Neurovascular coupling in primary auditory cortex investigated with voltage-sensitive dye imaging and laser-Doppler flowmetry

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Neurovascular coupling in primary auditory cortex investigated with voltage-sensitive dye imaging and laser-Doppler flowmetry Hiroshi Kameyama a , Kazuto Masamoto b,⁎, Yoichi Imaizumi a , Tetsuro Omura a , Takusige Katura c , Atsushi Maki c , Kazuo Tanishita d a

School of Fundamental Science and Technology, Graduate School of Science and Technology, Keio University, Yokohama 223-8522, Japan Molecular Imaging Center, National Institute of Radiological Sciences (NIRS), 4-9-1 Anagawa, Inage, Chiba 263-8555, Japan c Advanced Research Laboratory, Hitachi Ltd., Saitama 350-0395, Japan d Department of System Design Engineering, Keio University, Yokohama 223-8522, Japan b

A R T I C LE I N FO

AB S T R A C T

Article history:

The spatiotemporal dynamics of the neurovascular response to brief acoustic stimuli were

Accepted 16 September 2008

investigated in guinea pig primary auditory cortex. Neural activity and cortical tonotopic

Available online 30 September 2008

organization were measured with a voltage-sensitive dye (VSD) technique, whereas cerebral blood flow (CBF) response to neural stimulation was measured with laser-Doppler

Keywords:

flowmetry (LDF). The acoustic stimulus was given as a wide band sound (click), which

Neural activity

induced global activation or as one of two pure tones (1 kHz and 12 kHz), which induced

Cerebral blood flow

distinct localizations in the auditory cortex. The VSD imaging showed that the sound-

Tonotopic organization

induced activation area varied dynamically, and that the spatial extent had peaks at 37 ± 3 ms

Functional imaging

and 38 ± 8 ms after the onset of stimulation during 1-kHz and 12-kHz tones, respectively. We

Guinea pig

observed that the average CBF response had a similar peak intensity irrespective of the type of stimuli: 16 ± 9%, 18 ± 11%, and 16 ± 8% for click, 1-kHz, and 12-kHz tones, respectively. No significant differences in the CBF time course, time-to-onset (∼ 0.6 s), or time-to-peak (∼ 3.3 s) were found across the recording sites and stimulus types. These results showed that the CBF response measured with LDF produced a less specific spatial pattern relative to the neural map determined with VSD. The findings can be explained by the methodological limitations of LDF and/or neurovascular regulatory systems in the auditory cortex. © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

Hemodynamic-based neuroimaging techniques, such as functional magnetic resonance imaging (fMRI) and near-infrared spectroscopy (fNIRS), are widely used for neurological diagnosis and neuroscience research into human brain functions (Bandet-

tini et al., 1992; Kwong et al., 1992; Ogawa et al., 1992; Haglund et al., 1992; Hoshi and Tamura, 1993; Kato et al., 1993; Villringer et al., 1993; Maki et al., 1995). These imaging modalities measure neural activity indirectly via activity-induced changes in hemodynamic signals, such as cerebral blood flow (CBF), blood volume (CBV), and blood hemoglobin saturation. However, the fundamental issue of

⁎ Corresponding author. Fax: +81 43 206 0819. E-mail address: [email protected] (K. Masamoto). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.09.058

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how precisely organized these hemodynamic signals are with respect to the true sources of neural activity remains to be defined. A variety of optical imaging techniques have revealed that the hemodynamic signals measured in rat somatosensory cortex are finely localized to a single cortical columnar scale (Weber et al., 2004; Sheth et al., 2004; Berwick et al., 2008). Additionally, a sub-millimeter scale of cortical columnar organization was successfully imaged with fMRI and optical imaging in cat visual cortex (Vanzetta et al., 2004; Zhao et al., 2005; Fukuda et al., 2006; Moon et al., 2007). These studies suggest that the spatial specificity of hemodynamic signals must be controlled by neurovascular regulatory systems at the cellular level in the activated cortex (Chaigneau et al., 2003; Peppiatt et al., 2006; Takano et al., 2006). In the present study, the spatiotemporal pattern of hemodynamic signals induced by sound stimuli was examined in guinea pig primary auditory cortex. This tissue is known as a unique cortex in which columnar organization dynamically varies in response to sound tone, referred to as “tonotopic organization” (Horikawa et al., 2001; Eggermont et al., 2001; Mountcastle et al., 1997; Wallace et al., 2000; Redies et al., 1989; Bakin et al., 1996). This spatially-structured organization may provide a good model to investigate the relationship between localization of hemodynamic signals and neural sources with respect to a variety of sound processing mechanisms. Using optical imaging techniques with voltage-sensitive dye (VSD), the spatial map of tonotopic organization was imaged (Tokioka et al., 2000; Masamoto et al., 2003). The activity-induced hemodynamic signals were then measured with laser-Doppler flowmetry (LDF) at the multiple locations where evoked neural activity had been determined with VSD. The LDF has a high temporal resolution

