Histamine H3 Heteroreceptors Suppress Glutamatergic and GABAergic Synaptic Transmission in the Rat Insular Cortex

Histamine H3 receptors are autoreceptors that regulate histamine release from histaminergic neuronal terminals. The cerebral cortex, including the insular cortex (IC), expresses abundant H3 receptors; however, the functions and mechanisms of H3 receptors remain unknown. The aim of this study was to elucidate the functional roles of H3 in synaptic transmission in layer V of the rat IC. Unitary excitatory and inhibitory postsynaptic currents (uEPSCs and uIPSCs) were obtained through paired whole-cell patch-clamp recording in cerebrocortical slice preparations. The H3 receptor agonist, R-α-methylhistamine (RAMH), reduced the uEPSC amplitude obtained from pyramidal cell to pyramidal cell or GABAergic interneuron connections. Similarly, RAMH reduced the uIPSC amplitude in GABAergic interneuron to pyramidal cell connections. RAMH-induced decreases in both the uEPSC and uIPSC amplitudes were accompanied by increases in the failure rate and paired-pulse ratio. JNJ 5207852 dihydrochloride or thioperamide, H3 receptor antagonists, inhibited RAMH-induced suppression of uEPSCs and uIPSCs. Unexpectedly, thioperamide alone increased the uIPSC amplitude, suggesting that thioperamide was likely to act as an inverse agonist. Miniature EPSC or IPSC recordings support the hypothesis that the activation of H3 receptors suppresses the release of glutamate and GABA from presynaptic terminals. The colocalization of H3 receptors and glutamate decarboxylase or vesicular glutamate transport protein 1 in presynaptic axon terminals was confirmed through double pre-embedding microscopy, using a combination of pre-embedding immunogold and immunoperoxidase techniques. The suppressive regulation of H3 heteroreceptors on synaptic transmission might mediate the regulation of sensory information processes, such as gustation and visceral sensation, in the IC.


INTRODUCTION
The histaminergic system plays a critical role in physiological brain functions, such as the regulation of sleep and wakefulness, thermogenesis, and learning and memory (Haas et al., 2008;Panula and Nuutinen, 2013). The tuberomammillary nucleus (TMN) in the hypothalamus is the source of histaminergic neurons (Panula et al., 1984;Watanabe et al., 1984), which project to nearly the entire central nervous system (Köhler et al., 1985;Haas and Panula, 2003). Histaminergic neurons in the TMN abundantly express histamine H 3 receptors on the somata, dendrites, and axons (Haas et al., 2008). H 3 receptors are coupled to the G i/o protein (Clark and Hill, 1996), which reduces cAMP concentrations and modulates Ca 2+ conductance (Takeshita et al., 1998). As autoreceptors, TMN H 3 receptors critically regulate neural firing and histamine synthesis and release (Arrang et al., 1983(Arrang et al., , 1985. Several studies have reported another function of H 3 receptors as presynaptic heteroreceptors, which modulate the release of other neurotransmitters, i.e., acetylcholine, noradrenaline, glutamate, and GABA . The dysfunction of H 3 receptors causes various neurological disorders, including movement disorders, hyperphagia, neuroinflammation, and sleep disorders (Haas et al., 2008;Panula and Nuutinen, 2013). Therefore, H 3 receptors have recently received attention as potential targets for drug development against neurological disorders.
Histamine modulates higher brain functions processed in the cerebral cortex (Haas et al., 2008;Panula and Nuutinen, 2013). Among the histaminergic receptor subtypes, i.e., H 1 , H 2 , and H 3 , the functions of H 2 receptors have been well explored in the cerebral cortex. Intracellular recordings using slice preparations have demonstrated that H 2 receptors block Ca 2+dependent K + channels that regulate afterhyperpolarization (AHP) (McCormick and Williamson, 1989;McCormick et al., 1993), thereby increasing the frequency of repetitive spike firing . In contrast, little information is available about the functional roles of cortical H 3 receptors, although studies using autoradiography with [ 125 I]iodoproxyfan and H 3 receptor mRNA expression have demonstrated the high-density expression of H 3 receptors in the cerebral cortex (Pillot et al., 2002). In the insular cortex (IC), which plays an important role in gustatory and visceral information processing (Yamamoto, 1984), the injection of an H 3 receptor agonist, R-α-methylhistamine (RAMH), impairs aversive taste memory formation (Purón-Sierra et al., 2010). This physiological result corroborates the anatomical findings that the IC receives histaminergic projections from the TMN (Haas et al., 2008), and expresses abundant H 3 receptors throughout all layers (Pollard et al., 1993;Cumming et al., 1994;Pillot et al., 2002). Considering that H 3 receptors regulate excitatory synaptic transmission in the hippocampus (Brown and Haas, 1999), basolateral amygdala (Jiang et al., 2005), and striatum (Doreulee et al., 2001), it is likely that the H 3 receptor-mediated modulation of EPSPs plays a critical role in neuroplasticity in the cerebral cortex. In addition, GABAergic synaptic transmission, which regulates ocular dominance plasticity in the adolescent visual cortex (Hensch et al., 1998), might contribute to the induction of these changes. It is worth noting that H 3 receptor expression labeled with [ 3 H](R)α-methylhistamine increases from postnatal days 9-16, and reaches the adult level on postnatal days 23 in the cerebral cortex (Ryu et al., 1995). This developmental profile of H 3 receptor expression is contradict to that of histamine concentration showing very high content at birth and a steep decrease to the adult level by postnatal days 16. However, almost no information is available on the H 3 receptor-dependent modulation of synaptic transmission in the developing cerebral cortex.
The present study aimed to investigate H 3 receptor-mediated histaminergic modulatory mechanisms of excitatory and inhibitory local circuits comprising pyramidal and GABAergic cells in the developing IC using paired whole-cell patch-clamp recordings. The localization of H 3 receptors in the glutamatergic and GABAergic terminals was examined through electron microscopy and immunohistochemistry to examine the location of H 3 receptors in the IC. Furthermore, to elucidate the physiological function of H 3 receptors, the effects of an H 3 receptor agonist on excitatory propagation were examined using an in vivo optical imaging technique.

