NeuroImage 73 (2013) 113–120

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Ultra high-resolution fMRI and electrophysiology of the rat primarysomatosensory cortex☆

Yen-Yu Ian Shih a,b,c,d,⁎, You-Yin Chen e, Hsin-Yi Lai a,b, Yu-Chieh Jill Kao a,b,Bai-Chuang Shyu f, Timothy Q. Duong d,g,⁎⁎a Department of Neurology, University of North Carolina, Chapel Hill, NC, USAb Biomedical Research Imaging Center, University of North Carolina, Chapel Hill, NC, USAc Department of Biomedical Engineering, University of North Carolina, Chapel Hill, NC, USAd Research Imaging Institute, University of Texas Health Science Center at San Antonio, San Antonio, TX, USAe Department of Biomedical Engineering, National Yang-Ming University, Taipei, Taiwanf Institute of Biomedical Science, Academia Sinica, Taipei, Taiwang Department of Ophthalmology, Radiology, and Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

☆ Grant support: This work was supported in part by t(10POST4290091), Clinical Translational Science AUL1RR025767), and San Antonio Area Foundation toNS45879 and VA MERIT to TQD.⁎ Correspondence to: Y.Y.I. Shih, Experimental Neuroim

of Neurology and Biomedical Research Imaging Center, 13University of North Carolina, Chapel Hill, NC 27599, USA.⁎⁎ Correspondence to: T.Q. Duong, Research Imaging InstScience Center at San Antonio, 8403 Floyd Curl Drive, Sa+1 210 567 8152.

E-mail addresses: shihy@unc.edu (Y.-Y.I. Shih), duon

1053-8119/$ – see front matter © 2013 Published by Elhttp://dx.doi.org/10.1016/j.neuroimage.2013.01.062

a b s t r a c t

a r t i c l e i n f o

Article history:Accepted 28 January 2013Available online 4 February 2013

Keywords:fMRIHigh-resolutionCerebral blood volumeLocal field potentialCurrent source densitySomatosensory cortexRat

High-resolution functional-magnetic-resonance-imaging (fMRI) has been used to study brain functions at in-creasingly finer scale, but whether fMRI can accurately reflect layer-specific neuronal activities is less wellunderstood. The present study investigated layer-specific cerebral-blood-volume (CBV) fMRI and electro-physiological responses in the rat cortex. CBV fMRI at 40×40 μm in-plane resolution was performed on an11.7-T scanner. Electrophysiology used a 32-channel electrode array that spanned the entire cortical depth.Graded electrical stimulation was used to study activations in different cortical layers, exploiting the notionthat most of the sensory-specific neurons are in layers II–V and most of the nociceptive-specific neurons arein layers V–VI. CBV response was strongest in layer IV of all stimulus amplitudes. Current source density anal-ysis showed strong sink currents at cortical layers IV and VI. Multi-unit activities mainly appeared at layersIV–VI and peaked at layer V. Although our measures showed scaled activation profiles during modulationof stimulus amplitude and failed to detect specific recruitment at layers V and VI during noxious electricalstimuli, there appears to be discordance between CBV fMRI and electrophysiological peak responses,suggesting neurovascular uncoupling at laminar resolution. The technique implemented in the presentstudy offers a means to investigate intracortical neurovascular function in the normal and diseased animalmodels at laminar resolution.

© 2013 Published by Elsevier Inc.

Introduction

Recent technical improvements in magnetic resonance imaging(MRI) have helped to advance the spatial resolution of functionalMRI (fMRI) to study brain activities in fine cortical structures, includ-ing resolving hemodynamic functions from individual cortical col-umns (Duong et al., 2001; Fukuda et al., 2006; Kim and Fukuda,2008; Kim et al., 2000) and cortical layers (Harel et al., 2006; Jin

he American Heart Associationwards (CTSA, parent grantYYIS and the NIH/NINDS R01

aging Laboratory, Departments0 Mason Farm Road, CB# 7513,Fax: +1 919 843 4456.itute, University of TexasHealthn Antonio, TX 78229, USA. Fax:

gt@uthscsa.edu (T.Q. Duong).

sevier Inc.

