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1Department of Oral Physiology and 2Department of Orthodontics, Showa University School of Dentistry, Tokyo; and 3Department of Oral Anatomy and Neurobiology, Faculty of Dentistry, Osaka University, Osaka, Japan
Submitted 17 October 2007; accepted in final form 22 August 2008
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ABSTRACT |
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INTRODUCTION |
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; Mizuno and Sauerland 1970). The SupV receives abundant orofacial sensory inputs, including periodontal and spindle afferents (Dessem and Taylor 1989; Jerge 1963; Mizuno 1970; Nishimori et al. 1986; Nomura and Mizuno 1985; Shigenaga et al. 1989) and descending inputs from the cortical masticatory area (Hatanaka et al. 2005; Yasui et al. 1985). Retrograde axonal tracing studies have shown that premotor neurons targeting the MoV are present in the SupV (Landgren et al. 1986; Mizuno et al. 1983; Travers and Norgren 1983; Turman Jr and Chandler 1994a,b). Premotor neurons identified by antidromic activation of the MoV are also present in the SupV (Donga and Lund 1991; Inoue et al. 1992; Westberg et al. 1995). Therefore it is possible that the SupV is involved in controlling jaw-closing and jaw-opening muscle activity via sensory and descending inputs during mastication.
It is assumed that glycinergic SupV neurons inhibit jaw-closing motoneurons during the jaw-opening reflex (JOR) because the number of spike discharges evoked from SupV neurons by peripheral stimulation is proportional to the amplitude in the early phase of the inhibitory postsynaptic potentials (IPSPs) of jaw-closing motoneurons and the early phase of the IPSPs were decreased by strychnine (Kidokoro et al. 1968; Nakamura et al. 1973); SupV stimulation induced monosynaptic IPSPs in those motoneurons (Goldberg and Nakamura 1968; Kidokoro et al. 1968; Kolta 1997). In contrast, we previously showed that stimulus pulses to the SupV nearly always elicited masseter electromyographic responses at short latencies (Takamatsu et al. 2005). When the latency from the motoneuron to the muscle is considered, excitatory premotor neurons that target jaw-closing motoneurons are likely present in the SupV. However, the detailed mechanism of the synaptic transmission from SupV neurons to jaw-closing and jaw-opening motoneurons, particularly the neurotransmitters involved, is not clear. Furthermore, because feeding behavior changes dramatically from suckling to mastication during the early postnatal period, synaptic transmission might also change during postnatal development.
The aim of the present study was to characterize synaptic transmission from the SupV to jaw-closing and jaw-opening motoneurons and to examine postnatal changes in synaptic transmission in brain stem slice preparations from neonatal and juvenile rats. We found that inputs from the SupV excite jaw-closing and jaw-opening motoneurons through activation of glutamate, glycine, and 
-aminobutyric acid type A (GABAA) receptors in neonatal rats, whereas glycinergic and, most likely, GABAergic inputs to these motoneurons are inhibitory in juveniles.
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METHODS |
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Slice preparation
Experiments were performed with brain stem slices from postnatal day (P) P1–P12 Wistar rats (n = 107). The day of birth was defined as P0. Animals were anesthetized deeply with ether and decapitated. Each brain was removed rapidly and placed in cold oxygenated artificial cerebrospinal fluid (ACSF). The brain stem was cut into 400- or 500-µm transverse sections with a microslicer (Pro 7; Dosaka EM, Kyoto, Japan). For cutting P7–P12 rat brain stems, we used sucrose-based modified ACSF because it has been shown that the use of this ACSF during the slicing procedure is essential to maintain viable motoneurons in adult rats (Aghajanian and Rasmussen 1989). Modified ACSF contained (in mM) 260 sucrose, 3 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose. Viable motoneurons were obtained from P1–P6 rats by using normal ACSF during slice preparation, as previously shown in spinal (Takahashi and Berger 1990), hypoglossal (Berger et al. 1992), and trigeminal (Del Negro and Chandler 1998) motoneurons of neonatal rats. Normal ACSF contained (in mM) 130 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose. To compare membrane properties of motoneurons in slices cut in normal and modified ACSF, some slices from P1–P6 rats were prepared by using modified ACSF. There was no significant difference in the resting membrane potential between motoneurons obtained from the two kinds of slices in P1–P6 rats (normal ACSF, –68.2 ± 1.0 mV, n = 36; modified ACSF, –70.9 ± 0.9 mV, n = 8; P > 0.05). Furthermore, gramicidin-perforated patch-clamp recordings (see following text) from motoneurons (n = 8) in the slices prepared with modified ACSF in P1–P6 rats showed that all the eight motoneurons depolarized by pressure application of 1 mM glycine or bath application of 200 µM GABA similarly in the motoneurons prepared with normal ACSF.
Normal ACSF and modified ACSF were aerated continuously with a 95% O2-5% CO2 gas mixture. Slices from P1–P6 rats were incubated at 35°C for 1 h in normal ACSF. Slices from P7–P12 rats were incubated at 35°C in modified ASCF for 20 min followed by a 20-min incubation with a 50:50 mixture of modified and normal ACSF and then incubation in normal ACSF for an additional 20 min. All slices were maintained at room temperature (25–27°C) in normal ACSF. We also used normal ACSF for recordings.