MATERIALS AND METHODS
All experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and the procedures were approved by the Institutional Animal Care and Use Committee of Nihon University. All efforts were made to minimize the number of animals used and their suffering.

Cell Identification
The slices were transferred to a recording chamber continuously perfused with normal ACSF at a rate of 2.0-2.5 ml/min. Dual or triple whole-cell patch-clamp recordings were obtained from Venus-positive fluorescent neurons and pyramidal cells identified in layer V of the granular or dysgranular IC using a fluorescence microscope equipped with Nomarski optics (BX51, Olympus, Tokyo, Japan) and an infrared-sensitive video camera (Hamamatsu Photonics, Hamamatsu, Japan). The distance between Venus-positive and pyramidal cells was <100 µm. The electrical signals were recorded using amplifiers (Multiclamp 700B, Molecular Devices, Sunnyvale, CA, United States), digitized (Digidata 1440A, Molecular Devices) and observed on-line; the information was stored on a computer hard disk using Clampex software (pClamp 10, Molecular Devices).
The pipette solution used for recordings from interneurons and pyramidal cells contained the following (in mM): 70 potassium gluconate, 70 KCl, 10 HEPES, 15 biocytin, 0.5 EGTA, 2 MgCl 2 , 2 magnesium ATP, and 0.3 sodium GTP. The pipette solution had a pH of 7.3 and an osmolarity of 300 mOsm. The liquid junction potential for the current-and voltageclamp recordings was −9 mV, and the voltage was corrected accordingly. Thin-wall borosilicate patch electrodes (2-5 M ) were pulled on a Flaming-Brown micropipette puller (P-97, Sutter Instruments, Novato, CA, United States).
The recordings were obtained at 30-31 • C. The seal resistance was >5 G , and only data obtained from electrodes with access resistance of 6-20 M and <20% change during recordings were included in this study. The series resistance was 50% compensated. The membrane currents and potentials were lowpass filtered at 2-5 kHz and digitized at 20 kHz.
Before unitary excitatory/inhibitory postsynaptic current (uEPSC/uIPSC) or miniature EPSC (mEPSCs)/miniature IPSCs (mIPSCs) recordings, the voltage responses of preand postsynaptic cells were recorded after applying long hyperpolarizing and depolarizing current pulse (300 ms) injections to examine the basic electrophysiological properties, including input resistance, single spike kinetics, voltage-current relationships, repetitive firing patterns, and firing frequency.

Multiple Whole-Cell Patch-Clamp Recording
Because some cell pairs had mutual or ≥2 connections, most cells were recorded under voltage-clamp conditions (holding potential = −70 mV) during uEPSC/uIPSC recordings. Short depolarizing voltage step pulses (2 ms, 80 mV) were applied to presynaptic cells to induce action currents. Where indicated, RAMH (Sigma-Aldrich, St. Louis, MO, United States) was added directly to the perfusate.
Before starting the successive recording of uEPSCs/uIPSCs, we checked the stability of their amplitude for 5-15 min, and excluded the connections that exhibited a gradual decrease of uEPSC/uIPSC amplitude.
Unitary events were recorded in normal ACSF for 5-10 min; we applied RAMH for 7.5-10 min, followed by washing for 10 min. To examine the blocking effect of H 3 receptors, uEPSCs/uIPSCs were recorded under normal ACSF for at least 10 min, and JNJ 5207852 dihydrochloride (JNJ5207852; Tocris, Bristol, United Kingdom) or thioperamide maleate salt (Sigma-Aldrich), H 3 receptor antagonists, was applied for 7.5 min. Subsequently, RAMH was applied in combination with JNJ5207852 or thioperamide for 7.5 min.

Miniature EPSC and IPSC Recording
We recorded mEPSCs from layer V GABAergic interneurons under application of 1 µM tetrodotoxin (TTX) and 100 µM picrotoxin using the same internal pipette solution based on potassium gluconate and KCl as described above. The holding potential was −70 mV .
The RAMH application protocol during mEPSC/mIPSC recording was same as that used for unitary event recording.

Electrophysiological Data Analysis
Clampfit software (pClamp 10, Molecular Devices) was used to analyze the electrophysiological data. Input resistance was measured from slopes of least-squares regression lines fitted to V-I curves measured at the peak voltage deflection (current pulse amplitude up to −100 pA). By application of depolarizing step current pulses (300 ms), the action potential threshold was identified as the minimal potential from which the first action potential was elicited. The amplitude of action potential was measured from the resting membrane potential to the peak. The amplitude of AHP was measured from the negative peak to the action potential threshold. Repetitive firing in response to long (300 ms) depolarizing current pulses was evaluated by measuring the slope of least-squares regression lines in a plot of the number of spikes versus the amplitude of injected current (F-I curve; up to 250-300 pA). The amplitude of uIPSCs/uEPSCs was measured as the difference between the peak postsynaptic and baseline currents obtained during a 2-3 ms time window close to the onset. To measure the 20-80% rise time and decay time constants of uEPSCs/uIPSCs, single action currents were induced at 0.07-0.1 Hz, and 10-30 postsynaptic events were aligned to the peak of the presynaptic action currents and averaged.
To quantify the effect of RAMH on the amplitude, pairedpulse ratio (PPR) of the 2nd to 1st uEPSC/uIPSC amplitude, coefficient of variation (CV) and failure rate, we analyzed 15 events just before RAMH application and 225 s after drug application for control and RAMH data, respectively. The uEPSC/uIPSC amplitudes in the range of synaptic noise were considered as failures.
Miniature EPSCs/mIPSCs were detected at a threshold of three times the standard deviation of the baseline noise amplitude using event detection software (kindly provided by Dr. John Huguenard, Stanford University). To measure the amplitude, interevent interval, 20-80% rise time, half duration, and 80-20% decay time, mEPSCs/mIPSCs were analyzed from continuous 5 min recordings before and after RAMH application. To obtain cumulative plots of the interevent interval and the amplitude of mEPSCs/mIPSCs, >1000 events were sampled per each neuron (n = 11).