and Kim, 2008; Logothetis et al., 2002; Lu et al., 2004; Shen et al.,2008; Siero et al., 2011; Smirnakis et al., 2007; Tian et al., 2010;Zhao et al., 2006), but how well those fMRI signals can represent re-gional neuronal activities remains debated. At a coarse spatial resolu-tion, blood-oxygenation-level-dependent (BOLD) signal has beenfound to linearly correlate with evoked electrophysiological signals(Brinker et al., 1999; Disbrow et al., 2000; Goloshevsky et al., 2008;Huttunen et al., 2008; Masamoto et al., 2007; Ogawa et al., 2000;Van et al., 2006). Logothetis et al. (2001) demonstrated that localfield potential (LFP) shows better correlation and provides a betterestimate of the BOLD impulse response compared with multi-unitspike activity (MUA) by simultaneously recording BOLD and electro-physiology signals in the monkey visual cortex. It was later confirmedthat the tissue oxygen tension is evident in the cat visual cortex whenspike activity is absent and the LFP still exists (Viswanathan andFreeman, 2007). These data suggest that the BOLD hemodynamicscorrelate better with synaptic input (LFP) than output (spike). LFP ismainly generated from pyramidal cells whose dendrites extendthroughout the cortex. An electric dipole with two charges of

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opposite polarity usually generated from the pyramidal cells andspans across the entire cortical depth. At high resolution, the powerof LFP cannot be considered as a local measure of neuronal activitydue to the lack of laminar resolution of the dipole (Buzsaki et al.,2012; Kajikawa and Schroeder, 2011). Current source density (CSD)analysis converts LFPs at different laminar locations into electricalcurrent distribution, providing localization of electrophysiologicalsinks (inward membrane currents) and sources (outward membranecurrents), where sinks correspond to dendritic excitatory postsynap-tic potentials and sources correspond to passive efflux of charge fromneuronal cell bodies (Freeman and Nicholson, 1975; Mitzdorf, 1987).High-resolution fMRI has been used to resolve responses in differentcortical laminae. Most of the gradient-echo BOLD fMRI studiesshowed that the responses exhibited less spatial specificity to intra-cortical neuronal activities because the oxygenation changes were ev-ident in large draining vessels on the cortical surface. BOLD signalshave the shortest onset time in layers IV–V (Siero et al., 2011; Silvaand Koretsky, 2002; Silva et al., 2007), but with only a few exceptions(Koopmans et al., 2010; Logothetis et al., 2002; Olman et al., 2012),the gradient-echo BOLD signals were generally highest in the superfi-cial layer and the signals decreased monotonically with cortical depthin rats (Duong et al., 2000; Shen et al., 2008; Silva and Koretsky, 2002;Silva et al., 2000; Yu et al., 2012), cats (Harel et al., 2006; Jin and Kim,2008; Zhao et al., 2004, 2006), monkeys (Smirnakis et al., 2007), andhumans (Polimeni et al., 2010; Siero et al., 2011). In contrast,spin-echo fMRI (Duong et al., 2003; Lee et al., 1999; Zhao et al.,2006), similar to cerebral blood flow (CBF), cerebral blood volume(CBV), and oxygen metabolism fMRI (Harel et al., 2006; Jin and Kim,2008; Lu et al., 2004; Shen et al., 2008) has more similar layer speci-ficity to electrophysiology, showing activations predominantly in thecortical layers IV–V because this technique is less susceptible to largevessel contributions.

Albeit there are many studies showing spatial correlations betweenfunctional hyperemia and synaptic activities, neurovascular couplingwas found only at certain stimulus conditions (Nielsen and Lauritzen,2001) and the flow-neuronal activity relationship was shown to benonlinear after passing a certain threshold (Devor et al., 2003;Hewson-Stoate et al., 2005; Jones et al., 2004; Sheth et al., 2004), indi-cating that the hemodynamic response may not simply reflect the re-gional synaptic activities. The present study aimed to examine therelation between CBV fMRI signal and neural activity as a function ofcortical depth at ultra-high spatial resolution. We hypothesized thatCBV fMRI responses are not tightly coupled to electrophysiology mea-sures (MUA and CSD) across different cortical layers at high spatialresolution. Iron-oxide CBV fMRI was utilized because it has improvedfunctional contrast to noise ratio and laminar specificity. We alsohypothesized that graded electrical stimulation can evoke preferentialactivations in the middle and deep cortical layers based on the factthat cortical neurons driven by non-noxious stimulation were mainlyrecorded from layers II–V, while most of the nociceptive specific neu-ronswere found in layers V–VI (Lamour et al., 1983a).We implementedhigh-resolution CBV fMRIwith a custom-made small surface coil at highfield to achieve high signal-to-noise ratio and 40×40 μm in-planeresolution. LFP and MUA were recorded using a custom-designed32-channel electrode array covering the entire cortical depth withhigh spatial resolution (74 μm inter-electrode distance). These elec-trodes provided better sensitivity and accuracy because of closer prox-imity to sites of neural events.