Optical recordings
In all, 54 slices from 41 P1–P6 animals were stained in normal ACSF containing 100 µg ml–1 of fluorescent voltage-sensitive dye (Di-4-ANEPPS; Molecular Probes, Eugene, OR) as described by Tominaga et al. (2000) for 1 h under 0.4 kgf cm–2 of 95% O2-5% CO2 gas. After staining, unbound dye was removed by rinsing the sections in ACSF for 15 min. Slices were gently transferred into a recording chamber that was mounted on an upright fluorescence microscope (BX51WI; Olympus, Tokyo). The slices were superfused continuously with normal ACSF at a rate of 2.0 ml min–1 at room temperature with a peristaltic pump (EYELA MP-1000; Tokyo Rikakikai, Tokyo).
Stimulus-evoked responses in the slice preparations were measured as the fractional change in fluorescence of the voltage-sensitive dye with an optical imaging system (MiCAM01 or MiCAM Ultima; Brain Vision, Tsukuba, Japan) equipped with a 510- to 550-nm excitation filter, a dichroic mirror, and a 590-nm absorption filter (U-MWIG2 mirror unit, Olympus) and a 150-W tungsten-halogen lamp (MHF-G150LR; Moritex, Tokyo). The charge-coupled detector (CCD)–based camera head had a 3.0 x 2.0-mm2 imaging area (96 x 64 pixels, MiCAM01) and the CMOS camera head had a 10.0 x 10.0-mm2 imaging area (100 x 100 pixels; MiCAM Ultima), and the microscope magnification was adjusted to a x4 objective lens (0.28 NA, XLFluor4x/340; Olympus) so that an area of 0.75 x 0.5 mm2 (MiCAM01) or 2.5 x 2.5 mm2 (MiCAM Ultima) was covered by the image sensor. Each optical recording was acquired at a rate of 3.0 ms/frame for 512 frames. Fluorescence signals for each 1.5 s per trial, including 300 ms before stimulation, were recorded at 3-s intervals and averaged over 16–128 trials. A Teflon-insulated tungsten electrode (impedance 1 M
at 500 Hz, TOG204-045; Unique Medical, Tokyo) was used for single-pulse slice stimulation (0.33 Hz, 20–30 µA, 0.2-ms pulse duration). Fluorescence changes were expressed as a ratio (percentage fractional change) of the change in the intensity of fluorescence relative to that of the reference image. The differential image, processed with a 2 x 2 (pixels in the image plane) and x2 (time frames) software filter, was represented by a pseudocolor display in which red corresponded to fluorescence decrease and membrane depolarization. Optical data were collected and stored on a personal computer controlled by MiCAM01or MiCAM01 Ultima-associated software (Brain Vision). To represent the time course of fluorescence change, the optical signals were inverted with an upward deflection corresponding to depolarization. The MoV was visualized in the slices as an opaque bright oval region through the optical imaging system. The optical responses in the MoV evoked by stimulation were measured in the central region (3 x 3 pixels) of the MoV.
At the end of each experiment, brain stem stimulation sites were marked by passing a 10-s, 20-µA negative current through the electrodes in select slices. The slices were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 
1 day at –4°C. Slices were then rinsed with 0.1 M phosphate-buffered saline (PBS, pH 7.4) and incubated overnight in 5–25% sucrose/PBS at –4°C. Transverse 20-µm-thick frozen sections were then cut with a cryostat and stained with cresyl violet. The location of stimulation sites was confirmed by microscopic examination.
Photostimulation
We used laser photolysis of caged glutamate to determine whether SupV neurons send excitatory inputs to the MoV. Eight slices from five animals at P1–P5 were imaged with a monochrome CCD camera (C9100; Hamamatsu Photonics, Hamamatsu, Japan). A pulsed nitrogen laser (365-nm wavelength, 600-ps pulse duration; Micropoint, Photonic Instruments, Kawasaki, Japan) was directed into the epifluorescence attachment of the microscope, onto a dichroic mirror, and into the back aperture of the x4 objective. The location of the photostimulation was visualized directly through the objective and the laser beam was focused on an approximately 10-µm-diameter area of tissue. At the beginning of the experiment, 4-methoxy-7-nitroindolinyl-caged L-glutamate (Tocris Cookson, Ellisville, MO) was added to 25 ml ACSF to yield a concentration of 300 µM, which was circulated. All photostimulation experiments were started 15 min after the addition of caged glutamate. Single pulses of photostimulation were delivered to the MoV or SupV at 10-s intervals to trigger focal photolysis of caged glutamate. We varied the strength of photostimulation by neutral-density filters of varying optical densities.
Retrograde labeling of jaw-closing and jaw-opening motoneurons
In experiments that examined the nature and development of synaptic transmission between the SupV and MoV, we used a fluorescence-labeling technique to record from jaw-closing and jaw-opening motoneurons. One to 2 days before slices were prepared, animals were anesthetized with ether, and 2–5 µl of 10% 3,000 or 10,000 MW dextran-tetramethylrhodamine-lysine (DRL, Molecular Probes) in distilled water was injected bilaterally in the masseter or digastric muscles with a microsyringe (1010RN; Kloehn, Las Vegas, NV). After the animals recovered from anesthesia, they were returned to their mothers while the DRL diffused. Forty-one masseter motoneurons (MMNs) from 23 rats and 31 digastric motoneurons (DMNs) from 18 rats were retrogradely labeled by 3,000 or 10,000 MW DRL solution. MMNs or DMNs could be labeled 1 day after injection of 3,000 MW DRL solution. When 10,000 MW DRL was used, we waited for 2 days after DRL injection to prepare slices. Thus to prepare slices at P1, 3,000 MW DRL solution was injected into the muscles 1 day before slice preparation (i.e., at the day of birth, P0).