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(∼ 0.1 s) and measures red blood cell movements arising directly from microcirculatory activities, and has often been used to evaluate the temporal and quantitative couplings of neurovascular relations (Dirnagl et al., 1989; Royl et al., 2006).

2.

Results

Distinct localization patterns were clearly observed for neural signals responding to 1-kHz and 12-kHz tones (Fig. 1). The area of activation induced with the 1-kHz tone initially appeared in the rostral region and then expanded dorsally, whereas the activation caused by the 12-kHz tone was caudally located. The maximum extension of the activation area was observed to be 37 ± 3 ms and 38 ± 8 ms (mean ± SD) after the onset of stimulation for 1-kHz and 12-kHz tones, respectively. We observed that the spatial extension pattern differed among the sound tones presented. Figure 2 shows the timecourse for the number of the voxels for which the intensity surpassed the respective threshold levels (i.e., 20% to 90% of the peak maximum). The suprathreshold activity area induced with 1-kHz tone was relatively broad (e.g., a large number of voxels) and expanded quickly (e.g., a sharp rise), whereas the 12-kHz tone area was relatively restricted (e.g., a small number of voxels) and expanded slowly (e.g., a moderate rise). The 1-kHz and 12-kHz tonotopic areas were then determined by imaging the suprathreshold activity voxels, among which the signal change was determined for the thirty highest activities. Figure 3 depicts the tonotopic areas induced with the 1-kHz (black areas in Fig. 3A) and 12-kHz tones (light gray areas in Fig. 3B). Recording sites of the LDF and corresponding voxel locations were identified by referring to pial vasculature

Fig. 1 – Spatiotemporal dynamics of neural signals. The images represent typical spatiotemporal patterns of fluorescent intensity changes arising after the onset (at 0 ms) of 1 kHz (upper panels) and 12 kHz (lower panels) acoustic stimuli (0 to 78 ms after averaging all 15 iterations from the same animal). The peak signal change was set to 1.0 (dark red) and the intensity of each signal for a given recording time was normalized in each experiment (see color scale). Note that each stimulus induced different localization patterns, and the spatial extent varied dynamically over time.

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ferred stimulus was defined as the sound that induced a 1.5fold larger neural response as compared to the response to the other sound. For example, in 1-kHz preferred areas (n = 5), significantly higher neural activity was observed for 1-kHz stimuli (gray) relative to 12-kHz tones (white). In 12-kHz preferred areas (n = 7), higher neural activity was observed for 12-kHz stimuli relative to 1-kHz tones. No significant differences in the neural activity for 1-kHz and 12-kHz tones were observed in the overlapped areas (n = 8). However, none of these areas showed consistent results for the CBF responses to preferred vs. non-preferred stimuli (Fig. 5B).

3.

Fig. 2 – The spatial extent of stimulus-induced activation areas for 1-kHz and 12-kHz tones. Number of voxels in which the VSD signal was beyond the threshold level is shown for each time-point from the onset of stimulation (at 0 ms). The threshold level represents 90% (blue), 80% (red), 40% (green), and 20% (orange) of peak signal intensity across channels and over recording time, and data represent mean values of all animals (n = 6). The maximum spatial extent was observed at around 35 to 50 ms after the onset of stimulation, and the pattern of spatial extent was stimulus-dependent.