Electron Microscopic Immunohistochemistry
Four male Sprague-Dawley rats (350-400 g) were used for immunohistochemistry. The rats were deeply anesthetized using sodium pentobarbital (80 mg/kg, i.p.) and transcardially perfused with 100 ml of heparinized normal saline, followed by 400 ml of a freshly prepared mixture of 4% paraformaldehyde and 0.001% glutaraldehyde in 0.1 M phosphate buffer (PB; pH 7.4). The IC was removed and postfixed in the same fixative for 2 h at 4 • C. Transverse sections were cut on a microslicer (Vibratome, St. Louis, MO, United States) at 60 µm and cryoprotected in 30% sucrose in PB overnight at 4 • C. The sections were frozen on dry ice for 20 mins and thawed in phosphate buffer saline (PBS; 0.01 M, pH 7.4) to enhance penetration. The sections were pretreated with 1% sodium borohydride for 30 min to quench the glutaraldehyde and blocked with 3% H 2 O 2 for 10 mins to suppress endogenous peroxidases and 10% normal donkey serum (Jackson ImmunoResearch, West Grove, PA, United States) for 30 min to mask secondary antibody binding sites.
For double immunostaining for the H 3 receptor and vesicular glutamate transporter 1 (VGLUT1) or glutamic acid decarboxylase (GAD) 65/67, the sections were incubated overnight in a mixture of rabbit anti-H 3 receptor (1:50; AB5660P, Millipore, Billerica, MA, United States) and guinea pig anti-VGLUT1 (1:2,000; #135 304, Synaptic Systems, Göttingen, Germany) or mouse anti-GAD65/67 (1:1,000; MSA-225, Assay designs Stressgen, Ann Arbor, MI, United States) antibodies. After rinsing in PBS, the sections were incubated with a mixture of 1 nm gold-conjugated donkey anti-rabbit (1:50, EMS, Hatfield, PA, United States) and biotinylated donkey antiguinea pig or biotinylated donkey anti-mouse (1:200, Jackson ImmunoResearch) antibodies for 2-3 h. The sections were postfixed with 1% glutaraldehyde in PB for 10 min, rinsed several times in PBS, incubated for 5 min in silver intensification solution (IntenSE TM M, Amersham, Arlington Heights, IL, United States), and rinsed in 0.1 M sodium acetate and PB. Subsequently, the sections were incubated with ExtrAvidin peroxidase (1:5,000, Sigma, St. Louis, MO, United States) for 1 h, and the immunoperoxidase was visualized using nickel-intensified 3,3diaminobenzidine tetrahydrochloride (DAB). The sections were further rinsed in PB, osmicated (0.5% osmium tetroxide in PB) for 30 min, dehydrated in graded alcohols, flat-embedded in Durcupan ACM (Fluka, Buchs, Switzerland) between strips of Aclar plastic film (EMS), and cured for 48 h at 60 • C. Chips containing immunoreactivity for the H 3 receptor and VGLUT1 or GAD65/67 in the dysgranular IC region of the IC were cut from the wafers and glued onto blank resin blocks using cyanoacrylate. Thin sections were collected on Formvar-coated single-slot nickel grids and stained with uranyl acetate and lead citrate.
The grids were examined on a Hitachi H 7500 electron microscope (Hitachi, Tokyo, Japan) at 80 kV accelerating voltage. The images were captured using Digital Montage software driving a MultiScan cooled CCD camera (ES1000W, Gatan, Pleasanton, CA, United States) attached to the microscope and saved as TIFF files. To control for the specificity of primary antibodies, the sections were processed according to the above protocol, except that the primary or secondary antibodies were omitted to completely abolish specific staining.