Electrophysiological recordings
Whole cell and gramicidin-perforated patch-clamp recordings were performed with infrared videomicroscopy (BX51WI, Olympus) and a x40 water-immersion objective with differential interference contrast and epifluorescence optics. In preparations from 41 DRL-injected animals at P1–P12, the epifluorescence DRL-labeled MMNs and DMNs were quickly identified with the CCD camera. Patch electrodes were constructed from single-filament 1.5-mm-diameter borosilicate capillary tubing (GD-1.5, Narishige, Tokyo) with a microelectrode puller (P-97, Sutter Instruments, Novato, CA). Voltage-clamp experiments during whole cell recordings were done with an internal solution of (in mM) 103 K-gluconate, 27 KCl, 1 CaCl2, 10 HEPES, 11 EGTA, 2 MgCl2, 0.3 NaGTP, and 2 NaATP (pH 7.3, 285–300 mOsm). For current-clamp experiments in the whole cell configuration, electrodes were filled with a solution of (in mM) 115 K-gluconate, 25 KCl, 9 NaCl, 16 HEPES, 0.2 EGTA, 1 MgCl2, 3 K2ATP, and 1 NaATP. Biocytin (Sigma–Aldrich, St. Louis, MO) at a concentration of 5 mg ml–1 was added to the internal solution for recording from SupV neurons. For gramicidin-perforated patch-clamp recordings, an internal solution containing (in mM) 150 KCl, 10 HEPES, and 5 lidocaine N-ethylbromide (QX-314) was used. QX-314, a sodium channel blocker, is a large molecule and can enter the cell only when the membrane is ruptured. Voltage-activated sodium current was checked at the beginning and at the end of each cell recording. Gramicidin was dissolved in 10 mg ml–1 dimethyl sulfoxide and diluted in the pipette-filling solution to a final concentration of 20 µg ml–1 just before the experiment. Pipette resistance ranged from 2.5 to 5.0 M when the electrodes were filled. Stimulus-evoked postsynaptic currents (PSCs), postsynaptic potentials (PSPs) evoked by electrical stimulation of the SupV, and membrane potentials were recorded with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). A tungsten electrode (impedance 400 k; Unique Medical) was used for single-pulse stimulation (5–20 µA, 0.2-ms pulse duration). Series resistance compensation was set to 50–60% for gramicidin-perforated patch-clamp recordings and to 70–80% for whole cell recordings. Data were acquired at 2–20 kHz, filtered at 2–20 kHz, digitized with a Digidata 1332A interface (Molecular Devices), and stored and analyzed on a personal computer with pCLAMP 8.2 software (Molecular Devices). PSC data from five to eight trials were averaged and used for analyses, except for PSCs evoked by photostimulation, which were analyzed on a trial-by-trial basis. Liquid-junction potentials of 11 mV for voltage-clamp experiments and 12 mV for current-clamp experiments were subtracted from all membrane potentials in the whole cell patch configuration. No correction of liquid-junction potential for gramicidin-perforated patch-clamp recordings was made. All experiments were performed at room temperature.
Analyses of the morphology of SupV neurons projecting to the MoV
Whole cell recordings were obtained from SupV neurons of P1–P5 rats (n = 20) to examine their characteristics. To determine which SupV neurons projected to the ipsilateral MoV, the MoV was stimulated with a tungsten electrode (impedance 400 k; Unique Medical). The morphology of SupV neurons that met the following criteria was studied further: 1) the neuron responded to MoV stimulation <25 µA at a short and constant latency and 2) the neuron responded to 100-Hz triple shocks to the MoV when synaptic transmission was suppressed by substitution of external Ca2+ with Mn2+.
Neurons were filled by passive diffusion of biocytin from the patch pipette during and after the recording period (15–20 min) without application of current. After recording, patch pipettes were carefully detached from cells and the slices remained in the recording chamber for an additional 30 min to allow for biocytin transport into dendrites and axons. The slices were then fixed in 4% paraformaldehyde (Sigma) in 0.1 M PB at –4°C for 24 h and then left overnight in 30% sucrose in 0.1 M PB at –4°C. Transverse, 100-µm-thick serial sections were cut on a microslicer. Sections were incubated overnight at 4°C in streptavidin conjugated with horseradish peroxidase (1:800; Dako, Kyoto, Japan) in 0.05 M Tris-buffered saline (TBS) containing 1% Triton X-100. After several rinses with TBS, sections were reacted with 0.04% 3,3'-diaminobenzidine tetrahydrochloride, 0.002% H2O2, and 0.07% nickel ammonium sulfate in 0.05 M TBS and mounted on ovalbumin-coated slides. Stained cells were reconstructed from multiple sections with a camera lucida.
