both i;j¼1�8Vij � i

P

both i;j¼1�8Vij

Ycm ¼

P

both i;j¼1�8Vij � j

P

both i;j¼1�8Vij

such that Xcm ¼ center of signal source on the X-axis; Ycm ¼ centerof signal source on the Y-axis; Vij ¼ the LFP at time t recorded atelectrode position [i, j]; i ¼ electrode position on the X-axis (eightrows); j ¼ electrode position on the Y-axis (eight columns).

Only negative LFPs occurring at least 0.5 ms after the stimulationartifact and above the threshold voltage of 0.2 · the maximum peakpotential were calculated. Three segments of the negative LFP (N1,N2 and N3) were distinguished as discrete components. Serial pointsof the center of signal source in each time segment constituted thetrajectory path for each component. Lines connecting consecutivepoints were defined as vectors and the vectors within the time segmentof each component were summated to produce the vector representingeach component.

Current source density analysis

Approximately six–eight channels of evoked field potentials along thetrajectory path were selected from the 60-channel MEA arrayrecordings. LFPs evoked by thalamic stimulation were recorded for1 s in each channel per trial, and 10–20 sweeps were averaged foreach channel. Averaged LFPs were subjected to 1D current sourcedensity (CSD) analysis (Freeman & Nicholson, 1975). The fieldpotential values of some recording electrodes were obtained byinterpolating averaged responses from neighboring electrodes. CSDdata on upper and lower boundary sites were obtained by extrapola-tion. CSD color maps were generated using linear interpolation alongthe cortical layer axis.

Biocytin tracing in functional slices

Small crystals of biocytin (Sigma, St. Louis, MO, USA) were insertedinto the MT stimulation sites or the ACC recording sites of slices thatwere demonstrated by electrophysiological recording to have afunctional connection between the MT and ACC. Following thebiocytin incubation period (6–8 h), the slices were fixed with 4% PFAin 0.1 m PBS (pH 7.4) overnight. Fixed slices were washed several

times in PBS and then incubated in 1% H2O2 (in PBS) for 15 min toblock endogenous peroxidase activity. Blocked slices were transferredto vials containing 30% saccharose (in PBS) for 2 h for cryoprotectionand then were embedded and frozen in a cryosolidifiable medium(O.C.T. compound, Tissue-Tek, Hatfield, PA, USA). Fifty-micron-thick sections were collected on a freezing microtome (model CM3050, Leica Microsystems GmbH, Wetzlar, Germany). Biocytin-labeled structures could be visualized after sections were processed fordiaminobenzidine, ammonium-nickel-sulfate staining with a VECTA-STAIN Elite ABC kit (Vector Laboratories, Burlingame, CA, USA)according to the manufacturer’s protocol.

Biocytin labeling of recorded neurons

Intracellularly recorded cells were injected with biocytin using 2 nAdepolarizing pulses delivered with 300 ms on ⁄ off cycles for at least10 min. Recording electrodes were carefully withdrawn and the sliceswere left in the recording chamber for an additional 30 min to allowthe biocytin to diffuse. Slices were fixed by immersion in 4% PFA,0.1 m PBS for 24 h at 4 �C, and then cryoprotected by immersion in30% sucrose in 0.1 m PBS for 12 h. The tissue sectioning and biocytinimmunohistochemistry procedures employed were the same as thatdescribed above for biocytin tracing in functional slices.

Parvalbumin (PV) immunohistochemistry

PV immunohistochemistry was conducted in sections from sliceswithout biocytin-injected neurons. Sections were pretreated with0.5% hydrogen peroxide PBS solution for 30 min. After rinsing inPBS containing 0.1% Tween20, slices were incubated for 2 h inPBST (PBS with 0.3% Triton X-100). Non-specific binding wasblocked with a 2-h incubation in 10% normal horse serum (NHS,Vector Laboratories). Sections were then incubated at 4 �C for 48 hin primary antibody solution (1 : 500 dilution of monoclonal mouseanti-PV antibodies (pa-235, Sigma) in 0.1 m PBST, 2% NHS).Slices were then washed three times in PBST and transferred to abiotinylated secondary antibody solution (1 : 500 dilution of biot-inylated anti-mouse IgG in 0.1 m PBST, 2% NHS) for 1 h at roomtemperature. Sections were then processed for 3,3¢-diaminobenzidinestaining with the VECTASTAIN Elite ABC kit (Vector Laboratories)according to the manufacturer’s protocol, washed in PBS andmounted.

Results

Establishing the slice preparation procedure

The MT-ACC pathway was traced by following DiI signals (anterog-radely transported from the MT) through serial sagittal planes(Fig. 1A). The DiI signal was intense in the MT region, and longfiber tracts were visible through the striatum and the genu of the CC,which ultimately terminated in the ACC. We reconstructed the tractprojection by calculating the center of mass of the DiI signals in serialcoronal sections and assembling 3D images from these planes(Fig. 1B, and Supplementary material, Video S1). The trajectorythrough 2D X-, Y- and Z-planes was plotted and superimposed uponstandard mouse atlas plates (Fig. 1B). The 3D trajectory of thepathway between the thalamus and mPFC was a downward and thenupward sweeping curve with a turn of about 118� at the thalamus–striatum junction. It extended through the striatum and pierced throughthe genu of CC to the ACC.

