Pyramidal Neurons Are “Neurogenic Hubs” in the Neurovascular Coupling Response to Whisker Stimulation

Whisker stimulation.

For anatomical studies, to prevent c-Fos induction related to stress and/or novelty (Staiger, 2006) rather than to the stimulus itself, rats were habituated to the stimulation protocol by a daily ∼30–45 min handling period for 1 week preceding the experimental day. During each session, rats were gently restrained in a plastic cone and the whiskers manually displaced with a paintbrush (3–4 Hz) (Filipkowski et al., 2000). Two days before the experiment, all but three whiskers (C1, C2, and C3) (Fig. 1A) on the right side and all whiskers on the left side of the snout were clipped close to the skin under sodium pentobarbital anesthesia (60 mg/kg, i.p.). On the experimental day, awake restrained rats were subjected to a 20 min stimulation of the right C1–C3 vibrissae, returned to their cages and killed 1 h later for brain fixation (see below). CBF measurement studies (detail below) were performed in urethane-anesthetized rats and, to avoid neuronal adaptation related to long-lasting sensory stimulation (Heiss et al., 2008), a stimulus of higher frequency (8–10 Hz, electrical toothbrush) and shorter duration (3–6 stimulations of 20 s each interspaced by 40 s) was used. The identity of the recruited cells under such conditions of stimulation and anesthesia was anatomically analyzed in a separate group of rats (n = 1–3) perfused after three stimulation blocks separated by 20 min intervals, saline being administered intracisternally before the second and third blocks to best mimic pharmacological experiments (see below).

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Figure 1.

Increased CBF and c-Fos immunoreactivity induced by whisker stimulation. A, Schematic representation of the stimulation paradigm of whiskers C1, C2, and C3 used for immunohistochemical studies and of the corresponding barrels in the somatosensory cortex (modified with permission from Aronoff and Petersen, 2008). B, Representative cortical CBF recordings in the ipsilateral and contralateral cortices before and after whisker stimulation (20 s, illustrated by the black bar above the x-axis) of all whiskers on the right snout. CBF was increased significantly only in the stimulated contralateral cortex, with a minimal response on the ipsilateral side (n = 3). C, Overall distribution of c-Fos immunostaining in a coronal section of the stimulated contralateral (left) and unstimulated ipsilateral (right) barrel cortices following 20 min whisker stimulation. Increased c-Fos-positive nuclei were selectively located in specific layers of the contralateral cortex and, particularly, in layer IV where the target barrel is located. Note that very few c-Fos-positive nuclei were present in adjacent neighboring barrels (small arrows in left panel). High magnifications of the barrels in both contralateral and ipsilateral cortices are shown at the bottom. Scale bar, 100 μm.

Identification of activated cortical cells by double immunohistochemistry.

Rats were anesthetized with pentobarbital and their brains perfusion-fixed (500 ml of ice-cold 4.0% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4). The cells recruited by whisker stimulation were identified by double immunohistochemistry of c-Fos and markers of pyramidal cells, specific subtypes of GABA interneurons or glial cells on free-floating freezing microtome 25 μm thick coronal sections, as described previously (Kocharyan et al., 2008). Briefly, sections were first immunostained for c-Fos (rabbit anti-c-Fos, 1:15,000; Oncogene) detected with the ABC complex (45 min, Vectastain ABC kit, Elite PK-6100, Vector Laboratories) and visualized with the SG reagent (SK-4700, Vector Laboratories, blue-gray precipitate). Whereas a few single-labeled c-Fos sections were kept aside to delineate the location of activated cells within the barrel cortex, most were processed for double immunostaining with all other antibodies detected in the second position with species-specific secondary antibodies (1:200, Vector Laboratories, 45 min), and visualized with 3, 3′-diaminobenzidine (DAB, kit SK-4100, Vector Laboratories, brown precipitate). Primary antibodies were as follows: goat anti-choline acetyltransferase (ChAT, 1:250; Millipore Bioscience Research Reagents), anti-COX-1 or anti-COX-2 (1:500; Santa Cruz Biotechnology), guinea pig anti-VIP (1:5000; Peninsula), mouse anti-parvalbumin (PV, 1:40,000; Sigma), or rabbit anti-GFAP (1:300; DAKO), anti-neuronal nitric oxide synthase (nNOS, 1:10,000; Millipore), or anti-somatostatin (SOM, 1:2000; Peninsula Laboratories). Sections were observed under light microscopy (Leitz Aristoplan, Leica), and digital pictures taken (CoolPix 4500, Nikon), calibrated, and edited with Adobe Photoshop 7 (Adobe Systems).