In Vivo Optical Imaging
The method of optical imaging with a voltage-sensitive dye has been described previously (Fujita et al., , 2012(Fujita et al., , 2017Mizoguchi et al., 2011), and only a brief account of the methods employed will be given here. Five-week-old male Sprague-Dawley rats (Sankyo Labo, Tokyo, Japan) weighing 152.9 ± 4.5 g (n = 12) received atropine methyl bromide (5 mg/kg, i.p.) and were anesthetized with urethane (1.5 g/kg, i.p.). Anesthesia was maintained throughout the experiments by injecting additional urethane. The adequacy of anesthesia was gauged by the absence of the toe pinch reflex. Animals received a tracheotomy and intubation, and lidocaine (2% gel) was applied to the incisions to ensure complete analgesia. The anesthetized animal was mounted on a custom-made stereotaxic snout frame (Narishige, Tokyo, Japan), which was then tilted 60 • laterally to make the left IC accessible to the CCD camera system and electrodes. The left temporal muscle and zygomatic arch were carefully removed, and a craniotomy was performed to expose the IC and the surrounding cortices.
The voltage-sensitive dye, RH1691 (1 mg/ml, Optical Imaging, New York, NY, United States) in 0.9% saline, was applied to the cortical surface for approximately 1 h, residual dye was rinsed with saline for approximately 30 min, and fluorescent changes of RH1691 were measured by a CCD camera system (MiCAM02, Brainvision, Tokyo, Japan) mounted on a stereomicroscope (Leica Microsystems, Wetzlar, Germany). The cortical surface was illuminated through a 632 nm excitation filter and a dichroic mirror by a tungsten-halogen lamp (CLS150XD, Leica Microsystems), and the fluorescent emission was captured through an absorption filter (λ > 650 nm longpass, Andover, Salem, NH, United States). The CCD-based camera had a 6.4 mm 2 × 4.8 mm 2 imaging area consisting of 184 pixels × 124 pixels. To eliminate signal due to acute bleaching of the dye, the final image was obtained by subtracting an image without stimulation from an image with stimulation. The sampling interval was 4 ms. The acquisition time was set at 500 ms. Sixteen consecutive images in response to stimuli were averaged to reduce noise, including interference from the heartbeat, breathing, and spontaneous neural activity.
A tungsten concentric bipolar electrode (impedance = 500 K ; Unique Medical, Tokyo, Japan) was inserted 0.3 mm from the surface of the IC. The duration and intensity of stimulation pulse was set at 100 µs and 7 V. Five train pulses with a 50 ms interstimulus interval were applied. Each train pulse was delivered at 30 s intervals. After optical recording in control, RAMH (10 µM) was applied to the cortical surface for 60 min. Another optical recording was performed during the last 30 min of RAMH application.
The change in the intensity of fluorescence ( F) in each pixel, relative to the initial fluorescence intensity (F), was calculated ( F/F). For the measurement of amplitude and excitatory area, images of responses were processed with a spatial filter (9 pixels × 9 pixels) to avoid the influence of noise and brain movements on each pixel. A significant response was defined as a signal exceeding seven times the SD of the baseline noise. Optical imaging data were processed and represented using software (Brain Vision Analyzer, Brainvision, Tokyo, Japan). To quantify the spatiotemporal profiles of excitation, we set a region of interest (ROI) in the adjacent region of the stimulation site.
All chemicals, unless otherwise specified, were purchased from Sigma-Aldrich.

Statistics
The data are presented as the means ± SEM. Comparisons of the uEPSC/uIPSC amplitude and PPR between control and drug application were conducted using the paired t-test. The suppression rates of uEPSCs in pyramidal cell to pyramidal cell (Pyr→Pyr) or Pyr→GABAergic interneuron (Interneuron) connections, and uIPSCs in Interneuron→Pyr connections were analyzed using one-way ANOVA followed by post hoc Bonferroni test. Reductions in the amplitude of uEPSC/uIPSCs induced through five trains or paired-pulses were compared using Student's t-test. Failure rate was compared between control and RAMH using Wilcoxon signed-rank test. Box-and-whisker plots were made as follows: upper and lower limit of box indicates 75th and 25th percentile, respectively; line within box denotes the median; upper and lower limits of the capped lines indicate 90th and 10th percentile, respectively. The RAMHinduced suppression rates of uIPSC amplitude were compared between the 1st and the 2nd-5th uIPSCs using two-tailed multiple t-tests with Bonferroni correction. The amplitude and interevent interval of mEPSCs/mIPSCs were analyzed through non-parametric statistical analysis (Kolmogorov-Smirnoff test; K-S test) to assess the significance of the shifts in the cumulative probability distributions in control and RAMH application. Paired t-tests were used to compare the mean amplitude and interevent interval of mEPSCs/mIPSCs and the mean amplitude of excitation ( F/F) and excitation area in the optical imaging study. A level of P < 0.05 indicated statistical significance.

RESULTS
The IC is a rostrocaudally extended region that lies on the dorsal surface of the rhinal fissure. The results obtained in the present study comprised recordings obtained from granular and dysgranular IC neurons around the middle cerebral artery, which are part of the gustatory cortex (Yamamoto et al., 1980;Accolla et al., 2007;Kobayashi et al., 2010).
To explore the roles of H 3 receptors in the regulation of local circuits in the IC, we performed paired whole-cell patch-clamp recording from pyramidal cells and GABAergic neurons. VGAT-Venus line A transgenic rats were used to identify GABAergic neurons. In this study, GABAergic neurons in the IC were divided into fast spiking (FS) and non-FS neurons, which included late spiking and regular spiking neurons (Kawaguchi and Kubota, 1997;Koyanagi et al., 2010Koyanagi et al., , 2014Yamamoto et al., 2010Yamamoto et al., , 2015Kobayashi, 2011;Kobayashi et al., 2012). Table 1 summarizes the basic membrane properties of the recorded neurons. Before examining the synaptic connections between recording cells, we classified the recording GABAergic neurons into FS and non-FS neurons after the application of long depolarizing current pulses under current clamp conditions. Based on the results, the cell connection subtypes were classified as (1) pyramidal cell to pyramidal cell (Pyr→Pyr), (2) Pyr→FS, (3) Pyr→non-FS, (4) FS→Pyr, and (5) non-FS→Pyr. The present study did not examine connections between GABAergic neurons. Repetitive firing F-I slope c (spikes/pA) 0.05 ± 0.01 0.19 ± 0.02 * * * 0.07 ± 0.01 ## 0.14 ± 0.01 a Resting membrane potential; b Afterhyperpolarization. One-way ANOVA followed by post hoc Bonferroni's test. c The number of spikes for 300 ms is divided by the intensity of the injected current (pA). * , * * * P < 0.05, 0.001 in comparison with pyramidal cell. # , ## , ### P < 0.05, 0.01, 0.001 in comparison with fast spiking cell. † † P < 0.01 in comparison with regular spiking cell.