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ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 25, 2847–2861

A serial sectioning procedure based on the trajectory revealed by the3D reconstruction was established. Each mouse brain was quicklyremoved and chilled in oxygenated aCSF for 3 min. A parasagittal cut2.0 mm lateral to the midline was made in each hemisphere (Fig. 1C, a)to reveal the subcortical anatomy and guide subsequent cuts (Fig. 1C,b). The first cross-cut was made along the fissure between the frontalcortical surface and the olfactory bulb, parallel to the visible fiber tractin the striatum. The second cross-cut was made from the junctionbetween the cerebellum and visual cortex to the mid-point between theanterior commissure and the optic tract. The angle (118�) generatedfrom these two cross-cuts is the same as the pathway turning angle atthe thalamus–striatum junction. The brain block was glued onto anangled plate, which was transferred and fit onto a wedged block(Fig. 1C, c, blue). A 2-mm-deep cut was made with a surgical bladefrom the cortical surface directly above the vertex of the intersectingcross-cuts. The angled plate together with the brain block wasremoved from the wedged block and unfolded so that the lateralventricle was visible from above (Fig. 1C, d). The flattened plate withthe brain block was glued and immersed in cool oxygenated aCSF in avibratome (Fig. 1C, e). A few 500-lm slices were collected from eachbrain and incubated in oxygenated aCSF (Fig. 1C, f).

Confirming connectivity between the thalamus and ACC

To confirm preservation of the thalamocortical pathway in our slicepreparation, we traced biocytin-labeled fibers in slices used inelectrophysiological experiments (schematic diagram in Fig. 2A).Biocytin-labeled fibers and neurons were traced from the point ofinsertion, either the stimulation site (Fig. 2A) or the recording site(Fig. 2B). It was clearly evident in brightfield images of thinsections made from recorded slices that biocytin-labeled fibersoriginating from thalamic biocytin-injection loci (white asterisks)followed the major fiber bundle within the striatum (Fig. 2A, d),crossed the CC, entered cortical layers VI and V, and thenterminated in layer II ⁄ III (Fig. 2A, a–c). One intracellularly labeledneuron identified with electrophysiological recording was alsovisible within the ACC (Fig. 2A, a, arrow). Retrogradely labeledbiocytin could be traced from the cingulate cortical injection sites(Fig. 2B, white asterisks), through the striatum and then the reticularthalamic nucleus (Fig. 2B, a and b) to terminate in the MT (Fig. 2B,b and c). Neuronal somata and dendrites containing retrogradelylabeled biocytin were visible in the medial dorsal thalamic nucleus(Fig. 2B, d).

Fig. 1. Reconstruction of the thalamocingulate pathway. (A) DiI-labeled serial sagittal planes correspond to the lines drawn through the horizontal view of thebrain in the inset. A dot indicates the thalamic injection site. (B) A dashed red line connects the center of mass of the DiI signal (red dots) in each of a series ofcoronal planes. (C) Slicing procedure. (a) Two parasagittal cuts 2.0 mm from the midline. (b) Side view of the medial part of the brain block. Dashed lines indicatethe cuts in the following steps. (c) After the two angled ventral cuts, the brain block was glued onto an angled plastic plate (thick black line), and placed on a plasticwedge (blue). A dorsal cut was made through the cortex directly above the turning point of thalamocingulate pathway. (d) The angled plate was unfolded (curvedarrow), and the brain block was flattened. (e) The plate was glued onto the chamber stage of the vibratome and submerged in chilled oxygenated aCSF. Arrowsindicate the slicing direction of the vibratome. (f) A sample slice containing the intact thalamocingulate pathway (red trace). Abbreviations: ACC, anterior cingulatecortex; CC, corpus callosum; Hippo, hippocampus; St, striatum; Th, thalamus.

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ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 25, 2847–2861

Cortical layers and thalamic nuclei were distinguished by PVimmunohistochemistry. As our oblique sections did not conform to astandard atlas, we consulted the serial horizontal plates of the standard

atlas to identify the thalamic nuclei and cortical layers. We observedseveral discrete thalamic nuclei and clustered cell groups as well as cleardelineation of the mPFC layers in PV-labeled sections (Fig. 2B, inset).

Fig. 2. Anatomy of the thalamocingulate pathway revealed by retrograde and anterograde biocytin labeling in a functional MT-ACC slice. Biocytin-labeled fibersor neurons were visible in slices with biocytin inserted either in stimulating (A) or recording (B) sites. The schematic diagram in (A) shows the arrangement ofstimulating and recording electrodes in the experiment in which biocytin was inserted. Selected thin sections with biocytin were arranged in ventral to dorsal orderand used to trace the MT-ACC pathway in a recorded slice. (A) Biocytin-labeled fibers arose from the MT injection region (white asterisks), passed throughreticular thalamic nuclei (d), traveled from the striatum (d), cross the CC (d) and projected to cingulate cortical area (a–c). Biocytin-labeled fiber terminals weresparsely distributed in layers II ⁄ III, V and VI. An intracellularly injected neuron that was identified by electrophysiological recording to be responsive to MTstimulation is shown in (a) (arrow). Some biocytin-labeled fibers in the reticular thalamus region were branched (white arrows in d) and had local targets.(B) Retrogradely labeled biocytin fibers could be traced from the ACC, where the biocytin was injected (white asterisks), across the CC, back to the striatum andthrough the reticular thalamic nuclei, and finally to the MT. Dense biocytin-labeled fibers branched (white arrow) and terminated on local targets in the reticularthalamic nuclei (Ba and b). Biocytin-labeled fibers originated from medial dorsal thalamic nuclei (Bb and c). Some densely labeled neuronal somata and dendriteswere visible in the medial dorsal thalamic nucleus (Bd). Cortical layers, discrete thalamic nuclei and other subcortical structures could be clearly delineated inPV-immunolabeled sections from a slice with a confirmed functional connection of MT-ACC pathway (B, inset). Scale bar: 1 mm. Abbreviations: AM, anteromedialthalamic nucleus; MD, mediodorsal thalamic nucleus; Po, posterior thalamic nucleus group; Rt, reticular thalamic nucleus; Sp, septal nucleus; VB, ventrobasalthalamic nucleus.