Laser Doppler flowmetry of CBF.

Rats were anesthetized with urethane (1.1 g/kg, i.p., in saline) and a catheter filled with heparinized saline inserted in the femoral artery for monitoring of physiological parameters. Animals were fixed in a stereotaxic frame (David Kopf Instruments), body temperature maintained at 37°C using a temperature control system (FHC), and the whiskers on the right side trimmed to a length of ∼1.0 cm. The skull was exposed and a 3 × 3 mm region over the left barrel cortex (bregma coordinates AP: −3.0 mm; L: +/−7.0 mm) (Gerrits et al., 1998) was thinned to translucency with a drill while being intermittently cooled with saline. CBF was recorded from a laser Doppler flowmetry (LDF) needle-shaped probe (Transonic Systems) positioned in areas free of large blood vessels before, during, and after stimulation. Pilot experiments (n = 3) with two laser probes on corresponding areas of the left and right barrel cortex showed the selectivity of the contralateral CBF response (Fig. 1B); therefore, all subsequent measures were performed only in the left barrel cortex. Pilot experiments demonstrated an evoked CBF response of comparable intensity whether induced with a toothbrush (23.1 ± 3.1%) or manually with a Q-tip (19.1 ± 1.9%, ∼3–4 Hz, Niwa et al., 2000).

Pharmacology.

To investigate the contribution of glutamatergic and GABAergic neurons, as well as of astrocytes in the evoked CBF response, pharmacological blockade of receptors, inhibition of vasoactive messenger synthesis, or astroglial metabolism was achieved by intracisternal injection (3 μl, i.c.) under microscope monitoring (Kocharyan et al., 2008). Antagonists targeted group I metabotropic glutamate receptors (mGluR-1 and mGluR-5, MPEP + {"type":"entrez-nucleotide","attrs":{"text":"LY367385","term_id":"1257996803","term_text":"LY367385"}}LY367385, n = 5), NMDA (MK-801, n = 8), GABA-A (picrotoxin, n = 7), GABA-B (CGP 35348, n = 6), muscarinic acetylcholine (mACh, scopolamine, n = 4), or VIP [VIP(6–28), n = 5] receptors. Enzyme inhibitors targeted the rate-limiting prostanoid synthesizing enzymes COX (indomethacin, n = 3), COX-1 (SC-560, n = 4), and COX-2 (NS-398, n = 6), the vasodilatory epoxyeicosatrienoic acids (EETs) synthetic enzymes P450 epoxygenases (MS-PPOH, n = 5), and astroglial metabolism through inhibition of mitochondrial aconitase by the preferential uptake of fluoroacetate (n = 6) or fluorocitrate (n = 6) by glial cells (Fonnum et al., 1997). In each rat, only one compound or combination of two compounds was tested in addition to the no-drug and vehicle conditions. CBF measurements were recorded and averaged from three to six stimulations before and after either vehicle or drug administration (measured at 20, 40, and 60 min after injection). Heart rate and arterial blood pressure (PowerLab, ADInstruments) as well as blood gases and pH (Rapid Lab 348, Bayer) were monitored throughout the experiment and were comparable between all conditions (supplemental Table 1, available at www.jneurosci.org).