H 3 Receptor Activation Suppresses uEPSCs Obtained from Pyr→Pyr and Pyr→FS/Non-FS Connections
To examine the H 3 receptor-mediated effects on the excitatory synaptic connections (Pyr→Pyr, Pyr→FS, and Pyr→non-FS), 10 µM RAMH, an H 3 receptor agonist, was applied to the perfusate. The pyramidal cells were identified among Venusnegative pyramidal somata after repetitive firing with spike adaptations (Figure 1A). In Pyr→Pyr connections, the bath application of RAMH (10 µM) invariably suppressed the 1st uEPSC amplitude by 30.4 ± 4.6% (n = 17; P < 0.05, paired t-test; Figures 1B-D), which was not recovered after a 10 min washout ( Figure 1C). The RAMH-induced uEPSC suppression was accompanied by increases in the failure rate of the 1st uEPSCs (n = 17; P < 0.01, Wilcoxon test) and PPR (n = 17; P < 0.01, paired t-test), as shown in Figure 1D. In these Pyr→Pyr connections, the 2nd to 5th uEPSCs were less affected after the application of RAMH (Figure 1E), suggesting that the effect of RAMH is likely to be mediated by a presynaptic mechanism.
In contrast to the smaller uEPSC amplitude in response to the 2nd to 5th presynaptic action currents in Pyr→Pyr connections (Figure 1F), Pyr→Interneuron connections frequently showed paired-pulse facilitation. RAMH had little effect on the 2nd to 5th uEPSC amplitude in Pyr→Interneuron connections (n = 18; P > 0.1, paired t-test; Figure 2I).
These results suggest that a decrease in uEPSCs through RAMH might reflect presynaptic modulation via H 3 receptors.
Coefficient of variation analysis is another method to evaluate the synaptic efficacy changes occur at pre-or postsynaptic sites (Faber and Korn, 1991;Arami et al., 2013). According to a binomial model of synaptic transmission, functional changes in presynaptic sites are expected to be accompanied by a change in the CV of synaptic responses: Presynaptic changes result in the plot that CV −2 values against the efficacy changes are located on or below the diagonal line in the case of low release probability. Our CV analysis show in Figures 1E, 2H supports the hypothesis that the suppressive effect of RAMH on uEPSCS is likely to be mediated via a presynaptic mechanism.
H 3 Receptor Activation Suppresses uIPSCs Obtained from FS/Non-FS→Pyr Connections Figure 3 shows a typical example of the effects of RAMH on uIPSCs obtained from FS→Pyr (Figures 3A,C) and non-FS→Pyr (Figures 3B,D) connections. In both pairs, 10 µM RAMH almost consistently suppressed the amplitude of uIPSCs. RAMH reduced the 1st uIPSC amplitude by 20.0 ± 5.3% in FS→Pyr connections (n = 31; P < 0.01, paired t-test) and 35.7 ± 9.6% in non-FS→Pyr connections (n = 8; P < 0.05, paired t-test). Figures 3E,F are the time courses of uIPSC amplitude before, during and after the application of RAMH in FS→Pyr and non-FS→Pyr shown in Figures 3A-D, respectively. The recovery of uIPSC amplitude was not observed after a 10 min washout.
As there was no significant difference between these reduction rates (P = 0.11, Student's t-test), we combined the results obtained from FS→ and non-FS→Pyr synapses into one category, Interneuron→Pyr. Generally, RAMH suppressed uIPSC amplitude by 23.0 ± 4.7% (n = 39; P < 0.01, paired t-test), which was almost comparable to that of the uEPSC suppression observed in Pyr→Pyr or Pyr→Interneuron connections (P = 0.19, one-way ANOVA).
Similar to Pyr→Interneuron connections, H 3 receptors likely suppress uIPSC amplitudes via a presynaptic mechanism. The RAMH-induced uIPSC suppression was accompanied by increases in the failure rate of the 1st uIPSCs (n = 39; P < 0.05, Wilcoxon test) and PPR (n = 39; P < 0.05, paired t-test), as shown in Figure 3G. The result of CV analysis ( Figure 3H) also suggested that the suppressive effect of RAMH on uIPSCS is induced by a presynaptic mechanism. Interneuron→Pyr connections exhibited paired-pulse depression in the control, and the analysis of the 2nd and 5th uEPSC amplitude showed a significant suppression after the application of RAMH (n = 33; P < 0.05, paired t-test; Figure 3I). The RAMH-induced suppression rate of the 1st uIPSC amplitude was significantly larger than those of the 2nd and 5th uIPSCs (P < 0.001; twotailed multiple t-test with Bonferroni correction), suggesting that the effect of RAMH is likely to be mediated by a presynaptic mechanism.
These results suggest that H 3 receptors suppress the uIPSC amplitude between GABAergic and pyramidal cells in the IC, which is likely mediated via a presynaptic mechanism, i.e., the suppression of GABA release from FS and non-FS cells to pyramidal cells.

An H 3 Receptor Antagonist Increases the uIPSC Amplitude
To confirm that the suppression of uIPSCs through RAMH is mediated via H 3 receptors, we examined the effects of the preapplication of JNJ5207852, an H 3 receptor antagonist, on the . Filled and open circles indicate the normalized amplitude of uEPSCs in control and during RAMH application, respectively. * P < 0.05, * * P < 0.01, paired t-test. † † P < 0.01, Wilcoxon test. Figures 4A-C showed a typical example of the effects of the pre-application of 10 µM JNJ5207852 followed by 10 µM RAMH on uIPSCs obtained from FS→Pyr connections. Application of JNJ5207852 itself had little effect both on the amplitude of uEPSCs obtained from Pyr→Pyr/Interneuron connections (38.6 ± 8.8 pA to 26.4 ± 5.3 pA, n = 8, P = 0.14, paired t-test) and uIPSCs in Interneuron→Pyr connections (88.8 ± 46.5 pA to 89.3 ± 45.9 pA, n = 13, P = 0.15, paired t-test). RAMH had little effect of the amplitude of Pyr→Pyr/Interneuron connections under   application of JNJ5207852 (26.4 ± 5.3 pA to 25.9 ± 5.3 pA, n = 8; Figure 4D). Similarly, in 13 Interneuron→Pyr connections, RAMH (10 µM), in combination with JNJ5207852 (10 µM), had little effect on the amplitude of uIPSCs (89.3 ± 45.9 pA to 93.8 ± 45.7 pA; n = 13, P = 0.66, paired t-test; Figure 4D). These results suggest that the RAMH-induced suppression of uEPSCs and uIPSCs is induced through H 3 receptors.