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ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 25, 2847–2861

Evoked electrophysiological responses in the ACCIntracellular recordings revealed that bipolar thalamic stimulation-evoked EPSPs were intensity dependent (20–300 lA), as shown in thesuperimposed sweeps obtained from an ACC layer V cell, and thataction potentials (APs) were induced when stimulation was applied atintensities greater than 250 lA (Fig. 3B). Injection of depolarizingcurrent invoked regular spikes in this cell. Thalamus-evoked EPSPs andcurrent injection-induced depolarization were observed in cells in layerII ⁄ III (Fig. 3A) and layer VI (Fig. 3C). The EPSPs of these cells wereabolished after perfusion of a low-calcium (0.2 mm) aCSF solution.Eighty-seven cells downstream of the thalamic projections in

49 slices were recorded intracellularly (Vm ¼ )68 ± 0.8 mV, averagerecording time ¼ 30 ± 4.9 min). Data were determined from cells inwhich stable intracellular recordings were maintained for at least30 min. Voltage changes to the injected current pulses (500 msduration, increment of 0.1 nA, 0.5 Hz) were used to explore thepassive and active membrane properties of these ACC neurons(summarized in Table 1). Cells with regular or non-regular spike firing

patterns were compared (Table 2). Fifty-two cells were injected withbiocytin, and 34 cells were histologically verified. The recorded cellswere distributed mostly in layer V (n ¼ 20), and to a lesser extent inlayer II ⁄ III (n ¼ 10) and upper layer VI (n ¼ 4). All histologicallyverified neurons were identified as pyramidal neurons. The membraneproperties of the biocytin-labeled ACC neurons are given in Table 3. Acamera lucida drawing and photomicrograph of a typical layer Vpyramidal neuron intracellularly injected with biocytin are shown inFig. 3D. Note the arborizing branch points on the main axon(arrowheads) that were apparent as the main axon descended towardthe CC in this example neuron. This cell was classified as a regularspiking cell according to its firing pattern upon being subjected to adepolarizing current. Effective ACC-stimulating sites were distributedin the MT region and in the vicinity of the reticular thalamic nuclei;ineffective sites were more anterior and posterior (Fig. 3E). Consistentresponses were obtained when stimulation was applied along the fibertract pathway in the medial striatum, but not when stimulation wasapplied outside the pathway in the lateral striatum.

Fig. 3. Intracellular recording of anterior cingulate cortex (ACC) responses to thalamic stimulation and distribution of cingulate cortical neurons following thalamicstimulation. Evoked EPSPs in example cells in (A) layer II ⁄ III, (B) layer V and (C) layer VI of the ACC following bipolar stimulation of the thalamus. Higherintensity stimulation (250–300 lA) induced APs. The consecutive traces (from bottom to top) are membrane potential changes evoked by 500-ms, current pulsesapplied at 0.1-nA increments in stimulus intensity. (D) Camera lucida drawing and photomicrograph of an example layer V pyramidal neuron intracellularlyinjected with biocytin. The drawing was constructed from several photographs taken under a 40 · objective from two adjacent 60-lm sections. The intracortical axonarborized (branch points designated with arrows), and only the main axon descended into the corpus callosum (CC). (E) The distributions of the stimulating andrecording sites in all functional MT-ACC slices are shown in the schematic diagram. Abbreviations as for Fig. 2.

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Six cells in layer V and two cells in the border between layer V andVI appeared to be antidromatically activated, having short latencies(< 2 ms), APs that persisted in low-calcium aCSF and the ability tofollow high-frequency (100 Hz) stimulation (Fig. 4A). The evokedsynaptic responses were inhibited strongly by the AMPA receptorantagonist CNQX (15 lm) and partially by the NMDA receptorantagonist APV (30 lm, Fig. 4B, n ¼ 7 slices). CNQX and APVsignificantly decreased peak responses to 20.9 ± 3.5% and 71 ± 4% ofthe untreated control response, respectively. APV also affected thedecay phase (time point at falling phase and 50% of peak amplitude)of EPSPs and APs. Perfusion of the GABAA receptor antagonistpicrotoxin (30 lm in aCSF) induced fast spike trains with a largeEPSP mean amplitude (30.7 ± 6.9 mV, n ¼ 3); the picrotoxin-induced trains appeared 70–280 ms after stimulation. This finding

Table 2. Membrane properties of all recorded neurons in the ACC

Membranepotential (mV)

Input resistance(MW)

Current injection(nA)

AP duration(ms)

AP amplitude(mV)

AHP amplitude(mV) (n)

Regular spikes )68.4 ± 0.91 82.48 ± 13.89 0.26 ± 0.03 1.35 ± 0.06 52.30 ± 1.60 )8.52 ± 0.40 (42)Non- regular spikes )67.5 ± 3.10 86.73 ± 10.61 0.35 ± 0.08 2.56 ± 0.73 43.23 ± 3.18 )9.15 ± 1.53 (6)

AP and afterhyperpolarization (AHP) amplitudes were measured from the threshold of adapted APs. AP duration was measured at half of the peak amplitude. Thecurrent injected was the onset intensity of current that was sufficient to induce APs.

Table 3. Membrane properties of biocytin-labeled ACC neurons

ACC layer(n)*

Membranepotential (mV)

Input resistance(MW)

Current injection(nA)

AP duration(ms)

AP amplitude(mV)

AHP amplitude(mV)

Regularspikes

II ⁄ III (10) )68.44 ± 1.95 67.89 ± 18.23 0.27 ± 0.11 1.23 ± 0.17 58.21 ± 3.11 )9.46 ± 0.41 4V (20) )69.54 ± 1.52 74.97 ± 22.84 0.39 ± 0.08 1.09 ± 0.06 56.57 ± 2.15 )8.54 ± 0.80 11VI (4) )68.39 ± 1.09 63.94 ± 14.64 0.25 ± 0.06 1.04 ± 0.08 54.11 ± 1.75 )9.28 ± 1.27 4

*The number in parentheses indicates the total number of cells in each layer.