Drug applications
For optical imaging experiments, the following drugs were applied with pressure to the MoV at 1.5 ml min–1 through a microperfusion system (BPS-8 PR-10; ALA Scientific Instruments, Westbury, NY): 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM, Sigma), D-2-amino-5-phosphonopentanoic acid (APV, 20 µM, Sigma), strychnine (5–10 µM, Sigma), and bicuculline (20 µM, Sigma). For patch-clamp experiments, CNQX, APV, strychnine, 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl) pyridazinium bromide (SR95531, 5–10 µM, Sigma), bicuculline, and tetrodotoxin (TTX, 0.5 µM, Sigma) were applied to the bath. Glycine (1 mM, Sigma) was applied with pressure through a patch pipette to the soma of the recorded neurons with a Picospritzer III (General Valve, Fairfield, NJ).
Statistics
Values are presented as means ± SE. Data obtained before, during, and after drug application within groups were subjected to one-way repeated-measures ANOVA. Differences in data between groups were analyzed by the Student's t-test and two-way ANOVA. ANOVA was followed by Tukey's post hoc multiple-comparison test when appropriate. Probability values of <0.05 were considered significant. Statistical analyses were conducted with SPSS 13.0J and Microsoft Excel 2003.
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RESULTS |
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To examine which region, when stimulated, elicits excitatory optical responses in the MoV, single-pulse electrical stimulation was applied systematically to the reticular formation around the MoV every 250 µm in the mediolateral and dorsoventral directions in 40 brain stem slices from 32 rats (P1–P6) stained with Di-4-ANEPPS. Optical responses in the MoV were evoked by stimulation of the reticular formation dorsal to the MoV in 33 of 40 slices examined. Stimulus delivery is seen in the top two frames (arrowheads) of Fig. 1 A, and the optical response in the MoV is shown in the third frame. Histological analysis revealed that the stimulation sites were in the lateral SupV. Stimulation applied to sites outside of the SupV did not evoke optical responses in the MoV (data not shown). However, this does not indicate that excitatory premotor neurons are located only in the SupV in the slice preparations we used. Previous immunohistochemical studies revealed that glutamatergic premotor neurons for the trigeminal motor nucleus are also located in the principal sensory trigeminal nucleus and the intertrigeminal region (Kolta et al. 2000; Turman Jr and Chandler 1994b). It is possible that activation of premotor neurons in these areas did not cause excitation of trigeminal motoneurons large enough to be detected by our optical recording system.
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We then used focal laser photolysis of bath-applied caged glutamate to examine whether SupV neurons send excitatory inputs to the MoV. As mentioned earlier, electrical stimulation of the SupV evoked optical responses in the MoV. However, electrical stimulation may have activated axons of passage in the SupV and thus optical responses in the MoV may have been due to activation of neurons located in areas other than the SupV. To exclude this possibility, we examined whether selective activation of SupV neurons with caged glutamate photolysis induced excitatory postsynaptic currents (EPSCs) in trigeminal motoneurons of P1–P5 rats (n = 5). Whole cell patch-clamp recordings were obtained from trigeminal motoneurons from eight slices (n = 9; Fig. 2 A) and the lens was then switched to the x4 objective. When photostimulation was applied around the tip of the patch electrode, membrane depolarization was elicited at latencies of 0.7 ± 0.2 ms (n = 9; Fig. 2B). To minimize damage to the motoneurons or SupV neurons by the laser beam, the intensity of the laser was adjusted to be slightly stronger than the threshold for generating an action potential. One or two action potentials were consistently evoked in the motoneurons at latencies of 20.3 ± 3.5 ms (7.0–28.5 ms; n = 9) by each photostimulation under these conditions (Fig. 2B). Such long latency was probably due to the small amount of glutamate uncaged by the laser around the motoneurons. Next, the laser beam was focused on the SupV. In the presence of 5–10 µM strychnine, a glycine receptor antagonist, and 4–10 µM SR95531, a GABAA-receptor antagonist, photostimulation evoked rapidly rising inward currents in all motoneurons except one at latencies of 20.4 ± 3.8 ms (7.8–42.6 ms; n = 8) in voltage-clamp mode (Fig. 2C). The focus of the laser was set on the spot in the SupV where the rapidly rising inward currents were most frequently evoked. Some trigeminal motoneuron dendrites reportedly extend into the SupV in cats (Shigenaga et al. 1988) and thus uncaged glutamate in the SupV might have stimulated motoneuron dendrites. To exclude this possibility, we examined the effects of photostimulation of the SupV after addition of 0.5 µM TTX, which abolished the rapidly rising inward currents in all seven neurons tested (Fig. 2D). Thus the rapidly rising inward currents were most likely glutamatergic EPSCs. However, small, slowly rising inward currents were evoked immediately after photostimulation in three of the seven neurons (Fig. 2D, arrow). It is likely that they may have resulted from direct stimulation of the dendrites of recorded cells by uncaged glutamate.