Materials and methods

Subjects

A total of 16 adult male Sprague Dawley rats (weighing 250–300 g;Charles River Laboratories) were studied. All experimental procedureswere approved by the Institutional of Animal Care and Utilization

Committee, UT Health Science Center at San Antonio. Animals werehoused in the vivarium (12:12-h light–dark cycle, controlled humidityand temperature) with free access to food and water.

Animal preparation

Rats were initially anesthetized with 3% isoflurane and orotracheallyintubated for mechanical ventilation (Model 683, Harvard Apparatus,South Natick, MA). A PE-50 catheter was cannulated to the right femoralvein for subsequent drug administration. After the animalwas secured ina MRI compatible rat stereotaxic headset, isoflurane was discontinuedandα-chloralose (60 mg/kg first dose, followed by 30 mg/kg/h, i.v. infu-sion) was administered for anesthesia. The rat was then paralyzed withpancuronium bromide (3 mg/kg first dose, followed by 1.5 mg/kg/h,i.v.). End-tidal CO2 was continuously monitored via a capnometer(Surgivet, Smith Medical, Waukesha, WI). Non-invasive end-tidal CO2

values were previously compared to invasive blood-gas samplingsunder identical experimental conditions (Shih et al., 2012b) andkept in normal ranges (3–3.3%), corresponding to arterial pCO2 of30–40 mm Hg. Rectal temperature was maintained at 37.0±0.5 °Cwith warm-water circulating pad. Heart rate and blood oxygen satu-ration level were continuously monitored by a MouseOx system(STARR Life Science Corp., Oakmont, PA) andmaintained within nor-mal ranges. An established CBV fMRI technique was employed(Mandeville et al., 1998) by using 30 mg/kg monocrystalline ironoxide nanoparticles (MION) at 11.7 T to minimize the BOLD contri-bution (Shih et al., 2011, 2012c).

Forepaw stimulation

Electrical stimulation was achieved via a pair of needle electrodesinserted under the skin of the right forepaw (between digits 1 and 2and digits 3 and 4). The electrodes were then fixed with surgical tapeand the stimulation was confirmed by observing digit twitching. Gradedelectrical stimulation was applied using a stimulator (Model 2100, A-MSystems, Carlsborg, Washington, USA) with a constant current at 1, 2,6, and 10 mAwith a pulse-width of 0.3 ms at 3 Hz. Each stimulus ampli-tudewas presented at least once to each animal in a randomorder. Stim-ulation current above 2 mA is considered to induce nociception (Lowe etal., 2007; Shih et al., 2009, 2011, 2012a). A block-design stimulusparadigm was OFF–ON–OFF–ON–OFF for fMRI and OFF–ON–OFF forelectrophysiology, where OFF=100 s and ON=50 s.

fMRI data acquisition

MRI was performed in a group of six rats. MR images were ac-quired using an 11.7 T/16 cmmagnet and a 74 G/cm B-GA9S gradientinsert (Bruker, Billerica, MA). A custom-made small circular surfacecoil (ID~0.7 cm) was placed above the contralateral primary somato-sensory cortex of the forelimb (S1FL). Magnetic field homogeneitywas optimized using standard FASTMAP shimming with first ordershims on an isotropic voxel of 7×7×7 mm encompassing the imag-ing slices. A T2-weighted pilot image was taken in the mid-sagittalplane to localize the anatomical position by identifying the anteriorcommissure (bregma−0.8 mm). Imaging slice was oriented perpen-dicular to the cortical surface for better cortical layer alignment(Fig. 1). CBV fMRI was acquired with FLASH sequence using spectralwidth=14.8 kHz, TR/TE=26/10 ms, FOV=0.768×0.768 cm, slicethickness=2 mm, matrix=192×192, yielding in-plane resolu-tion=40×40 μm and temporal resolution=5 s. The flip angle ofthe FLASH sequence was adjusted to maximize the signal in theS1FL, which was the Ernst angle (~10°). High in-plane resolution isneeded to unambiguously resolve layer-specific responses becausethe thickness of cortical layer IV in the S1 area is only about 250 μm(Sun et al., 2006).