Table 1. Properties of cingulate cortical neurons receiving thalamic inputs

Values (n)

Passive membrane propertiesResting membrane potential (mV) )68.5 ± 0.8 (38)Input resistance (MW) 85.93 ± 15.43 (23)

Active membrane propertiesCurrent to generate action potential (nA) 0.3 ± 0.05 (21)Action potential amplitude (mV) 57.08 ± 1.61 (21)Action potential duration (ms) 1.13 ± 0.04 (21)

MT-evoked responseEPSP (mV; stimulus intensity ¼ 100 lA) 3.53 ± 0.57 (21)Action potential (mV) 54.97 ± 4.09 (12)

Data are presented as mean ± SEM.

Fig. 4. Antidromic activation and glutamatergic effect on evoked responses of cingulate cortical neurons. (A) Persistence of APs in an ACC neuron under low-calcium aCSF conditions; this neuron could follow high-frequency (100 Hz) stimulation. (B) Effects of d,l-2-amino-5-phosphonopentanoic acid (APV) and APVplus 6-cyano-7-nitroquinoxaline (CNQX) on evoked excitatory postsynaptic potentials (EPSPs) in the ACC, and the effect of APV on evoked decay phase EPSPsand APs in the ACC. The bar graph summarizes the effects of CNQX and APVon evoked EPSP peak amplitude, decay phase and initial slope in seven slices. CNQXaffected all parameters, whereas APVonly affected the decay phase and the peak amplitude. *P < 0.01 vs control. Data are means ± SEM. n ¼ 9 (control); n ¼ 7(CNQX); n ¼ 6 (APV).

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suggests that GABAergic interneurons may mediate the late phaseof intracortical polysynaptic transmission activated by thalamicinputs.

We orientated the slices such that the ACC was positioned directlyabove the MEA (Fig. 5A, inset, red square ) MEA recording area; redasterisk ) thalamus stimulating site). LFPs recorded via the MEA are

C D

BA

Fig. 5.

Fig. 6.

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shown superimposed on the ACC in Fig. 5A, and representativeexample sweeps in aCSF from one channel are shown in Fig. 5B (redtraces). A fast negative component N1 (peak amplitude)28.14 ± 6.01 lV at 8.32 ± 0.72 ms), a slow-rising positive compo-nent P (22.06 ± 2.68 lV at 11.45 ± 0.78 ms), a negative componenttailing the positive component N2 ()18.17 ± 3.07 lV at27.77 ± 1.00 ms) and a late negative component N3()16.70 ± 3.51 lV at 43.68 ± 3.38 ms) were observed (n ¼ 11slices). P, N2 and N3 were attenuated under low-calcium conditions(Fig. 5B, blue trace), indicating that these components were synap-tically activated. Meanwhile, application of aCSF with a highconcentration of divalent ions strongly influenced N3 (Fig. 5B, greentrace), indicating that it may be polysynaptic.

Evoked temporal and spatial activity in the ACC

To determine the spatial distribution of thalamus-evoked ACCresponses, the isopotential contour of these four LFP components atspecific time points were plotted against overlaid ACC cortical layers(Fig. 5C and D, color maps). N1 was strongly activated in layer VI andcoupled with a complementary positive potential in more superficiallayers. N2 was generated mostly in layer V and extended into layerII ⁄ III. N3 covered most of layers V and VI. These spatial distributionswere consistent in all 11 slices tested. The effect of low-calciumconditions on the spatial distribution of the evoked LFP components P,N2 and N3 was clearly demonstrated in serial isopotential maps(Fig. 5C). The late N3 component was selectively suppressed in thepresence of a high concentration of divalent ions as demonstrated byan altered spatial distribution of the N3 component as shown inFig. 5D.

A trajectory path calculated from the center of signal source of LFPsconsisting of sequential N1 (black), N2 (blue) and N3 (green)segments was constructed from one experiment (Fig. 6A). N1 traveledfrom ventral-lateral to dorsal-medial, and started in layer VI near thecingulum and ended in layer V. N2 emerged at the edge between layerII ⁄ III and layer V and descended to dorsal layer V. N3 progressedopposite to N1, from dorsal-medial to ventral-lateral, starting in layerV and ending in layer VI. The component vectors were constructedand overlaid on a biocytin tracing image. The resultant trajectory ofevoked responses was found to be initiated in deep cortical layers, toproject up to more superficial layers and then back down to deepcortical layers (Fig. 6B, inset). The progression of the evoked LFPs,the CSD data and the trajectory path can be found in theSupplementary material, Video S2. The trajectories of the threenegative components had similar extensions and angles in 11

experiments, and the average of these trajectories formed a loop froma region near the cingulum, through the dorsomedial ACC and back tothe ventrolateral ACC (Fig. 6C).Three columns in the recording array along the trajectory vectors of

the negative components (Fig. 6D) were selected to generate LFP andCSD profiles (Fig. 6E). The N1 sink current (blue) coupled with acomplementary source current (red) andmoved upward from deep in theACC. The N2 sink current swept downward slowly from superficiallayers and the N3 sink current traveled down from superficial to deeplayers (dashed lines and white arrows in Fig. 6E, CSD color maps). Thesimilarity of the field potential and CSD profiles in these three columnsand the singularity of the responses in the vertical dimension indicatethat current flows along the trajectory path through the layers and isconsistent with a columnar organization of signaling.