Because it was recognized that simultaneous blockade of several pathways, even with high inhibitor or antagonist concentrations, did not fully inhibit the hyperemic response to sensory stimulation (Koehler et al., 2009; Leithner et al., 2010), we did not attempt to reach maximal inhibition but aimed to provide evidence for the contribution of specific pathways. Hence, we used a 10⁻⁴ m concentration for most compounds except for fluoroacetate (10⁻³ m), fluorocitrate (3 × 10⁻⁴ m), MK-801 (4 × 10⁻³ m), and MSPP-OH (10⁻³ m), and presented the most efficacious incubation time for each compound. Concentrations and incubation times were determined from previous (Kocharyan et al., 2008) or from pilot studies based on demonstrated efficacy of the compounds after intracerebroventricular injection in other CNS-mediated functions. GABA receptor antagonists were used at doses that did not induce epileptic activity (Kocharyan et al., 2008; see below).

Drugs.

Unless otherwise stated, drugs were prepared in 0.5 m PBS. MK-801, {"type":"entrez-protein","attrs":{"text":"CGP35348","term_id":"875599329","term_text":"CGP35348"}}CGP35348, and picrotoxin (vehicle 0.5% ethanol in 0.5 m PBS) were purchased from Tocris Bioscience. Fluoroacetate sodium, fluorocitrate, indomethacin (vehicle 0.2% ethanol in 0.5 m PBS), MPEP, LY-367385 (vehicle 10⁻³ m NaOH in 0.5 m PBS equilibrated to pH 7.4 with 1N HCl), scopolamine, and VIP(6–28) were from Sigma. MS-PPOH [N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide, vehicle 0.5% ethanol in 0.5 m PBS], NS-398 (N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide, vehicle 1.5% DMSO in 0.5 m PBS) and SC-560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole, vehicle 0.5% ethanol in 0.5 m PBS] were obtained from Cayman Chemicals.

Electrophysiological recordings.

This approach was used to measure the concurrent effects of picrotoxin on somatosensory evoked potentials (SEP) and CBF. Rats were prepared as indicated above for pharmacological studies with the following modifications. The bone over the somatosensory cortex was removed to expose the dura mater. A chamber made of silicone was created on top of the surrounding bone, filled with saline, and a 16-channel electrocorticography (ECoG) array specifically designed for the rat barrel cortex (NeuroNexus Technologies) was positioned. The LDF probe was positioned above the array and, once a reproducible stimulus-evoked CBF response was detected, the positions of the array and the LDF probe were secured with 1% agar. Whiskers were deflected (4 Hz) using an air-puff stimulator (Sanganahalli et al., 2008) during 10 stimulation blocks (20 s on interspaced by 40 s off) following intracerebroventricular injection (3 μl) of either vehicle or picrotoxin (10⁻⁴ m), with a 20 min interval between the two conditions. Electrophysiological data were acquired at 24 kHz using a RZ2 multichannel recording system (Tucker-Davis Technologies) and analyzed using analysis scripts written in MATLAB (MathWorks). SEP data were obtained after resampling at 2 kHz and filtering between 0.5 and 250 Hz. SEP amplitudes were measured by averaging twenty 1 s epochs within each stimulation block and identifying the four pairs of trough (P deflection) and peak (N deflection) associated with the 4 Hz stimulation. The average trough-to-peak amplitude was then computed for each of the 10 stimulation blocks, in each of the 16 channels of the ECoG array. The 10 resulting SEP amplitudes (1 per each stimulation block) were used to identify channels that demonstrated statistically significant SEP after vehicle injection (one-tailed t test, comparison between trough-to-peak amplitudes during stimulation epochs and corresponding times during the baseline epochs). In one rat, six of the channels did not show statistically significant SEP and were therefore excluded from the analysis. The SEP amplitudes were averaged over ECoG contacts to obtain the overall SEP amplitudes under vehicle or picrotoxin injection.

Closed cranial window testing.