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To characterize the nature of the transmitters involved in synaptic transmission between the SupV and the MoV in developing rats, we tested the effects of several receptor antagonists of fast synaptic transmission on SupV stimulation-induced optical responses in the MoV. Seventeen slices from 15 neonatal animals (P1–P6) and 14 slices from 9 juvenile animals (P7–P12) were used in this experimental series. In P1–P6 rats, exposure of the MoV to a combination of the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist CNQX (20 µM) and the NMDA receptor antagonist APV (20 µM) for 20 min significantly reduced the peak value of optical responses in the MoV by 42.8 ± 20.7% (n = 17, P < 0.01) compared with control responses (Fig. 3, A and G). The optical responses recovered to 89.8 ± 20.7% of the control level after the tissue was washed for 20 min with the normal ACSF. Interestingly, application of strychnine (10 µM) to the MoV significantly suppressed optical responses in the MoV by 55.4 ± 19.8% (n = 16, P < 0.01) (Fig. 3, B and G). The responses recovered to 82.8 ± 11.8% of control values after the tissue was rinsed for 20 min with the normal ACSF.
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SupV stimulation-evoked postsynaptic potentials and currents in developing jaw-closing and jaw-opening motoneurons
Immature neurons have relatively high intracellular Cl– concentrations ([Cl–]i) (DeFazio et al. 2000; Plotkin et al. 1997; Shimizu-Okabe et al. 2002). Thus glycinergic excitatory synaptic transmission from the SupV to the MoV in P1–P6 rats might be due to high [Cl–]i in trigeminal motoneurons. To confirm that glycinergic or GABAergic PSCs evoked by SupV stimulation were excitatory, we performed gramicidin-perforated patch-clamp recordings of MMNs (jaw-closing motoneurons) or DMNs (jaw-opening motoneurons) that left [Cl–]i undisturbed (Kyrozis and Reichling 1995). We also examined the contribution of glutamatergic receptors to SupV stimulation-evoked PSCs in MMNs and DMNs.
For recording from MMNs at P1–P4 or P7–P12, 20 slices from 15 neonatal animals and 11 slices from 8 juvenile animals were prepared, respectively. For recording from DMNs at P1–P4 or P7–P12, 18 slices from 10 neonatal animals and 10 slices from 8 juvenile animals were prepared, respectively. The resting membrane potentials of MMNs were –69.6 ± 1.0 mV for P1–P4 (n = 24) and –68.1 ± 3.0 mV for P9–P12 (n = 10); the resting membrane potentials of DMNs were –67.0 ± 1.6 mV for P1–P4 (n = 20) and –70.2 ± 3.4 mV for P9–P12 (n = 10). There were no significant between-group differences in resting potential (P > 0.05).
At P1–P4, electrical stimulation of the SupV evoked short-latency PSCs at 2.8 ± 0.4 ms in all MMNs (n = 6) and at 3.6 ± 0.3 ms in all DMNs (n = 5) tested (Fig. 4 A). Because all PSCs were observed as transient inward currents at a holding potential of –60 mV, SupV stimulation elicited EPSCs at this age. Combined bath application of CNQX (20 µM) and APV (20 µM) significantly reduced the amplitude of EPSCs in MMNs by 66.2 ± 8.0% (n = 6, P < 0.01) and in DMNs by 69.4 ± 2.6% (n = 5, P < 0.05). The remaining PSCs were inward. Subsequent addition of strychnine (10 µM) almost completely abolished the remaining PSCs in MMNs and DMNs (Fig. 4A). Partial recovery of EPSCs was observed in MMNs and DMNs after the antagonists were washed out for 20 min with normal ACSF. In current-clamp mode, SupV stimulation evoked depolarizing PSPs in MMNs (n = 6) and DMNs (n = 4) at membrane potentials of –60 mV in the presence of 20 µM CNQX and 20 µM APV (Fig. 4B). Under these conditions, an action potential (arrowhead) could be triggered by an EPSP evoked by SupV stimulation alone (Fig. 4B) or SupV stimulation with simultaneous injection of a depolarizing subthreshold intracellular current pulse (see Gulledge and Stuart 2003). Addition of 10 µM strychnine completely abolished the remaining PSPs. Pressure application of 1 mM glycine to MMNs (n = 6) and DMNs (n = 5) induced membrane depolarization and a remarkable decrease in membrane resistance in all neurons tested at P1–P4 in the presence of 20 µM CNQX and 20 µM APV (Fig. 4C). Bath application of 200 µM GABA to MMNs (n = 4) and DMNs (n = 5) induced membrane depolarization and a remarkable decrease in membrane resistance in all neurons tested (data not shown).