Input-specific short-term plasticity in the ACC

Six neurons were subjected to a minimal stimulation protocol in thepresence of APV to compare the synaptic characteristics of ACCneurons receiving different inputs. Their EPSP properties weredetermined by averaging successful evoked response traces (Table 4).The major differences between these input sites were amplitude, initialslope and EPSP half-width. EPSPs evoked with MT stimulation had asmaller amplitude and initial slope, but a longer half-width and decaytime in the minimal stimulation condition, than EPSPs evoked bystimulation in other sites.We investigated whether short-term plasticity in the ACC responds to

stimulation originating in theMT in vitro like it does in vivo (Pirot et al.,1994; Herry et al., 1999; Gemmell & O’Mara, 2002; Kung & Shyu,2002). The intensity dependence of evoked EPSPs was tested withTungsten electrodes placed in the thalamus, layer VI of the ACC and thecontralateral CC (Fig. 7A). We found that MT-stimulation producesmarked short-term facilitation in the ACC. Paired pulses were deliveredwith varied (50, 100, 150, 300, 500, 800 ms) interpulse intervals (IPIs)at 80% of the intensity that induced maximum responsive activity(Fig. 7B). Maximal potentiation was obtained with a 50-ms IPI, andPPF with 50-ms and 100-ms IPIs was significantly greater when thestimulation was delivered inside the thalamus rather than in a deep ACClayer or the contralateral CC (Fig. 7C). Tetanus applied to the thalamusenhanced EPSPs at all frequencies tested (10–200 Hz), and themaximal potentiated response was obtained with 50-Hz stimulation(20-ms IPI, Fig. 7D). The thalamus-evoked potentiated responses with20 Hz and 50 Hz stimulation were significantly greater than thatevoked by stimulating at the other sites (Fig. 7E).

Fig. 5. Spatial ⁄ temporal distribution of evoked LFPs in the ACC. (A) Inset shows a model slice including the thalamus, striatum and ACC. The small red squarerepresents the MEA recording location and the asterisk indicates the stimulating site. Sixty sweeps of evoked field potentials are shown overlaid on the anatomicalpositions of their recording leads. (B) Control sweeps (representative example shown in red) were abolished under low-Ca2+ artificial cerebrospinal fluid (aCSF)conditions (blue), except for the initial negativity. The late part of the negative component was diminished in high divalent aCSF conditions (green). Serialcomponents of the sweeps were designed as N1, P, N2 and N3. (C) Isopotential contour maps representing the presence of each component at different time pointswhen recorded in aCSF or in low Ca2+ are shown in the upper panel and lower panel, respectively. The time points of respective peaks are shown in the upper controlsweep of (B). (D) Isopotential contour maps representing the presence of each component at different time points when recorded in aCSF or in high divalent aCSFare shown in the upper panel and lower panel, respectively. The time points of respective peaks are shown in the lower control sweep of (B).

Fig. 6. The temporal progression of evoked local field potentials (LFPs) and current source density (CSD) profiles in the ACC. (A) A typical sweep selected from60 sweeps recorded via one channel. (B) The tracing movement of the center of signal source of components N1, N2 and N3 are shown in different colors torepresent the progression of the activities. The trajectory vectors calculated for each component are shown superimposed on an image of a biocytin-labeled ACCsection (Fig. 5B, inset). Note that the vectors are aligned with the fiber tract projecting from deep to superficial layers. (C) The trajectory vectors of the N1, N2 andN3 components from 11 experiments were overlapped, and the averages of the components are shown overlaid on the cortical layout. (D) Sweeps corresponding tothree columns aligned with the trajectory vector were selected for CSD analysis. (E) Sweeps of LFPs, CSD and the corresponding CSD profiles (right, color maps)in each column after stimulation are shown against time. The dashed white arrows in the CSD color maps follow the depth–time course of the sink currentscorresponding to N1, N2 and N3, respectively.

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To illustrate the spatial distribution of the potentiation of LFPsevoked by thalamic inputs, isopotential maps calculated from specifictime points representing the P, N2 and N3 components are shown inFig. 8A. Strong potentiation of the N2 and N3 components bythalamic afferents occurred with 50-, 100- and 150-ms IPIs relative tothe responses observed with deep layer stimulation. Potentiatednegative potentials expanded from superficial layers to deeper layers.The CSD profiles were generated along the axis of the trajectorypathway to illustrate the influence of the potentiation on the spatialdistribution of the responses (Fig. 8B). When a 100-ms IPI stimulationof the MTwas used, N2 and N3 were enhanced in layer V; meanwhilesimilar stimulation applied to the deep layers of the ACC failed toproduce a clear potentiation effect.Tetanic potentiation was also evident in the LFP data. Comparisons

across various frequencies of the potentiation and expansion of the N2component with stimulation via thalamic inputs and the contralateralCC are illustrated in Fig. 9A. The columnar CSD profiles evoked by10-Hz tetanic stimulation of the thalamus or contralateral CC werecompared to illustrate the differential effects (Fig. 9B). Tetanicstimulation of the thalamus enhanced N2 in layer V and attenuated N3after the third shock. On the contrary, tetanic stimulation of thecontralateral CC depressed the development of both N2 and N3.Nociceptive-associated unit activities were recorded in vivo in a

region of the MT confirmed to project to the ACC by the spike-triggered averaging technique (see experimental set-up in Supple-mentary material, Fig S1). EPSPs were recorded from ACC neuronswhen this thalamic bursting unit pattern was transformed intotransistor–transistor logic pulses that triggered stimulation in thethalamus, deep cortical layers or contralateral CC (Fig. 10A). Theamplitude of EPSPs evoked by the first four stimuli from differentstimulation sites were measured and normalized relative to the firstresponses. The thalamus-evoked fourth EPSPs showed greater poten-tiation than that produced by contralateral CC or deep layerstimulation (Fig. 10B and C). We further measured the maximalresponse that occurs during the train of thalamus bursting. Analyses ofboth the maximal to control ratios of the EPSPs and the number ofevoked APs showed that thalamic stimulation efficiently delivered thebursting unit pattern to the ACC and elicited substantial cingulateneuron firing (Fig. 10D). EPSP potentiation following thalamic burststimulation was diminished following perfusion of aCSF with APV(30 lm) or a high concentration of divalent ions (Fig. 10E and F).These results indicate that short-term plasticity in the thalamocingulatepathway may provide a specific means by which the MT can signal tothe ACC. This transmission may be mediated by polysynapticpathways, and the NMDA receptor may play an important role inmediating information flow to this area.