Fig. 1. High resolution MRI and CBV-weighted fMRI. Data were acquired by using a custom-designed coil (ID~0.7 cm) at an 11.7 T MRI scanner. The coil was placed above the leftS1FL to provide better signal-to-noise ratio. Post-MION (A) axial and (B) sagittal images with 20 mm FOV. (C) Horizontal image was acquired parallel to the surface of the left S1FLas shown in A & B. (D) Horizontal image with the same position as C but with smaller FOV (7.68 mm). Forepaw stimulation at 2 mA, 0.3 ms pulse width, and 3 Hz was applied toidentify the activation area. (E) Axial image perpendicular to the cortical surface and parallel to the cortical penetrating vessels with 7.68 mm FOV as shown in A, B, and D. This sliceposition was used for the rest of the fMRI studies.

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Electrophysiology recording

Electrophysiology was performed in a separate group of five rats. Astereotaxic frame (Model 900, David Kopf Instruments, Tujunga, CA,USA) was used to position the head. The animal physiological condi-tions were kept the same as the MRI experiment, except that cranioto-mies were performed over the S1FL area contralateral to the stimulatedforepaw to implant the recording electrode. A stainless steel screw wasplaced 2 mm caudal to lambda as a reference electrode. After the durawas removed, a custom designed polyimide-based 32-channel micro-electrode array (Chen et al., 2009) was implanted at the S1FL (coordi-nates from Bregma: AP, +0.5 mm; ML, −4 mm; and VD, −2. mm;approachedwith a coronal angle of 22.5°). The spacing between two ad-jacent channels was 74 μm. The diameter of the electrode was 16 μm.Data were acquired using a Cerebusmulti-channel data acquisition sys-tem (BlackrockMicrosystems, Salt Lake City, Utah, USA). The data wereamplified and filtered into: LFP (0.3–500 Hz and sampled at 1 kHz) andMUA (150 to 5000 Hz and sampled at 20 kHz). At the end of the exper-iment, a 30 μA direct-current was delivered to the deepest contact leadfor 10 s to label the recording site. The rats were then given an overdoseof anesthetic and perfused with a mixture of 150 ml ice-cold PBS solu-tion and 500 ml 4% paraformaldehyde at the flow rate of 50 ml/min.The brainswere carefully removed from the skulls and kept in amixtureof 4% paraformaldehyde and 30% sucrose at 4 °C for two days. After-wards, each brainwas sliced into 50 μmcoronal sectionswith a freezingmicrotome (CM 1800, Leica, Germany). Nissl staining of brain tissuesections with 0.1% cresyl violet was used to confirm the recordinglocations.

Data analysis

Image analysis was performed using a custom-written program inMatlab (Math-Works, Natick, MA), STIMULATE (University of Minneso-ta), and Statistical Parametric Mapping (SPM). Time series MRI datawere co-registered to align slight image drift overtime using the spatialrealignment function in SPM5 (Duong and Muir, 2009; Muir andDuong, 2011). Spatial translation and rotationwere applied automatical-ly to co-register images showing difference from the mean image aver-aged across the entire time-series using mutual information. Thestimulus-evoked ΔR2⁎ value, which varies linearly with the stimulus-evoked CBV fraction, was calculated as follows (Mandeville et al., 1998)after MION injection: ΔR2

� ¼ 1TE ln Sctrl=Sstimð Þ, where Sctrl and Sstim are

the MR signal intensities before and during stimulation, respectively.The stimulus-evoked percent-CBV change was further computed by

dividing the basal CBV: %CBV ¼ ln Sctrl=Sstimð Þln Spre�MION=Sctrlð Þ, where Spre-MION is the

MR signal intensity before MION injection. Intensity profiles across theentire cortical depth (2 mm) were obtained from flattened images byprojecting lines perpendicular to the cortical surface on a T2-weightedimage coregistered to the time-series data. Profiles were then averagedalong the length of the S1FL (~1.3 mm) and plotted as a function oftime. The signals along the cortical depth were assigned to fivepre-defined cortical layers according to Nissl staining, where thepercent-thickness of layers I, II–III, IV, V, and VI were 12%, 20%, 14%,18%, and 36% of the total cortical depth, respectively (Sun et al., 2006).Layer-specific stimulus-evoked percent CBV changes to different stimu-lus amplitudes were tabulated.