(Fig. 3C). In some locations, the VSD signal showed robust suprathreshold activities for both stimuli. These areas were defined as “overlapped” (dark gray areas in Fig. 3D). The peak intensity of the CBF response was compared across multiple sites for each subject (Fig. 4). A circle in Fig. 4B represents the actual LDF recording site. The numbers in the figure correspond to the locations (#1 and #2) at which the CBF data were obtained (Fig. 4A). The figure shows that similar CBF responses were observed irrespective of the recording sites in the case of the click stimulus. The peak intensity of the CBF response to 1-kHz and 12-kHz tones, however, showed statistically significant differences (⁎P < 0.05) at specific locations. After averaging all population data (n = 20 recording sites), the CBF peaks were observed to be 16 ± 9%, 18 ± 11%, and 16 ± 8% for click, 1-kHz, and 12-kHz tone stimuli, respectively, showing no significant differences between the sound types. Additionally, neither sound types nor recording sites caused significant differences in the time-to-onset (0.6 ± 0.4 s) or timeto-peak (3.3 ± 0.4 s) of the CBF response. The neural and vascular responses were compared for preferred and non-preferred sound tones (Fig. 5). The pre-

Discussion

The present study showed that CBF responses measured with LDF failed to discriminate the tonotopic organization determined by VSD imaging in the guinea pig primary auditory cortex (Fig. 5). This result can be explained by the methodological limitations of LDF and/or physiological effects, such as global changes in CBF distinct from the active regions in the auditory cortex. The LDF sampling volume is determined by the distance between emission and detection fibers. In this study, we used a needle probe in which the fiber diameter was 0.1 mm and the distance between fibers was 0.14 mm. Ideally, LDF detects localized changes in CBF arising in the target areas where the probe is placed, but it is also likely to be affected by neighboring areas as well as the probe location, due to strong light scattering of brain tissue. Indeed, two-dimensional imaging with laser-Doppler CBF measurement (in-plane resolution of 0.11 mm × 0.11 mm) showed a large spatial extension of the CBF response apart from the activated columns (Kannurpatti and Biswal, 2006). Thus, the possibility that LDF measurements may be contaminated by signals arising from outside the location where probe was placed cannot be ruled out. The VSD imaging technique is known to be sensitive to membrane potentials, including subthreshold and suprathreshold synaptic potentials as well as output action potentials, and thus the signal represents the variable activity arising from the integration of various neural compartments (e.g., dendrite, axon, and cell somata) (for reviews: Grinvald and Hildesheim, 2004). In rat barrel cortex, it was shown that the VSD signal induced by a single whisker deflection spreads into neighboring barrels along the preferential rows (Petersen et al., 2003). In the present study, we observed that the spatial extent of VSD signals varied within the field of view (3 mm by 3 mm) with relatively low signal intensities (Figs. 1 and 2). This widely spread subthreshold neural activity may contribute to the physiological source of non-specific changes in CBF. Another explanation for the non-specific CBF response is that remote vasodilation of arterial networks results in a nonspecific CBF response distant from the activated areas (Iadecola et al., 1997; Erinjeri and Woolsey 2002). The remote vasodilation mediated by rapid signal propagation along the vessel trees was also observed in studies of isolated cerebral arterioles (Dietrich et al., 1996; Horiuchi et al., 2002). In the present study, no detectable differences in the temporal dynamics of the CBF response were observed between the recording sites. These results were supported by a previous

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Fig. 3 – The tonotopic map induced with 1-kHz and 12-kHz tones. Each panel represents the location of the 30 channels that showed the highest signal changes during 1-kHz (A) and 12-kHz stimuli (B) in one representative animal. (C) The activation map was then compared with cortical vasculature to identify the placement of the LDF probe. Each grid represents a 0.25 mm × 0.25 mm photo-diode array (effective channels = 128). (D) Some areas showed activation for both the 1-kHz and 12-kHz stimuli (dark gray, overlap).

report that showed a similar time-course of CBF responses over broad areas of activated cortex (Durduran et al., 2004). These findings may therefore indicate that the vasodilatory signal generated from the activation foci quickly propagates along the vessel, extending to a point several millimeters away from the activated neural spots. Possible contributors to this rapid propagation of the vasodilatory signals are changes in membrane potentials that run through endothelial cells, vascular smooth muscle cells, and/or glial gap junctions (Emerson and Segal, 2000; Xu et al., 2008).