Discussion

Our innovative slice preparation preserves a physiologically intactthalamocingulate connection. It can used to elucidate the organizationof ACC circuitry and input-specific short-term plasticity in thethalamocingulate pathway. Examination of ACC synaptic plasticity isparticularly important as it may underlie nociceptive learning andmemory.

Methodological considerations

Unlike thalamo-sensory cortical pathways, which can be preserved byslicing at a specific angle (Agmon & Connors, 1991; Cruikshank et al.,2002; MacLean et al., 2006), the thalamocingulate pathway can not beT

ab

le4.

Responses

intheACCby

minim

alstim

ulationon

differentinputs

Inpu

tMinim

alstim

ulation

streng

th(l

A)

Risetime10

–90%

(ms)

Risingslope

(lV

⁄ms)

Decay

time

(ms)

Half-width

(ms)

Latency

(ms)

Amplitude

(lV)

MT

23.1

±4.8(8.5–5

0)6.4±1.4(3–14.1)

104.8±25

.2*(25.4–

214.6)

24.8

±5.3(10.1–

44.8)

30.9

±5.5(14.1–

52.9)

8.5±0.8(5.6–1

3)64

2±54

#(465

–877)

Con

tra-

lateralCC

19.7

±3.3(4.5–3

0)4.3±0.5(2.5–6

.4)

259.5±44

.6(145

.2–466

.2)

20.8

±3.8(11.1–37

.7)

22.4

±3.8(10–

36.9)

10.3

±0.7(7.9–1

3)12

27±112(895–1

850)

Deeplayers

9.4±1.3(4.5–1

4)5.1±1.2(1.6–8

.8)

307.0±66

.9(114

–508

.7)

23.2

±5.4(14.6–

49)

22.1

±4.6(10.1–

42.3)

5.3±0.4(3.8–7

)1155

±19

5(503

–17

10)

Dataareexpressedas

mean±SEM

(n¼

6),withrangegivenin

parenthesesbelow.*P

<0.05

vscontralateralCCanddeep

layer.

#P<0.01

vscontralateralCC.

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ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 25, 2847–2861

obtained by a single cut at any angle because it is not contained in anysingle plane (Krettek & Price, 1977; Groenewegen, 1988; Wang &Shyu, 2004). We demonstrated herein that the 3D pathway can befunctionally conserved in a slice if it is transformed into a 2D plane byintroducing cross-cuts that intersect at the precise angle of thepathway’s curvature and unfolding the brain block. Importantly, thepreservation of multiple long-distance pathways in the preparationmeans that it can be used to study information processing between theACC and other areas such as the basal ganglia, CC and reticularthalamic nuclei. Furthermore, once the precise trajectory paths aredetermined this plane-transforming slice technique can be used topreserve the pathways between the mPFC and other subcortical areas.

The stimulation we employed was confined to a region within350 lm of the stimulating electrode tip (Kung & Shyu, 2002). Thus,non-specific excitation of extra-thalamic regions could be excluded.We found that the most effective sites were restricted to the MTregion. Orthodromic and antidromic responses could be distinguishedby stimulating close to the medial core of the MT and reducing theapplied current. Although we were able to selectively excite thalamic-ACC afferents, it was difficult to distinguish among mPFC afferentsfrom different thalamic nuclei (Groenewegen, 1988; Wang & Shyu,2004). The topographic arrangement of corticopetal thalamic projec-tions should make it possible to distinguish among the nuclei byinjecting florescent dyes into different target cortical regions toretrogradely label the distinct thalamic nuclei.

Synaptic transmission of the thalamocingulate pathway

Consistent with findings revealing extensive MT terminals in themPFC (Krettek & Price, 1977; Berendse & Groenewegen, 1991;Kuroda et al., 1996; Gioanni et al., 1999; Wang & Shyu, 2004), wefound that responses to MT stimulation were primarily in the ACC.CSD analysis confirmed that cingulate neurons driven by MT inputsproduce maximal synaptic activation in layer II ⁄ III of cingulate cortex

area 1 (Sun et al., 2006). Synaptic activation of cingulate neurons bypaired-pulse stimulation of MT afferents induced homosynaptic short-term facilitation consistent with in vivo findings (Sikes & DeFrance,1985; Gemmel & O’Mara, 2002; Kung & Shyu, 2002). Thepotentiation sites are likely the initial terminal fields of MT afferentdendrites of deep layer pyramidal neurons (Vogt & Miller, 1983;Horikawa et al., 1988; Kuroda et al., 1996).Blockade of EPSPs and APs by CNQX and APV indicates that both

AMPA ⁄ kainate and NMDA receptors mediate thalamic excitation ofACC neurons. Indeed, MT inputs are the primary source ofglutamatergic input to cingulate neurons (Greengard et al., 1991;Kirkwood et al., 1993; Pirot et al., 1994; Hedberg & Stanton, 1995).In addition to their direct axo-dendritic synaptic contacts on principalcingulate neurons, thalamocingulate afferents also terminate onGABAergic interneurons (Kuroda et al., 1998, 2004). Thus, GAB-Aergic neurons may inhibit cingulate neurons via feedforwardinhibition. Moreover, the presence of both pre- and postsynapticGABAergic elements would allow for disinhibition of inhibitoryinterneurons following activation of thalamocingulate afferents (Ku-roda et al., 2004). Furthermore, our findings of antidromic responsesare consistent with previous demonstrations of ACC layer V beinghighly reciprocally connected with the thalamus (Hsu & Shyu, 1997;Kuroda et al., 2004). This finding implies that thalamic stimulationmay initiate reverberatory excitation between ACC and MT neurons.GABAergic innervation of layer V corticothalamic projection neuronshas also been observed (Kuroda et al., 2004). Thus, inhibitoryinterneurons are intimately involved in modulating signaling betweenthe ACC and MT.