Electrophysiology data were analyzed by using custom-writtencodes in MATLAB. The 50 s LFP and MUA data during stimulationwere split into 150 trials of 333 ms post-stimulus periods based onthe stimulus time stamps (3 Hz). Averaged LFPs and MUAs wereobtained by averaging over 150 trials. ∑MUAs were then calculatedas an integration of the area under the response curve during the100 ms post-stimulus period. Current source-density (CSD) analysis(Freeman and Nicholson, 1975; Mitzdorf, 1987) processed averagedLFPs using a numerical approximation of the second finite differencesas described previously (Wojcik and Leski, 2010). The relationshipbetween LFP and CSD is described by a Poisson equation as follows(Freeman and Nicholson, 1975):

∇⋅ J ¼ −Im ¼ −σ∇2ϕ

where σ is the conductivity of the conducting medium and ψ is the FP.The CSD units should be multiplied by the unit of conductivity. How-ever, if the recording area is small, the tissue conductivities along allof the axes can be assumed to be homogeneous (McAllister andWells, 1981). The equation can be simplified as follows:

∇⋅ J ¼ −Im ¼ −σ∇2ϕ:

And the approximation of trans-membrane current is

Im ¼ − 1kh2

� � Xnm¼−n

amϕ xþmhð Þ

whereϕ is the extracellularfield potential, h is the distance between ad-jacent recording sites (74 μm), and x is the coordinate perpendicular tothe cortical layer. The D2-filter was performed for spatial smoothingand the parameters n, k, and am are constants as follows: n=2, k=4,

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a0=−2, a±1=0 and a±2=1 (Freeman andNicholson, 1975; Sun et al.,2006; Tenke et al., 1993). The brain tissue conductivity of 0.3 S/m wasused (Bédard and Destexhe, 2011; Goldenholz et al., 2009) and was as-sumed isotropic and homogeneous in the cortex (Chapman et al., 1998;Csicsvari et al., 2003). To compare with the CBV fMRI (reflecting overallmetabolism-related hyperemia after stimulus) and ∑MUA, the peakamplitude of the first sink component after the stimulus onset wascalculated.

Data were then averaged across subjects to provide group-averagedlaminar profiles of evoked sink distribution and MUAs in the S1FL. Sta-tistical analysis was performed by SPSS software (IBM, Armonk, NY,USA). Repeated-measures ANOVA with LSD post-hoc test was used.Homogeneity of the variances was assessed by Levene's test. The signif-icance level was set at P b 0.05. All data are presented as mean± SEM.

Results

Representative high-resolution CBV fMRI of the rat S1FL (40×40 μmin-plane resolution) is shown in Fig. 2A. A thick slice was used toincrease the signal-to-noise ratio andminimize individual vessel contri-bution to the laminar CBV profiles. Nevertheless, column-like activa-tions were still observed in the cortex which may originate frompenetrating vessels perpendicular to the cortical surface. The cortexwas linearized to facilitate layer analyses (Fig. 2B). Fig. 3A shows CBVprofile time courses of 1, 2, 6, and 10 mA stimulation (n=6). No appar-ent spatial shift in the mediolateral direction was observed at differentstimulus intensities; therefore data were averaged along the length ofthe cortex (~1.3 mm) within the ROI (Fig. 2B) and plotted over time.The percent CBV changes varied with the cortical depth. The greatestsignal changes occurred in cortical layer IV and decreased continuouslytoward both the cortical surface and corpus callosum. The basal CBVfraction at different cortical layers is shown in Fig. 3B. Cortical layer IVshows the higher CBV fraction compared with layers I and VI(P b 0.05). Percent CBV changes to graded forepaw electrical stimulationexhibited similar distribution as the basal CBV fraction (Fig. 3C), where asignificantly stronger CBV response was observed at cortical layer IVcompared with layers I and VI at all stimulus amplitudes (P b 0.05).

Fig. 4A shows LFP traces from a representative subject and Fig. 4Bshows corresponding CSD traces calculated from the LFP data. The am-plitudes of the first CSD sink peak were quantitatively expressed as abar graph shown in Fig. 6A. Cortical layers IV andVI showed the greatestsink currents among all stimulus conditions. Fig. 5 shows MUA tracesfrom a representative subject response to graded forepaw electricalstimulation. ∑MUAs were stronger in cortical layer V compared to allother layers at 6 and 10 mA stimuli (P b 0.05; Fig. 6B). Higher stimulusamplitude evoked greater MUA responses compared to 1 mA at all