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In P9–P12 rats, gramicidin-perforated patch-clamp recordings revealed that SupV stimulation evoked EPSCs at a holding potential of –60 mV with a latency of 2.7 ± 0.3 ms in all MMNs (n = 7) and 2.8 ± 0.3 ms in all DMNs (n = 6) examined (Fig. 5 A). However, combined application of CNQX (20 µM) and APV (20 µM) revealed outward PSCs in all neurons tested (MMNs, n = 6; DMNs, n = 6). The remaining PSCs in MMNs and DMNs were almost completely abolished by subsequent addition of strychnine. We then performed whole cell recordings to examine the effects of SR95531 on MMNs and DMNS in P9–P12 rats. In four of five MMNs, SupV stimulation-evoked PSCs were significantly decreased by 10 µM SR95531 application by 36.5 ± 6.3% (n = 4, P < 0.05). In four of five DMNs, SR95531 also significantly reduced SupV stimulation-evoked PSCs by 56.1 ± 11.3% (n = 4, P < 0.01). In the remaining one MMN and one DMN, SupV stimulation-evoked PSCs were not altered by SR95531. Addition of 10 µM strychnine abolished the remaining PSCs in all five MMNs and five DMNs. These results suggest that some MMNs and DMNs of P9–P12 rats also received GABAergic and glycinergic inputs from the SupV similarly in P1–P4 rats. To determine the reversal potentials of the PSCs in the presence of CNQX and APV, we evoked PSCs by SupV stimulation at various holding potentials (Fig. 5B). In slices from P1–P4 rats, reversal potentials of strychnine-sensitive PSCs in MMNs and DMNs were –36.1 ± 5.2 mV (n = 6) and –27.4 ± 8.7 mV (n = 5), remarkably more depolarized than the resting potentials for both (Fig. 5, C and D). In contrast, the reversal potentials of the strychnine-sensitive PSCs for MMNs and DMNs at P9–P12 were –74.1 ± 3.9 mV (n = 6) and –75.6 ± 3.2 mV (n = 5), significantly more hyperpolarized than their resting potentials (P < 0.05) and the reversal potentials at P1–P4 (P < 0.01, Fig. 5, C and D). These results suggest that glutamatergic, glycinergic, and GABAergic PSCs in MMNs and DMNs evoked by SupV stimulation were excitatory at P1–P4; and glycinergic and, most likely, GABAergic PSCs changed from excitatory to inhibitory as the rats developed.
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We examined the physiological and morphological properties of SupV neurons projecting to the MoV. Whole cell patch recordings were made from 143 SupV neurons in 23 slices from 20 P1–P5 animals. Twelve (8.4%) neurons were antidromically activated by MoV stimulation at intensities of <25 µA and at constant latencies of 3.0 ± 0.1 ms (Fig. 6 Aa). Subtracting the 0.2-ms utilization time for initiation of the spike potential from and adding a 0.5-ms synaptic delay to the mean antidromic latency of 3.0 ms yields a 3.3-ms latency that approximated the 2.8-ms mean latency for EPSCs in MMNs and 3.6 ms in DMNs that were evoked by electrical stimulation of the SupV. Ten of the 12 antidromically activated SupV neurons tested showed tonic firing after we injected a 1-s depolarizing current pulse at the resting membrane potential (Fig. 6Ab). The remaining two neurons showed a rapid adaptation of firing in response to the current pulse injection. The responses of SupV neurons to serial hyperpolarizing and depolarizing current pulses are shown in Fig. 6Ac. Nine neurons had no depolarizing sag and showed a relatively linear I–V relation over hyperpolarized potentials. Three neurons had small depolarizing sags during step hyperpolarization.
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; Voogd et al. 1998). In rats, the dorsolateral division exists at the whole rostrocaudal level of the MoV, whereas the ventromedial division is found only at the caudal half of the MoV (Jacquin et al. 1983; Sasamoto 1979; Uemura et al. 2007). The neuron shown in Fig. 6 was recorded in the slice from the rostral part of the motor nucleus where only the dorsolateral division exists. Therefore this neuron likely projected to the jaw-closing motoneuron pool. The axon of one other neuron that traveled to the MoV in the lateral–ventral direction mainly reached the dorsolateral division. Axons of the remaining two neurons projected to the MoV in the medial–ventral directions and ramified broadly within the MoV; however, it was difficult to judge from our histological results whether their axons entered the ventromedial division as well as the dorsolateral division of the MoV. Thus all four of the biocytin-labeled neurons likely projected to the jaw-closing motoneuron pool, although further studies are necessary to confirm whether the SupV premotor neurons project to either jaw-closing or jaw-opening motoneurons, or both. |
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DISCUSSION |
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Glutamatergic inputs from the SupV to the MoV
Electrical stimulation of the SupV evoked optical responses and PSCs in the MoV. The stimulus intensities delivered to the SupV were 20–30 µA for optical recording and the area that was directly activated by electrical stimulation was confined within the SupV (see Fig. 1). For patch-clamp recordings the SupV was stimulated at intensities (5–20 µA) weaker than those used for optical recordings; therefore the stimulus field produced by the stimuli was likely to be confined within the SupV. Antidromic optical responses could be evoked in the SupV after MoV stimulation when synaptic transmission was suppressed by substitution of Mn2+ for external Ca2+. Twelve SupV neurons were antidromically activated by MoV stimulation and 4 of the 12 neurons made synaptic contacts with the motoneurons. These results suggest that a certain number of premotor neurons in the SupV target the MoV.
Turman Jr and Chandler (1994b) showed histochemically that the SupV contains glutamatergic premotor neurons that target the MoV. In the present study, application of CNQX and APV attenuated optical responses in the MoV and EPSCs in MMNs and DMNs that were induced by electrical stimulation of the SupV. Furthermore, in the presence of strychnine and SR95531, activation of SupV neurons by focal laser photolysis of bath-applied caged glutamate evoked the rapidly rising inward currents in P1–P5 trigeminal motoneurons. The focus of the laser beam was set on the spot in the SupV where the rapid inward currents were most frequently evoked. Photostimulation became less effective to evoke the rapid inward currents, when the focus of the laser was moved away from the spot (data not shown). These results suggest that glutamatergic excitatory premotor neurons projecting to MMNs and DMNs are likely located in the SupV.