Synaptic organization and intracortical circuitry

CSD analysis revealed that early deep sink currents (components N1and P) were accompanied by a more superficial source current. Thiscurrent configuration suggests that MT afferents terminate on vertic-

Fig. 7. PPF and tetanic potentiation of the anterior cingulate cortex (ACC) evoked by stimulation of the thalamus, deep cortical layers or the contralateral corpuscallosum (CC). (A) Schematic diagram showing stimulation and recording sites. The amplitudes of evoked excitatory postsynaptic potentials (EPSPs; normalizedwith maximal response) are presented graphically as a function of stimulus intensity. (B) Example traces of EPSPs evoked by different inputs at varying pairedpulse intervals (50–800 ms). (C) A greater paired-pulse potentiation ratio (second EPSP ⁄ first EPSP) at the 50- and 100-ms intervals was observed with stimulationin the thalamus than in the deep layers or contralateral CC (*P < 0.01 vs stimulation in deep layers or in contralateral CC). Data are means ± SEM. n ¼ 5(thalamus); n ¼ 5 (deep layers); n ¼ 4 (contralateral CC). (D) Example traces of tetanic potentiation in the ACC evoked by stimulation of the thalamus, deep layersor contralateral CC. (E) Potentiated responses (Max ⁄ Control EPSP) were greater with thalamic stimulation than with deep layer or contralateral CC stimulation, at20 and 50 Hz (*P < 0.01 vs deep layer and vs contralateral CC stimulation). Data are means ± SEM. n ¼ 12 (thalamus); n ¼ 11 (deep layers); n ¼ 9 (contralateralCC).

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ally orientated basal dendrites of layer V pyramidal neurons, creatinglocal sink currents and a distal source current at their apical dendrites(Cauller & Connors, 1994; Castro-Alamancos & Connors, 1996;

Kuroda et al., 1996). Sinks located in layer II ⁄ III (component N2)have successively longer latencies than that of the initial sink current.The recurrent axonal collaterals evident in our biocytin-labeled layer

Fig. 9. Tetanic potentiation of LFPs and current source density (CSD) in the ACC evoked by stimulation in the thalamus or contralateral corpus callosum (CC).(A) Sixty sweeps of evoked LFPs are shown overlaid on the anatomical positions of their recording leads. Example sweeps from one recording lead, indicated by asquare, are displayed and the potentiations by tetanic stimulation at different frequencies in the thalamus are shown. Isopotential contour maps represent the presenceof the N2 component when the control pulse or pulses with different frequencies were applied in the thalamus. (B) Tetanic stimulation-evoked LFPs from oneelectrode are shown as sweep profiles. The changes of the evoked activities in the ACC after the first and third shock of a train of tetanic stimulation were comparedin the CSD color maps.

Fig. 8. PPF of LFPs and current source density (CSD) in the ACC evoked by stimulation of the thalamus (Th) or the deep cortical layers. (A) Sixty sweeps ofevoked LFPs are shown overlaid on the anatomical positions of their associated recording leads. Example sweeps chosen from one recording lead, indicated by asquare, are displayed and the PPF at different IPIs with thalamic and deep layer stimulation are shown. Isopotential contour maps representing the appearance of theP, N2 and N3 components when activated by the control pulse or pulses with different IPIs delivered in the thalamus or in the deep layers are shown. (B) Traces ofLFPs evoked by paired pulses stimulation from the thalamus or deep layers recorded at the electrode highlighted in the inset were superimposed. The CSD colormaps of different time points were constructed from the field potential data along the axis of the trajectory paths. CC, corpus callosum.

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V pyramidal neurons suggest that intracortical synaptic currents sweepup from layer V to layer II ⁄ III.

Sink currents in layer II ⁄ III may also be excited directly by MTinputs. There are major thalamic projections to layers II, III andV (Krettek & Price, 1977; Groenewegen, 1988; Berendse &Groenewegen, 1991; Kuroda et al., 1998), and anterogradely labeledMT afferents terminate predominantly in layer II ⁄ III (Wang & Shyu,2004). Moreover, MT stimulation strongly activates layer II ⁄ III sinkcurrents (Sikes & Defrance, 1985; Sun et al., 2006).

Our current flow and vector analysis showed that the late sinkcurrents (N3) progress in an opposite direction advancing fromsuperficial to deep layers. The high divalent ion concentration effecton N3 was consistent with polysynaptic activation from layer II ⁄ IIItraveling to layers V and VI. Altogether our analysis of the temporal-spatial progression of thalamus-evoked current flow revealed a ‘deep–superficial–deep’ trajectory across the ACC layers. These resultsprovide evidence for functional columnar organization of processingin the ACC where vertically organized minicolumns are derived fromdendritic bundles of layer II ⁄ III, V and VI mPFC pyramidal neurons(Gabbott et al., 2005). This organization has been associated withsynaptic interactions in specific corticoafferent, interareal and inter-modular synaptic networks (Conde et al., 1995; Rao et al., 1999;Gabbott et al., 2003).

Input-specific synaptic transmission and plasticity

The enhanced PPF and tetanic potentiation of thalamic synapses oncingulate pyramidal neurons makes them unique in the ACC.

Interestingly, thalamocingulate synapses undergo bursting frequencyfacilitation resulting in differential increases in synaptic strength.Thalamocingulate synapses show much more facilitation than ACCsynapses formed by axons projecting from deep layers or thecontralateral cortex. There are several factors that may contribute tothis differential enhancement of ACC synaptic plasticity: (1) inherentor acquired synaptic differences; (2) differences in cell morphology –especially neurite arborization; and (3) input-specific induction ofchanges in the electrical response properties of different classes ofneurons.The neurotransmitter release probabilities within ACC synapses

may be affected by the variable morphological arrangements that existbetween different synaptic inputs, e.g. thalamic vs callosal (Vogt et al.,1981). Furthermore, NMDA NR2B receptor subunits are upregulatedin ACC synapses following persistent peripheral noxious stimulation.Thus, increased NR2B-mediated currents may underlie some activity-driven plasticity in the ACC (Wu et al., 2005).There are hyper-reciprocally connected pyramidal neurons in the