Fig. 2. High resolution CBV-weighted fMRI responses. (A) Column-like activationwas ob-served in the contralateral S1FL which may originate from penetrating vessels. Stimula-tion paradigm was 2 mA, 0.3 ms pulse width, and 3 Hz. (B) Linearized cortical image.To achieve laminar analysis, the cortex was linearized via finding the edge of the corticalsurface and radially projecting lines perpendicular to the edge (inset). Cortical surface andcorpus callosum can be more clearly identified on the pre-MION FLASH image. The depthof the S1FL is generally 2 mm.

cortical layers (P b 0.05). Fig. 6C shows 32 equally spaced contacts ofthe electrode array that spanned the cortex for electrophysiological re-cording. At the end of the experiment, a small lesion was induced bypassing 30 μA anodal current to the last contact lead for 10 s to confirmthe recording site by Nissl staining.

Discussion

The present study demonstrated high-resolution CBV fMRI and elec-trophysiological responses to graded forepaw electrical stimulation inthe rat S1FL. The major findings were: (i) Column-like activationswere observed using CBV fMRI at 40×40 μm in-plane resolution.Those column-like activations may result from vasodilation of smallvessels located nearby the cortical microcolumns. (ii) CBV responsewas strongest in layer IV of all stimulus amplitudes. The activation pro-files across cortical layers were similar among different stimulus ampli-tudes. (iii) CSD sink components that peaked at layers IV and VI weresignificantly stronger than other layers among all stimulus amplitudes.(iv) MUAmainly appeared in layers IV–VI and peaked at layer V duringnoxious stimuli. There appears to be discordance between CBV fMRIand electrophysiological peak responses, suggesting neurovascularuncoupling at laminar resolution. These data supported our hypothesisthat fMRI does not accurately reflect electrophysiology measures athigh spatial resolution.

Laminar-specific neuronal activity

Unmyelinated C primary afferentfibers have been found to carry no-ciceptive signals in rats (Lynn and Carpenter, 1982), monkeys (Croze etal., 1976), and humans (Torebjork, 1974; Van Hees and Gybels, 1972).Nociceptive neurons in the ventroposterior thalamic nuclei respond toelectrical stimulation of cutaneous C-fibers (Guilbaud et al., 1980) andproject to the S1 (Schouenborg et al., 1986). Aδ-fibers also respond tointense mechanical stimuli and nociception (Julius and Basbaum,2001). In contrast, myelinated Aβ primary afferent fibers primarily de-tect innocuous stimuli and consequently do not contribute to pain(Djouhri et al., 1998). Previous fMRI and electrophysiology studiesshowed that the responses of Aβ-, Aδ-, and C-fibers reached their max-imum amplitude and saturated when the nerve-stimulating currentswere 10, 15, and 20 times higher than the minimal current to evokeaction potential, demonstrating that the graded electrical stimulationhas potential to differentiate brain areas associated with nociception(Chang and Shyu, 2001). The rat cortex exhibits high laminar specificityto different stimulations. Cortical neurons driven by non-noxious cuta-neous stimulationweremainly recorded from layers II–V (98%), where-as most of the nociceptive specific neurons were found in layers V–VI(92%) (Lamour et al., 1983a). A previous study using a combination oflatency measurements, anodal block of A fiber conduction, and gradedstimulation showed that the strong field potentials in layers V and VIwere primarily evoked by nociceptive C fiber input (Kalliomaki et al.,1993). By electrically stimulating the median nerve, Jellema et al.(2004) found that specific thalamic afferents first depolarized layer IIIand layer V pyramidal cells. After that, superficial pyramidal cells weredepolarized via supragranular intracortical projections, and followed bythe generation of spikes in layer V pyramidal cells, which enhancedsupragranular pyramidal cell activation. Sun et al. further showed thatnoxious electrical stimulation of the rat hindpaw primarily evoked sinkcurrents at layer IV and spread transynaptically upward to layers II–III.In addition, laser stimuli evoked a delayed sink current occurred at theboundary of layers V and VI and swept downward to deep layer VI(Sun et al., 2006). In short, the major excitatory intracortical circuits in-clude: (i) the feed-forward circuit starting from layer IV to layer II/III,and then to layers V and VI. (ii) The feed-back circuit starting from layersV andVI to layers III and IV, and from layer III to layer IV (Bannister, 2005;Lubke and Feldmeyer, 2007; Schubert et al., 2007). Although CSD repre-sents synaptic distribution in cortical layers, it reveals a static wiring

Fig. 3. Group averaged CBV responses to graded forepaw electrical stimulation. (A) Averaged CBV fMRI time course of different stimulus amplitudes (n=6). The ROI was 1.3×2 mm2

(shown in Fig. 2B) and centered to the activation site. Data were averaged across the lateral to the medial side and plotted over time. The X-axis indicates imaging time period, whilethe Y-axis represents cortical depth. The black boxes indicate stimulation epochs. (B) Basal CBV fraction (ΔR2*) at different cortical layers. (C) Percent CBV changes to graded forepawelectrical stimulation at different cortical layers (n=6). *Significantly different from layer IV; Significant level was set at Pb0.05. Error bars are s.e.m. values.