In rat barrel cortex, previous reports showed that a single cortical column is fed by one or more penetrating arterioles (Cox et al., 1993; Woolsey et al., 1996). Also, microvascular density varies in accordance with the layer-specific neural organization (Masamoto et al., 2004). These studies indicate that anatomical connections between neural and vascular systems may exist for blood flow controls at the columnar scale. Since the columnar organization is dynamically varied in the primary auditory cortex, it is unsurprising that the geometric structure of vascular networks differs from those of other cortices, such as the barrel

Fig. 4 – CBF response to acoustic stimuli. (A) Averaged peak CBF change is shown for click (C), 1-kHz (1) and 12-kHz (12) tones for two probe locations (#1 and #2, corresponding to the number enclosed by a circle in panel B). Note that no significant differences in the peak intensity of the CBF response were observed for the click stimulus across the recording sites. *P < 0.05: statistically significant difference in peak intensity following 1-kHz vs. 12-kHz stimuli. Error bar: ±1 SD (n = 10 iterations for each stimulus and in each location of one representative animal). (B) Probe location was identified according to the cortical vasculature. Scale bar: 0.5 mm.

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ketamine hydrochloride (46 mg/kg) and xylazine hydrochloride (23 mg/kg). Supplementary injections of a half dose of the initial administration were performed every hour. The animal was tracheotomized and mechanically ventilated with a mixture of 30% oxygen and 70% nitrogen at a rate of 60 to 80 cycles per minute. The animal was placed on a stereotaxic apparatus, and a piece of the skull (7 mm × 7 mm) overlying the auditory cortex of the left hemisphere was removed (Redies et al., 1989; Wallace et al., 2000). During the animal preparation and subsequent experiments, body temperature was maintained at 35± 1°C with a heating pad (ATB-1100, Nihon Koden, Tokyo, Japan). The heart rate, arterial oxygen saturation level, and end-tidal carbon dioxide level were maintained within physiological limits. All experiments were performed in a sound-shielded room.

4.2.

Fig. 5 – Comparison of VSD and LDF responses across the three groups of recording sites. The measured data were divided into three groups; 1-kHz preferred (n = 5 recording sites), both overlapped (n = 11), and 12-kHz preferred (n = 4) (see text). (A) The normalized VSD signal was significantly different in both 1-kHz preferred and 12-kHz preferred groups for 1-kHz (gray bar) vs. 12-kHz tones (white bar). (B) In contrast, none of the groups showed significant differences in the CBF responses to 1-kHz vs. 12-kHz tones.

cortex where columnar organization is fixed. Local differences in microcirculatory structures between the somatosensory cortex and auditory cortex are currently investigated. To date, most neurovascular coupling studies have been performed in the somatosensory cortex. However, it remains to be seen whether the findings obtained in one cortex are applicable to other brain systems. The primary auditory system offers a promising setting for further experiments that aim to link the spatiotemporal systems across neurometabolic as well as neurovascular couplings with a variety of neural activities induced by specific sound classes.

4.

Experimental procedures

4.1.

Animal preparation

All experiments were carried out in accordance with the regulations and guidelines set by the Animal Experimental Committee of Keio University and the Advanced Research Laboratory, Hitachi Ltd. A total of six Hartley guinea pigs (390 to 470 g body weight, five to six weeks old) were used in the experiments, for which the methods were previously described (Tokioka et al., 2000; Masamoto et al., 2003). The animal was anesthetized with an intramuscular injection of a cocktail of

Acoustic stimulation

Acoustic stimulation was presented binaurally with an approximately 60-dB sound pressure level (SPL) via a circumaural earphone (Tucker Davis Technologies, USA). Three stimuli, click (wide-band sound up to ∼ 50 kHz with 0.02 ms duration), 1-kHz, and 12-kHz pure tones (50 ms plateau duration), were applied to induce variable levels of activation. Since the literature on the hearing ability of guinea pigs suggests similar sensory thresholds (e.g., 10–20 dB SPL) across the 1 to 12 kHz frequency range (Heffner et al., 1971), the stimulus intensity was fixed in all experiments. For CBF experiments, each sound pulse was applied with a 3 s duration at a rate of 5 Hz (i.e., a total of fifteen stimuli), and repeated for ten trials with an inter-run time of approximately 27 s. For VSD imaging, each sound pulse was repeatedly applied at a rate of 5 Hz for a 3 s duration (i.e., a total of fifteen stimuli). The presentation order of the three sound types was randomized across the animals.

4.3.