mPFC that display pronounced synaptic augmentation and post-tetanicpotentiation (Wang et al., 2006). These neurons have an elaboratemorphology, able to support strong recurrent activation. MT-respon-sive mPFC pyramidal neurons and nociceptive-specific neurons in theACC in particular exhibit extensive intracortical dendritic and axonalarborization and axonal recurrent activity (Yamamura et al., 1996;Onn & Wang, 2005). The complex morphology of ACC neurons andtheir potential for recurrent circuit activity may support sustainedactivity in MT-ACC synapses following stimulation.Our observation of input pathway-dependent facilitation suggests that

different cortical afferents alter activity patterns in local prefrontal

Fig. 10. Effects of MT bursting stimulation on anterior cingulate cortex (ACC) neurons. (A) Excitatory postsynaptic potentials (EPSPs) were recorded from ACCneurons when the in vivo bursting stimulus pattern was applied at the thalamus, deep cortical layers or contralateral corpus callosum (CC) in the slice (seesupplemental data Fig. S1 for the in vivo and in vitro stimulation and recording set-ups.) (B) Normalized EPSPs calculated from the first four responses evoked bybursting stimuli applied at different locations. The fourth EPSP evoked by thalamic stimulation was larger than that evoked by contralateral CC stimulation(*P < 0.05). Data are means ± SEM. n ¼ 7 (thalamus); n ¼ 4 (contralateral CC and deep layers). (C) The normalized EPSPs were plotted as a function of theinstant input frequency, which was calculated from the interval between the subject and the proceeding stimuli. (D) Results from six slices showed that burstingthalamic stimulation enhanced the delivery of thalamic inputs to the ACC and evoked more APs in ACC neurons when stimulation was delivered to the thalamusthan when it was delivered to the deep layers or the contralateral CC (**P < 0.01 vs deep layers and vs contralateral CC). Data are means ± SEM. n ¼ 9 (thalamusand deep layers); n ¼ 6 (contralateral CC). (E) EPSP potentiation by MT bursting stimulation is shown in the upper sweeps. The EPSPs evoked by the same stimuliduring perfusion of high divalent artificial cerebrospinal fluid (aCSF) or d,l-2-amino-5-phosphonopentanoic acid (APV). The effects are illustrated withsuperimposed sweeps obtained during control (black lines) and in high divalent and APV conditions (red lines). (F) Summary of high divalent aCSF or APVeffectson MT bursting-induced potentiation. Both high divalent aCSF and APV suppressed the potentiation effect. *P < 0.05 vs control, **P < 0.01 vs control; data aremeans ± SEM; n ¼ 4 (high divalent); n ¼ 5 (APV). MD, mediodorsal thalamic nucleus; Th, thalamus.

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circuits by producing complex dynamic changes in EPSPs and affectingtemporal summationwithin different classes of neurons. Consistent withfindings in primary sensory cortex (Shin et al., 2001; Rose&Metherate,2005), we observed decreased facilitation in the presence of highdivalent ion concentrations and APV, which suggests that NMDAreceptor-mediated long-lasting polysynaptic activity within the ACCcontributes to MT stimulation-induced facilitation. Our minimalstimulation experiment showed that content size but not decay timediffered betweenMTand other inputs.MT-patterned stimulation appliedat minimal intensity did not differentially affect facilitation amongstimulation sites (n ¼ 3, data not shown). Thus, this facilitation can notbe attributed to differences in synapse strength, and it can be deducedthat APV-sensitive facilitation of the MT-ACC pathway is likely due todifferential receptor compositions between these synapses.

Frequency modulation and sensory information processing

Short-term modifications in ACC synapses could regulate theprocessing and gating of amplitude- and frequency-modulated MTinputs. For effective transmission, thalamic inputs must be modulatedwithin a certain frequency range (Buonomano & Merzenich, 1995;Zucker & Regehr, 2002). Activities in this frequency range areamplified and better able to stimulate the cortex.Our thalamocingulate slice preparation can be used to investigate

the nature of ACC processing of thalamic signals. The finding that MTbursting stimulation can elicit differential responses in ACC neuronsprovides a critical assessment of the importance of MT input inrelaying information to the ACC. Bursting thalamic impulses canpotently activate cortical circuits, and the later impulses within eachburst are more likely to elicit APs (Swadlow & Gusev, 2001).

Supplementary material

The following supplementary material may be found onwww.blackwell-synergy.comFig. S1. Experimental set-up in vivo.Video S1. Animated 3-D imaging of DiI florescence tracing in mousebrain.Video S2. Time series progression of theLFPs,CSDand trajectory path.

Acknowledgements

The authors thank Dr Sergiy Sylantyev for technical assistance. The authorswould also like to thank Chu-Fang Chan for her help with the histology. Multi-channel silicon probes used in in vivo experiments (supplementary information)were provided by the University of Michigan Center for Neural Communica-tion Technology, sponsored by NIH NIBIB grant P41-RR09754. This studywas supported by grants from the National Science Council and the AcademiaSinica, Taiwan, ROC.

Abbreviations

ACC, anterior cingulate cortex; aCSF, artificial cerebrospinal fluid; AMPA,a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; AP, action potential;APV, d,l-2-amino-5-phosphonopentanoic acid; CC, corpus callosum; CNQX,6-cyano-7-nitroquinoxaline-2,3-dione disodium; CSD, current source density;DiI, 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate; EPSP,excitatory postsynaptic potential; GABA, c-aminobutyric acid; IPIs, interpulseintervals; LFPs, local field potentials; MEA, multi-electrode array; mPFC,medial prefrontal cortex; MT, medial thalamic nucleus; NHS, normal horseserum; NMDA, N-methyl-d-aspartate; PBS, phosphate-buffered saline; PFA,paraformaldehyde solution; PPF, paired-pulse facilitation; PV, parvalbumin.

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