Bourque and Kolta (2001), however, suggested interconnection between the SupV, the intertrigeminal region, and the medial peritrigeminal area in which premotor neurons targeting the MoV are located. Thus photostimulation of the SupV might have evoked EPSCs polysynaptically in trigeminal motoneurons and the shortest latency of EPSCs in trigeminal motoneurons evoked by SupV photostimulation (7.8 ms) was longer than the mean latencies for EPSCs in MMNs (2.8 ms) and DMNs (3.6 ms) evoked by electrical stimulation of the SupV. However, this discrepancy in the latencies was likely due to a delay in spike generation in SupV neurons after photostimulation because it took 7.0 ms to generate an action potential in trigeminal motoneurons by direct photostimulation at the intensities used in the present study. Therefore the EPSCs in the motoneurons were likely evoked monosynaptically by the photostimulation, although we cannot rule out the possibility for involvement of polysynaptic pathways, especially in cases of long latencies of the EPSCs.
Glycinergic and GABAergic inputs from the SupV to the MoV
The SupV has been shown histochemically to contain glycinergic and GABAergic premotor neurons that target the MoV (Li et al. 1996; Turman Jr and Chandler 1994a). In the present study, application of strychnine, bicuculline, and SR95531 altered optical responses in the MoV and EPSCs in MMNs and DMNs that were induced by electrical stimulation of the SupV.
The PSCs that remained after CNQX and APV application were almost completely abolished by strychnine. Bicuculline and SR95531 reduced the remaining PSCs by 52% in five of eight MMNs and by 34% in five of seven DMNs from neonates and by 37% in four of five MMNs and by 56% in four of five DMNs in juveniles. Therefore 10 µM strychnine reduced GABAergic and glycinergic PSCs. However, strychnine abolished the remaining PSCs after the addition of bicuculline and SR95531 to CNQX and APV; apparently, all MMNs and DMNs receive glycinergic inputs from the SupV. In contrast, bicuculline and SR95531 did not abolish the remaining PSCs after application of CNQX and APV and they had little effect on the remaining PSCs in three MMNs and two DMNs in the neonates and in one MMN and one DMN in the juveniles. Apparently, MMNs and DMNs receive GABAergic inputs that are weaker than glycinergic inputs from the SupV. The number of glycine-immunoreactive boutons synapsing on MMNs and DMNs in cats is close to twice the number of GABA-immunoreactive boutons (Bae et al. 1999; Shigenaga et al. 2005). The SupV may be the origin of glycinergic and GABAergic axon terminals at MMNs and DMNs. Our observation of stronger glycinergic than GABAergic inputs from the SupV to the MoV in the present study is consistent with the abundance of glycine-immunoreactive boutons at MMNs and DMNs reported in the studies cited earlier.
Glycine and GABA evoke membrane depolarizations during fetal and early postnatal life in many areas of the nervous system (Ben-Ari et al. 1989; Luhmann and Prince 1991; Owens et al. 1996), including the spinal cord (Reichling et al. 1994; Wu et al. 1992) and brain stem (Kandler and Friauf 1995; Singer et al. 1998), as well as adult spinal laina I neurons following peripheral nerve injury (Coull et al. 2003). In the present study, strychnine reduced optical responses evoked in the MoV by SupV stimulation and the CNQX/APV-insensitive inward currents of MMNs and DMNs in perforated patch-clamp recordings were almost abolished by strychnine in the brain stem of P1–P6 neonatal rats. The mean reversal potentials of strychnine-sensitive PSCs in neonate MMNs and DMNs were –36.1 and –27.4 mV, respectively, that were much more depolarized than their resting membrane potentials. Application of glycine and GABA depolarized neonate MMNs and DMNs. However, strychnine enhanced the optical responses in the MoV and strychnine-sensitive PSCs in MMNs and DMNs became outward in the brain stems of P7–P12 juvenile rats. These results indicate that glycinergic and GABAergic inputs from the SupV depolarize MMNs and DMNs in neonates, whereas SupV glycinergic and likely GABAergic inputs hyperpolarize MMNs and DMNs in juvenile rats. Such differences in the synaptic transmission between neonates and juveniles were not due to the different use of the ACSFs during slice preparation because application of glycine or GABA also evoked depolarization in all motoneurons tested, obtained from the slices prepared using modified ACSF, similarly in the motoneurons prepared with normal ACSF.
GABA- and glycine-induced membrane depolarization has been shown to be caused by Cl– efflux due to high [Cl–]i maintained in immature cells (Ben-Ari 2002; Owens et al. 1996). Increased expression of K-Cl cotransporter isoform 2 (KCC2) is believed to underlie the functional conversion of GABA from excitatory to inhibitory (DeFazio et al. 2000; Rivera et al. 1999). Because KCC2 mRNA is expressed in the MoV of adult rats (Toyoda et al. 2005), developmental up-regulation of KCC2 may occur in MMNs and DMNs and lead to a transition from membrane depolarization to membrane hyperpolarization in response to glycine and/or GABAA receptor activation.
Roles of premotor neurons in the SupV
The present study showed that a wide variety of inputs (i.e., glutamatergic, glycinergic, and GABAergic) is sent to MMNs and DMNs from the SupV. Intracellular staining of four SupV premotor neurons also shows that there were at least three patterns of axonal projection to the MoV. Recently, Hsiao et al. (2007) reported a morphological and electrophysiological diversity in SupV neurons, although the SupV neurons recorded in their study were not identified as premotor neurons for targeting the MoV. Such diversities of SupV premotor neurons may indicate that those neurons play a wide variety of roles in orofacial motor function.