CBF measurement

Local CBF was measured with LDF (TBF-LN1, Unique Medical, Tokyo, Japan). The LDF light source was a laser diode with 780-nm excitation (2 mW at output). The LDF probe (0.5 mm diameter and 0.14 mm fiber separation, LP-N, Unique Medical) was adjusted on the dura. Two to six locations were selected as potential recording sites in each animal by avoiding visible pial vessel areas. The probe location was recorded with a video camera with a reference to pial vasculature for later analysis. The analog output of the LDF signal was recorded with the aid of data acquisition software (MDLOG-UT, Japan Think Net Co. ltd., Tokyo) at a rate of 40 Hz. The low-pass filter (<0.3 Hz) was applied to reduce systematic noises (Masamoto et al., 2003). Time series data were averaged across all ten trials in each experiment. The average data were then normalized by the baseline (a mean of the 5 s pre-stimulus level) and a peak amplitude and time-to-peak were recorded. Time-to-onset was determined as the intercept of the upward line passing through two points (10% and 90% of the peak) at the prestimulus baseline.

4.4.

Neural recording

Neural activity was measured with a voltage sensitive dye (VSD) imaging technique, as described previously (Fukunishi

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et al., 1992; Tokioka et al., 2000). Briefly, the dura was removed after completion of LDF recording and the exposed cortex was stained with voltage-sensitive dye RH1838 (0.2 to 0.3 mg/ml in saline). Supplementary dye solution (60 to 100 μl) was applied as needed. The cortical surface was then rinsed with saline solution. The VSD signal was detected with a custommanufactured optical system that consists of a 12 × 12 photodiode array (effective channels = 128) installed in an eye port of a microscope. The field of view was 3 mm × 3 mm, and each pixel detected an area of 0.25 mm × 0.25 mm (see Fig. 3C). All signals of the photodiode array (128 channels) were inputted in parallel at 1 kHz into a computer. Independent component analysis (ICA) was performed to remove physiological noises caused by respiration and heartbeat (Maeda et al., 2001). Time series data were obtained on a per-voxel basis, and averaged across all fifteen trials. The averaged data were then normalized to the mean of the 20 ms pre-stimulus levels. An activation map was generated by scaling voxel intensity by the maximum intensity across all voxels. Note that the maximum intensity of VSD signal was observed around 30 to 50 ms after the onset of each stimulus, indicating low contamination by neural activityinduced physiological signals (e.g., oxygen consumption and hemodynamic response), because these physiological changes have been characterized as slow signal components originating from intrinsic tissue properties (Tsytsarev et al., 2008). Neural activity at the recording site of CBF was evaluated by averaging data from four adjacent voxels corresponding to the location of the LDF probe (0.5 mm in a diameter). The VSD signal represents a region of neural activity to a depth of ∼ 0.5 mm from the cortical surface (i.e., cortical layer II/III by Song et al., 2006), which is relatively comparable to the large sampling volume of LDF (a hemisphere of ∼ 1 mm diameter).

4.5.

Comparison of neural and CBF responses

To further compare the spatial specificity of the neural activity and CBF responses induced by preferred and non-preferred tones, the recording sites were classified into three groups: 1kHz preferred, both overlapped, and 12-kHz preferred groups, based on the tonotopic activity measured with VSD. The 1-kHz preferred designation was given to sites in which neural activity was induced more than 1.5-fold greater by the 1-kHz tone relative to the 12-kHz tone. Similarly, sites labeled as 12kHz preferred had more than a 1.5-fold stronger neural response to the 12-kHz tone as compared to the 1-kHz tone. The remainders of the recording sites were categorized as the both overlapped group. It should be noted that the click, 1-kHz, and 12-kHz stimuli induced similar peak amplitudes of evoked VSD signals (0.6 ± 0.3%, 0.5 ± 0.3%, and 0.5 ± 0.3%, respectively) across the recording sites (n = 20 sites), indicating that the impact of stimulation on the auditory cortex is comparable across all stimulation conditions. Thus, the peak intensity induced by the click sound, which induced non-localized robust activation, was used to normalize the signal changes in each animal and allow for inter-subject comparisons. Off-line analysis was performed with Matlab 6.5 (MathWorks Inc., Natick, MA, USA). Statistical significance was tested with Student's t-test (P < 0.05).

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Acknowledgments We thank Ms. Kyoko Yamazaki and Ms. Mariko Katura for technical support. This study was partially supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan, Grant-in-Aid for Scientific Research A.

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