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organization instead of dynamic neuronal responses evoked by gradedstimuli (Einevoll et al., 2007). Therefore, increasing the stimulus intensi-ty would result in scaling of the amplitude of the same laminar profile asshown in our data.

MUA describes the aggregates of extracellular spikes and may bemore suitable to address laminar responses to stimulus (Einevoll etal., 2007; Krupa et al., 2004; Sakata and Harris, 2009). According tothe laminar architecture of the cortex, most of the cortical neurons arelocated in layers IV and VI (Helmstaedter et al., 2007; Meyer et al.,2010). Dense thalamocortical projections were also found in theselayers (Bernardo and Woolsey, 1987; Herkenham, 1980; Keller et al.,1985; White, 1979). Although the number of neurons in layer V is lessthan layer IV or VI, the primary output action potentials were emittedby the thick-tufted cells in layer Vb (de Kock et al., 2007; Feldmeyer,2012; Helmstaedter et al., 2007). High MUAs in layers IV–VI in ourdata were in good agreement with others reported previously (Meyer

Fig. 4. (A) Representative LFP averaged from 150 trials during graded forepaw electrical stiulation. The sink and source current traces were derived from averaged LFP. Zero current baward directed and marked with shade. The CSD traces showed apparent sink currents at la

et al., 2010) and substantiates the cortical laminar cytoarchitecture.Our original hypothesis was that graded electrical stimulation at therat forepaw was able to modulate laminar activation profile of MUA.However, our data failed to detect specific recruitment at layers V andVI during noxious electrical stimuli. Instead, a scaled activation profilewas observed during graded electrical stimuli. This indicates that elec-trical stimulus at the forepaw is non-selective and the response in thecortical laminae may be contaminated by a variety of peripheral input,which eliminates the modality specificity across layers. Another poten-tial explanation is that the amount of nociceptive neurons is relativelysmall in population. It has been shown that 29% of the neurons in the so-matosensory cortex are sensory-related and only 8% are nociceptive-specific out of 694 neurons recorded in 26 rats (Lamour et al., 1983b).An alternative and more specific noxious stimulation, such as usingCO2 laser to selectively evoke nociception without affecting other sen-sory modalities, or using optogenetics to activate a specific group of

mulation. (B) Corresponding CSD laminar responses to graded forepaw electrical stim-se lines were drawn to divide the sink and source currents. The sink current was down-yers IV and VI.

Fig. 5. Representative MUA traces during graded forepaw electrical stimulation. Theresponses mainly located at layers IV–VI.

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neurons in the defined cortical layers may be more suitable to studyintracortical neurovascular coupling in the future.

Laminar-specific CBV fMRI response

High in-plane resolution is needed to resolve layer-specific responsesbecause the thickness of cortical layer IV in the S1 area is only about250 μm (Sun et al., 2006). The hemodynamic responses have beenshown to better reflect the input of the neural population rather thanits output (Jones et al., 2004; Logothetis et al., 2001; Viswanathan andFreeman, 2007). Our CBV fMRI response only peaked at layer IV acrossall stimulus amplitudes and better correlated to the major synapticinput revealed by CSD. The sink currents in cortical layer VI and MUApeaked at layer V were not reflected by CBV fMRI. This may be due tothe distribution of vascular network and the control of microcirculationin the cortex. In general, neurons inside the cortical layer IV have bettersomatotopic representation (Woolsey et al., 1996). High vascularity inlayer IV (Fonta and Imbert, 2002; Lauwers et al., 2008; Patel, 1983;Smirnakis et al., 2007; Zheng et al., 1991)may be to accommodate great-er metabolic needs compared with other layers (Kennedy et al., 1976;Kossut et al., 1988). The activation-induced CBV change was highly cor-related with the basal CBV. In the rat cortex, the highest vessel densitywas found in layer IV. The vessel density in layer VI was only 10% thatof layer IV and the vessels also decreased in size along the depth and di-minished in layer V or VI (Patel, 1983). A lower basal CBV indicated less