Periodontal and spindle afferents (Dessem and Taylor 1989; Jerge 1963; Mizuno 1970; Nishimori et al. 1986; Nomura and Mizuno 1985; Shigenaga et al. 1989) and orofacial afferents (Goldberg 1972; Sessle 1977) facilitate jaw-closing muscle activity and the SupV is a recipient of such afferents. Therefore it is possible that excitatory glutamatergic premotor neurons in the SupV are involved in increasing jaw-closing muscle activity via sensory inputs during mastication.
Glycinergic interneurons that inhibit jaw-closing motoneurons during the JOR are assumed to exist in the SupV (Kidokoro et al. 1968; Nakamura et al. 1973). This assumption is strongly supported by our present finding that SupV stimulation elicits IPSCs in MMNs of juvenile rats. The JOR contributes to protecting the mouth by inhibiting jaw-closing muscle activity when a hard piece of food or a stone is bitten or when sharp material, such as a fish bone, pierces the oral mucosae during mastication. However, inhibitory glycinergic and GABAergic effects from the SupV emerged after P7 in the present study and this emergence preceded the initiation of immature mastication at around P12 (Westneat and Hall 1992). At around P7, the teeth begin to erupt (Asahito et al. 1999; Nakakura-Ohshima et al. 1993) and the tongue or buccal mucosa may then be bitten by the erupting teeth. Thus inhibition of jaw-closing muscle activities by elicitation of the JOR is likely needed to prevent the oral mucosa from bites after tooth eruption. In fact, latency of the JOR decreases drastically after P6 (Iriki et al. 1983), suggesting that the JOR comes into function on tooth eruption. In the present study, glycinergic and GABAergic inputs from the SupV to the MoV were excitatory in neonatal rats. Because neonates feed by suckling and ingest only milk, inhibition of the jaw-closing muscles may not be necessary. Instead, such excitatory glycinergic and/or GABAergic synaptic transmission to the MoV might promote the maturation of local neural circuits by Ca2+ influx in immature neurons through voltage-gated Ca2+ channels (see Flint et al. 1998; Hsiao et al. 2005; Lo et al. 1998). Thus the developmental switch of glycinergic and GABAergic effects from the SupV to MMNs and DMNs could play an important role in the smooth transition from suckling to mastication by function development of the JOR prior to the initiation of mastication.
SupV stimulation evoked glutamatergic EPSCs and EPSPs in DMNs of neonates and juveniles in the present study. SupV neurons, however, do not appear to be responsible for excitation of DMNs during the JOR because lesions of the oral and interpolar parts of the spinal trigeminal nucleus that spared the SupV abolished the JOR induced by stimulation of the inferior alveolar nerve (Sumino 1971) and because application of lidocaine to the caudal part of the spinal trigeminal nucleus significantly suppressed temporomandibular joint-evoked reflex of the digastric muscles (Cairns et al. 2001).
Stimulation of the SupV also evoked glycinergic and GABAergic IPSCs in DMNs of juvenile rats. When there is a low level of continuous background activity in the jaw-opening muscles in cats and humans, the jaw-jerk elicited by tapping on the chin occasionally inhibits the jaw-opening muscles (Matthews 1975). Because the SupV receives spindle afferents from the jaw-closing muscles, SupV neurons might inhibit DMNs in a way similar to the Ia inhibitory interneurons for limb muscles. However, stimulation of the mesencephalic trigeminal nucleus, which contains the spindle cell bodies, does not evoke short-latency PSCs in DMNs of anesthetized cats (Kidokoro et al. 1968). The inhibition of DMNs during the jaw-jerk reflex might be easily masked in anesthetized animals.
Kogo et al. (1996) induced rhythmic activity in the motor branches of the trigeminal nerves in isolated brain stem preparations from neonatal rats by application of N-methyl-D-aspartate (NMDA). They suggested, by transection of the preparations, the existence of rhythmogenetic circuits between the trigeminal and facial motor nuclei, which include the SupV. Enomoto et al. (2002) developed slice preparations that consist of islands attached to a 300-µm area surrounding the MoV and including the SupV. They recorded rhythmic motoneuronal activity during NMDA application and proposed that premotor neurons that drive NMDA-induced rhythmic activity are located in the 300-µm area surrounding the MoV. In anesthetized adult rats, the rhythmic neuronal activity corresponding to the masticatory rhythm during cortically induced fictive mastication can be recorded from the SupV (Inoue et al. 1992). Furthermore, Hsiao et al. (2007) showed that some of these neurons have intrinsic burst-generating capabilities. Thus it is possible that SupV neurons are involved in generation of suckling and/or masticatory rhythm. It is also possible that SupV neurons transmit trigeminal rhythm inputs from the rhythm generator possibly located in the SupV or another area. The SupV may be involved in facilitating or inhibiting activity of the jaw-closing and jaw-opening muscles to ensure appropriate jaw movement during feeding.
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Address for reprint requests and other correspondence: T. Inoue, Department of Oral Physiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan (E-mail: inouet{at}dent.showa-u.ac.jp)
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ABSTRACT
INTRODUCTION