This approach was used to compare the vascular effect of picrotoxin after cortical superfusion with that observed after intracerebroventricular injection and to measure picrotoxin effects on EEG activity. Rats were anesthetized with isoflurane (5% in 30% oxygen for induction, 2–2.5% for maintenance) and placed under artificial ventilation via tracheotomy (50 bpm, SAR 830/P ventilator, CWE). Respiratory parameters, including tidal pressure, O₂ saturation, and end-tidal CO₂ levels, were monitored via a capnograph (BCI 300 Capnocheck, BCI), and rectal temperature was kept stable with a heating blanket (∼37°C, TC-1000 temperature controller, CWE). The femoral artery and vein were cannulated for arterial blood pressure recordings and blood analysis (pH, pCO2, and pO2, ABL 80 FLEX, Radiometer) and pancuronium bromide infusion (0.5 mg/kg/h), respectively. Rats were then placed in a stereotaxic frame, the bone and dura overlying the right barrel cortex (coordinates as above) were removed (∼2 mm diameter), and the exposed cortex was covered with 1% agarose in artificial CSF (aCSF) (Harvard Apparatus). A piece of standard #1 glass coverslip was sealed over this region with dental cement, through which three tubes were placed for aCSF circulation, vehicle or picrotoxin (10⁻⁶ m) superfusion (5 μl/min, 0–30 min), or insertion of the EEG recording electrode. Two reference electrodes were placed on the thinned skull on the contralateral side and over the cerebellum. Anesthesia was then switched to urethane (1 g/kg, i.p., 2 injections over 30 min) and, after recovery (30 min), the CBF responses to left whisker stimulation were measured by laser Doppler (Moor Instruments) using a probe positioned on the coverslip. EEG potentials were amplified 10,000-fold, digitized at a sampling rate of 2.0 kHz, transferred to a computer, and analyzed off-line using scripts written in MATLAB between (0–30 Hz) under aCSF, vehicle, and picrotoxin and presented as spectrum analysis (Lévesque et al., 2009). Physiological parameters were unaltered (supplemental Table 1, available at www.jneurosci.org).

Whole-cell recordings and single-cell RT-PCR.

Young rats were decapitated, their brains quickly removed, and coronal slices (300 μm thick) containing the barrel cortex were cut using a vibratome (Karagiannis et al., 2009). Individual slices were transferred to a recording chamber and perfused (1–2 ml/min) with oxygenated aCSF (30–34°C). Patch pipettes (4–8 MΩ) were filled with 8 μl of autoclaved K-gluconate-based RT-PCR internal solution. Astrocytes and pyramidal neurons were visualized in the slice using infrared videomicroscopy with Dodt gradient contrast optics, and astrocytes were also stained by superfusion (20 min) of aCSF containing 1 μm sulforhodamine 101 hydrate (SR101, Sigma) (Nimmerjahn et al., 2004). SR101 was excited at 535 nm with a light-emitting diode system (CoolLED, Precise Excite) and red fluorescence collected at 607/34 nm using a bandpass filter (Semrock). Images were captured with a digital camera (CoolSnap HQ2, Roper Scientific) and Imaging workbench 6.0 software (Indec). Whole-cell electrophysiological properties were recorded in voltage clamp mode for astrocytes and in current clamp mode for pyramidal neurons. Input resistance of astrocytes was determined by plotting a voltage/current curve of the current response at steady state and was corrected for series resistance determined by plotting a voltage/current curve of the capacitive transients. Input resistance of pyramidal neurons was measured as described previously (Karagiannis et al., 2009). Membrane potential values were not corrected for liquid junction potential. At the end of the recording, the cell cytoplasm was aspirated in the recording pipette, expelled into a test tube, and reverse transcription (RT) performed (Lambolez et al., 1992). Two single-cell RT-PCR (scRT-PCR) protocols were designed to detect the expression of COX-1 and COX-2 isoforms simultaneously with GFAP and the calcium-binding protein (S100β) in astrocytes or with the vesicular glutamate transporter 1 (VGlut1) in pyramidal cells. Two amplification steps were performed essentially as described (Cauli et al., 1997), with 10 μl of the RT reaction products using the primer pairs listed in Table 1.