Fig. 6. Group averaged CSD and MUA responses to graded forepaw electrical stimulation. (Aplitude (inward current) was quantified at different cortical layers. *Significantly different(B) Group-averaged MUA (n=5) of different stimulus amplitudes.∑MUAs were calculatedulus onset. *Significantly different from layer V; #Significantly different from 1 mA; Significahead) was observed at the boundary of the cortical layer VI and corpus callosum. Lesion was iof the experiment to confirm the recording site and ensure that the 32 recording channels s

contrast enhancement and hence less sensitivity. It is likely that thosesparse arterial terminals in layer VI respond minimally to adjacent neu-ronal activity or generate very weak CBV changes and thus resulting inpoor spatial specificity of the CBV responses to the strong sink currentsin layer VI. fMRI activation at high resolution has been suggested to spa-tially relate to functional vascular unit (Duong et al., 2001; Duvernoy etal., 1981; Harel et al., 2010; Kim et al., 2000). Themicrovascular structurein the rat cortex is highly organized but is not completely isolated. In cor-tical layer IV, dense vascular connections have been found between thecortical columns by micro-injecting dye into the arteriole (Woolsey etal., 1996). These microvessels in layer IV possess finer tuning of flowchanges and can nourish the neighboring columns (Woolsey et al.,1996), whichmay lead to functional hyperemia occurring predominant-ly in layer IV during stimulation.

Potential limitations

Potential drawbacks of the present study were: (i) a relatively thickslicewas used to increase the signal-to-noise ratio andminimize poten-tial contribution from large penetrating vessels to the laminar CBV pro-files. Therefore, the CBV fMRI signals may be susceptible to partialvolume effect. The S1FL has reasonably uniform layer thickness andsimilar distribution of neurons/vasculature within each layer. We ad-justed our slice angle to make it perpendicular to the cortical surfaceto reduce the error in layer assignment (Fig. 1). (ii) Electrophysiologywas not performed simultaneouslywith fMRI and the recording volumewas different. Future studies will focus on developing MR-compatiblemultichannel electrode arrays for simultaneous recording to eliminatethe physiological variations in different subjects. (iii) Short TR may in-duce in-flow effect, which could reduce the negative MION fMRI signalchange and result in incorrect estimate of CBV changes. Small flip angle(~10°) was used in this study which minimized in-flow effects. Similarstudy that used long TR in MION fMRI showed that the layer-specificCBV response patterns were similar (Shen et al., 2008), suggestingthat inflow effect with our TR was likely small. A high dose of MIONused in this study also minimized the in-flow effect because the intra-vascular blood T2* value is dramatically shortened and thus invisible(Lu et al., 2004). (iv) In our CSD analysis, the extracellular medium ispresumed to be uniform and resistive (Mitzdorf, 1985; Pettersen et al.,2006). The mismatch between CBV and CSD profiles could potentiallybe due to the negligible estimation of the surrounding extracellularme-dium in the CSD analysis since the ionic flow and polarization effectmay alter the expression of current source (Bédard and Destexhe,2011). (v) The borders of the layers were not individually identifiedand percent depth ratio was used for layer assignment. This may affectthe quantitation of the layer responses.

) Group-averaged CSD (n=5) of different stimulus amplitudes. The first CSD sink am-from layer IV. xSignificantly different from layer I. #Significantly different from 1 mA.by integrating the area under the response curve during the first 100 ms after the stim-nt level was set at Pb0.05. Error bars are s.e.m. values. (C) Reference lesion site (arrow-nduced by passing an anodal current (30 μA for 10 s) to the 32nd contact lead at the endpanned the entire cortical depth.

119Y.-Y.I. Shih et al. / NeuroImage 73 (2013) 113–120

Conclusions

The present study developed ultra-high-resolution fMRI and electro-physiology techniques to study neurovascular responses in the rat so-matosensory cortex. The spatial resolution was 40×40 μm in-plane forCBV fMRI. The spacing between two adjacent recording sites for electro-physiologywas 74 μm. CBV fMRI and electrophysiological data showdif-ferent functional activation profiles across the cortical depth, peaking atdifferent locations. These findings indicate that hemodynamic responsemay not exclusively reflect local neuronal activity, and may depend onthe distribution and the reactivity of regional microvasculature. Thistechnique offers a means to investigate intracortical neurovascular func-tion in the normal and diseased animal models.

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