Thioperamide Increases the uIPSC Amplitude
We also examined the effects of thioperamide, an H 3 receptor antagonist, which is known as an inverse agonist (Morisset et al., 2000). Figures 5A-C showed a typical example of the effects of the pre-application of 10 µM thioperamide followed by 10 µM RAMH on uIPSCs obtained from FS→Pyr connections. Under thioperamide application, RAMH had little effect on the uIPSC amplitude. In 14 Interneuron→Pyr connections, RAMH (10 µM), in combination with thioperamide (10 µM), had little effect on the amplitude of uIPSCs (244.9 ± 59.7 pA to 224.4 ± 38.7 pA; n = 8, P = 0.48, paired t-test; Figure 5D). Similarly, RAMH also showed little effect of the amplitude of Pyr→Pyr connections under application of thioperamide (14.7 ± 6.8 pA to 15.0 ± 7.3 pA, n = 2). These results indicated that the RAMH-induced suppression of uIPSC amplitudes was mediated through H 3 receptors. In addition to the blockade of RAMH-induced suppression of uIPSCs by thioperamide, we observed that the application of thioperamide alone increased the uIPSC amplitude compared with the control (Figures 5B,C), though thioperamide had no significant effect on uEPSCs in Pyr→Pyr connections (n = 5; P = 0.58, paired t-test; Figure 5E). In 10 Interneuron→Pyr connections, thioperamide (10 µM) significantly increased the amplitude of uIPSCs by 102.3 ± 37.1% (P < 0.05, paired t-test; Figure 5F). The enhancement of uIPSCs through thioperamide application was not accompanied by significant changes in the failure rate of the 1st uIPSCs (n = 10, P = 0.18, Wilcoxon test) and the PPR (n = 10; P = 0.57, paired t-test; Figure 5F), and the facilitative effect of thioperamide was larger in the 1st uIPSCs compared with the 3rd uIPSCs (P < 0.05; two-tailed multiple t-test with Bonferroni correction; Figure 5G).
These results suggest that thioperamide facilitates GABAergic synaptic transmission via postsynaptic mechanisms in the connection of Interneuron→Pyr.

mEPSC and mIPSC Frequencies Are Suppressed through an H 3 Receptor Agonist
The frequency of synaptic events under the application of the Na + channel blocker, TTX, reflects the release probability of neurotransmitters, including GABA and glutamate. To strengthen the hypothesis that H 3 receptors suppress GABA/glutamate release via presynaptic H 3 heteroreceptors, mEPSCs and mIPSCs were recorded from pyramidal cells and GABAergic interneurons, respectively, under the application of TTX and glutamatergic/GABAergic receptor antagonists. Figure 6 shows the typical effect of RAMH on mEPSCs obtained from GABAergic interneurons in the layer V IC. In addition, 100 µM of picrotoxin was applied to block GABA A receptor-dependent currents. The interevent interval of mEPSCs was reduced through RAMH (Figures 6A-C; P < 0.001; K-S test) without changing the amplitude (Figures 6A-C; P = 0.16; K-S test). A total of 11 neurons were analyzed to compare the mean interevent interval and amplitude of mEPSCs recorded under control and RAMH conditions. RAMH significantly increased the interevent interval of mIPSCs (0.40 ± 0.06 s vs. 0.48 ± 0.07 s, n = 11, P < 0.01, paired t-test) without affecting the mIPSC amplitude (11.7 ± 0.9 pA vs. 11.9 ± 0.9 pA, n = 11, P = 0.66, paired t-test; Figure 6D).
These miniature recordings support the hypothesis derived from the results of unitary synaptic event recordings, suggesting that H 3 receptors in glutamatergic and GABAergic terminals regulate the release of glutamate and GABA, respectively.

Electron Microscopy and Immunohistochemistry
The electrophysiological findings in the present study strongly suggest the presynaptic regulation of glutamate and GABA release through H 3 receptors. Electron microscopy and immunohistochemistry are potent tools for providing direct evidence of the existence of H 3 receptors in glutamatergic or GABAergic terminals. We performed pre-embedding electron microscopy and immunohistochemistry for the H 3 receptor and GAD, a GABA-synthesizing enzyme that catalyzes the decarboxylation of glutamate, or for H 3 receptors and VGLUT1, a suitable marker for immunolabeling glutamate terminals. Figures 8A,B show two examples of the observed localization of H 3 receptors on the GAD-immunopositive terminals in the dysgranular IC. H 3 receptors and GAD were labeled with silvergold (arrowheads) and immunoperoxidase (arrows), respectively. Similarly, H 3 receptors labeled with silver-gold (arrowheads) were occasionally observed on VGLUT1-immunopositive terminals, as shown in Figures 8C,D. Most presynaptic GAD/VGLUT1-immunopositive axons with H 3 receptors terminated to middle-sized dendrites.

RAMH-Induced Modulation of Excitatory Propagation in the IC
To elucidate the functional role of H 3 receptors in cortical information processing in vivo, we examined the effects of RAMH on excitatory propagation in the IC by the optical imaging technique using a voltage-sensitive dye. As previously we reported, repetitive electrical stimulation of the DI rostral to the MCA, i.e., the gustatory cortex, consistently evokes excitation that propagates rostrocaudally, which is principally mediated by AMPA and GABA A receptors (Fujita et al., , 2012Mizoguchi et al., 2011). Therefore, we used the same protocol to evaluate the effects of RAMH on glutamatergic and GABAergic synaptic transmission in the IC.
Electrical stimulation of the dysgranular IC (5 train of pulses at 20 Hz, 7 V) evoked excitatory propagation that spread parallel to the rhinal fissure ( Figure 9A). The amplitude of excitation was largest around the stimulation electrode ( F/F = 0.25 ± 0.03%, n = 12; Figure 9B). Application of RAMH (10 µM) to the cortical surface decreased the amplitude between the bottom to peak of optical signals responding to the 1st stimulus (n = 12; P < 0.001, paired t-test; Figures 9A-C). Although the RAMHinduced suppression of the amplitude was also observed in the responses to the 2nd to 5th stimuli, the 1st responses showed the most prominent suppression by RAMH and the suppression was gradually recovered in the following responses ( Figure 9D). In parallel to the RAMH-induced suppression of the excitation amplitude, the area of excitation was decreased in the 1st responses from 11.1 ± 0.8 mm 2 to 8.3 ± 0.9 mm 2 (n = 11, P < 0.01, paired t-test), and the latter responses showed less decrease in the excitation area (P = 0.06-0.41, paired t-test; Figure 9D). These results suggest that RAMH suppresses excitatory propagation especially responding to the 1st stimulation, though the RAMH-induced suppression is less in the latter responses.

DISCUSSION
In the present study, we examined the functional roles of H 3 receptors in local circuits in the rat IC and investigated how the H 3 receptor-dependent synaptic modulation regulates the excitatory propagation in vivo preparations. An H 3 receptor agonist suppressed both uEPSCs and uIPSCs, regardless of presynaptic and postsynaptic neuron subtypes, and these effects were likely mediated through H 3 heteroreceptors in glutamatergic and GABAergic presynaptic terminals. These H 3 receptor-mediated modulations of synaptic transmission suppress excitatory propagation in the IC, and thus, H 3 heteroreceptors likely regulate cortico-cortical information processing in coordination with H 3 autoreceptors.

H 3 Receptors in the IC
Histaminergic axons derived from the TMN widely project to the cerebral cortex, including the IC (Haas et al., 2008 (Pollard et al., 1993), and the IC shows the highest abundance of H 3 receptor expression in the cerebral cortex (Pollard et al., 1993;Cumming et al., 1994). Consistently, the in situ hybridization of H 3 receptor mRNA combined with autoradiographic mapping of the H 3 receptor antagonist, [ 125 I]iodoproxyfan, demonstrated high concentration of H 3 receptors in the cerebral cortex, particularly in the IC (Pillot et al., 2002). Therefore, the IC is considered as the best target to examine the physiological roles of H 3 receptors in the cerebral cortex.
In contrast to the subcellular distribution patterns of H 3 receptors in the TMN, the distinct spatial patterns of H 3 receptors in cerebrocortical neurons remain unclear. The present study corroborates previous reports of H 3 receptor expression in the IC and further demonstrates that H 3 receptors are expressed in VGLU1-or GAD-immunopositive terminals. Several studies have demonstrated the expression of H 3 heteroreceptors in glutamatergic terminals of the dentate gyrus (Brown and Reymann, 1996;Doreulee et al., 2001) and striatum (Doreulee et al., 2001) or GABAergic terminals in the hypothalamus (Jang et al., 2001). However, these electrophysiological findings only implicate the presynaptic functions of H 3 receptors. Thus, electron microscopy and immunohistochemistry performed in the present study provides a direct anatomical evidence of H 3 heteroreceptors in the cerebral cortex.

H 3 Heteroreceptor-Mediated Modulation of Synaptic Transmission
An in vitro whole-cell patch-clamp study in the rat dentate gyrus demonstrated that histamine or RAMH reduces the amplitude of evoked EPSCs and increases in PPR (Brown and Reymann, 1996) through a reduction in Ca 2+ influx (Brown and Haas, 1999). This finding is applicable in vivo, as RAMH decreases evoked EPSP amplitude, which is blocked through thioperamide, in the dentate gyrus (Manahan-Vaughan et al., 1998). Similarly, the H 3 heteroreceptor-mediated suppression of evoked EPSPs/EPSCs has been reported in the striatum (Doreulee et al., 2001) and basolateral amygdala (Jiang et al., 2005). Despite abundant H 3 receptor expression in the cerebral cortex, there is little evidence showing the functional roles of H 3 receptors. Microdialysis in the prefrontal cortex demonstrated that thioperamide does not affect the basal release of glutamate and GABA (Welty and Shoblock, 2009). However, GABA release through strong stimulation, such as high potassium or NMDA application, is suppressed with thioperamide (Dai et al., 2006;Welty and Shoblock, 2009).
There are at least three subtypes of GABAergic interneurons in the IC Koyanagi et al., 2010;Yamamoto et al., 2010), and the kinetics of postsynaptic currents are different among interneuron subtypes, e.g., FS cells induce much larger uIPSCs in amplitude than LTS cells (Xiang et al., 2002;Kobayashi et al., 2008;Koyanagi et al., 2010). Therefore, the identification of presynaptic and postsynaptic neuron subtypes provides valuable information regarding EPSCs/uIPSC recordings. We consider that paired whole-cell recording has a large advantage in the precise analysis of glutamate/GABA-mediated synaptic transmission, as presynaptic and postsynaptic neuron subtypes can be identified. The present study demonstrated that the H 3 receptor agonist consistently suppressed the amplitude of uEPSCs/uIPSCs, regardless of presynaptic or postsynaptic neuron subtypes, suggesting that H 3 receptors are widely distributed in the IC, rather than expressed in specific neurons.

Intracellular Cascade of Synaptic Modulation through H 3 Receptors
H 3 receptors are metabotropic receptors coupled to G i/o proteins (Clark and Hill, 1996). H 3 receptors reduce intracellular cyclic AMP (cAMP) concentrations through the suppression of adenylyl cyclase (Torrent et al., 2005;FIGURE 9 | Effects of RAMH on excitatory propagation induced by repetitive stimulation in the IC. (A) Color-coded optical signals induced by a train of 5 pulses at 20 Hz in control (upper panels) and during application of RAMH (10 µM). Note that RAMH suppressed optical signal amplitude response to the 1st stimulus but increased those to the 2nd to 5th stimuli proximal to the stimulation site. A template of cortical subdivisions of the IC and surrounding regions is shown in the right panel of control. Blue circles in the AI, the rostral DI (rDI) and the caudal DI (cDI) indicate the locations of regions of interest (ROIs). (B) Optical responses in AI, rDI, and cDI in control (black) and during RAMH application (red). The ROIs are shown in the top left panel of (A). (C) Spatiotemporal maps along the horizontal black line shown in the top panel at the 3rd in (A). Arrowheads on the left indicate the timing of electrical stimulation. Note that RAMH suppressed optical signals responding to the 1st stimulus. (D) Normalized amplitude and area of the effect of RAMH on excitatory propagation responding to the 1st to 5th stimuli. Note that RAMH-induced suppression of amplitude and area was prominent in the 1st response. AI, agranular IC; DI, dysgranular IC; DLO, dorsolateral orbital cortex; Fr3, frontal cortex (area 3); GI, granular IC; MC, motor cortex; Pir, piriform cortex; SI, primary somatosensory cortex; SII, secondary somatosensory cortex. * P < 0.05, * * P < 0.01, * * * P < 0.001, paired t-test. -Delgado et al., 2006). The reduction in the cAMP concentration decreases Ca 2+ currents via L, N, and P/Q channels (Takeshita et al., 1998), and in turn, H 3 autoreceptor activation reduces histamine release from presynaptic terminals. Therefore, it is reasonable to speculate that similar mechanisms underlie the regulation of glutamate or GABA release through H 3 heteroreceptors in the cerebral cortex. The findings of RAMH-induced suppression of uEPSCs/uIPSCs imply that adenylyl cyclase, in glutamatergic or GABAergic terminals, is constantly activated to maintain intracellular cAMP concentrations, thereby contributing to the facilitatory effects on neurotransmitter release from these terminals. Although several studies supported the findings that H 3 receptor activation suppresses spontaneous GABA release by inhibiting P/Q channels in the hypothalamus (Jang et al., 2001) and striatum (Jang et al., 2001;Arias-Montano et al., 2007), they suggest an independent mechanism of the adenylyl-cAMP pathway. Further studies are necessary to clarify the second messenger systems involved in H 3 receptor activation.

Moreno
The effects of thioperamide on synaptic transmission are controversial. As described above, thioperamide is considered a typical H 3 receptor antagonist (Arrang et al., 1987), whereas several studies have reported thioperamide as an inverse agonist (Morisset et al., 2000). The present study demonstrates that the application of thioperamide slightly enhanced the uIPSC amplitude.
There are at least two possibilities that explain the thioperamide-induced enhancement of uIPSCs. If histamine is spontaneously released and H 3 receptors are consistently activated in the slice preparations, then the application of thioperamide might suppress the H 3 receptor-dependent decrease in the uEPSC/uIPSC amplitude. Second, thioperamide behaves as an inverse agonist. Little effect of JNJ5207852 on uEPSCs and uIPSCs suggests that the 1st possibility is unlikely. The contradictory effects of thioperamide on uEPSCs and uIPSCs also supports the second possibility. Furthermore, in a previous study of IC pyramidal cells, we showed that histamine increases spike firing via H 2 receptors and RAMH does not change the frequency, suggesting that spontaneous histamine release is less likely in the IC slice preparations . Therefore, the facilitative effects of thioperamide on the amplitude of uEPSCs/uIPSCs might indicate a role for this molecule as an inverse agonist.

Functional Significance of H 3 Receptors in Vivo
H 3 receptors are involved in higher brain functions, such as cognition, learning and memory, sleep, appetite, and energy metabolism, and the disruption of H 3 receptors induces related neurological disorders (Haas et al., 2008). Based on this evidence, H 3 receptors have recently been considered as promising targets for the treatment of several neuronal disorders, including hypersomnia (Leurs et al., 2005), eating disorders (Leurs et al., 2005;Steffen et al., 2006), schizophrenia (Ito, 2009), and pain (Cannon et al., 2007). It is interesting to focus on the study that the activation of H 3 receptors in the IC impairs aversive taste memory formation (Purón-Sierra et al., 2010), because the functional roles of the IC have been implicated in gustatory, visceral, and nociceptive information processing (Yamamoto et al., 1981;Yasui et al., 1991;Ogawa and Wang, 2002;Jasmin et al., 2003;Kobayashi, 2011). Our present findings that activation of H 3 suppresses glutamate and GABA release from presynaptic terminals may be involved in this process. In addition, recent studies have indicated additional functions for the IC in attention, reasoning, planning, and decision-making processes associated with smoking and drug abuse (Naqvi et al., 2007;Naqvi and Bechara, 2009). Thus, H 3 antagonists are potential candidates for the improvement of the physiological functions of the IC.

AUTHOR CONTRIBUTIONS
MK designed this study. HT, KY, and Y-CB contributed to the acquisition, analysis, and interpretation of the data in this study. TS contributed to the interpretation of the data. KY and MK drafted and wrote the manuscript. All authors have approved the final version of the manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.